Skin POMC peptides: their actions at the human MC-1 receptor and roles in the tanning response.

The melanocortin 1 (MC-1) receptor is a key control point in the regulation of skin pigmentation. Alpha-MSH is an agonist at this receptor and through its activation regulates melanocyte function. alpha-MSH is cleaved from pro-opiomelanocortin (POMC) in the pituitary, but in humans the skin is a more important source of the peptide. Skin pigmentation is therefore regulated by locally produced alpha-MSH rather than that of pituitary origin. alpha-MSH acts as a paracrine and/or autocrine mediator of UV induced pigmentation. However, the predominant alpha-MSH in human skin is desacetyl alpha-MSH and, compared to the acetylated form, is a relatively weak agonist at the human MC-1 receptor. By acting as a partial agonist desacetyl alpha-MSH may even oppose the actions of acetylated alpha-MSH and other MC-1 receptor agonists. The most abundant MC-1 receptor agonist in human epidermis is ACTH1-17. This POMC peptide, which is produced by keratinocytes, is more potent than acetylated alpha-MSH in stimulating melanogenesis in human melanocytes and, in contrast to the latter, produces a biphasic dose-response curve. This is probably a consequence of its activation of both the cAMP and IP3/DAG signalling pathways. alpha-MSH peptides, on the other hand, selectively activate the cAMP pathway. Compared with alpha-MSH, ACTH1-17 could have the more important role as a paracrine mediator of melanogenesis and other melanocytic processes. However, ACTH1-17 is not the only POMC peptide in the skin and may interact with related peptides at the MC-1 receptor. These interactions are likely to represent important determinants of melanocyte function and skin pigmentation.

Melanin-concentrating hormone, melanocortin receptors and regulation of luteinizing hormone release.

Melanin-concentrating hormone (MCH) is a neuropeptide, identified by its ability to either mimic or antagonize the melanin-dispersing action of alpha-melanocyte stimulating hormone (alphaMSH) on skin melanophores. MCH and alphaMSH also have antagonistic actions in the brain affecting feeding behaviour, aggression, anxiety, arousal and reproductive function through the release of luteinizing hormone (LH). It is not clear, however, how they exert their opposite effects in the central nervous system (CNS). One possibility is that they act via a common receptor. In this study we have examined the effect of a number of MC receptor antagonists, with relative selectivity for the MC3, 4 and 5 subtypes, on the actions of MCH on LH release. We confirmed that bilateral administration of MCH (100 and 200 ng/side) into the medial preoptic area of oestrogen-primed (oestradiol benzoate 5 microgram) ovariectomized anaesthetized rats, stimulated the release of LH. This effect was blocked by the concomitant administration into the medial preoptic area of the MC4/5 antagonist ([D-Arg8]ACTH(4-10) and the MC3/5 antagonist ([Ala6]ACTH(4-10)-both at 500 ng/side-but not by the MC3/4 antagonist, SHU9119 (200 ng/side). Furthermore, the MC3 agonist [Nle3]-gamma2 MSH failed to affect LH release. These results indicate that the MC3 and MC4 receptors are not involved in mediating the action of MCH but are consistent with an action via the MC5 subtype. Preputial glands, which express MC5 receptors, were also stimulated by MCH which is in keeping with this idea. In HEK293 cells transfected with the MC5 receptor MCH increased the production of IP3. However, it was much less potent than alphaMSH and unlike alphaMSH, had no effect on the production of cAMP. MCH (10-10 to 10-5 M) also failed to displace I125NDP-MSH from cells transfected with MC5 receptors indicating that it was not acting as a competitive antagonist and its binding site was distinct from that of alphaMSH. Thus while MCH may function as an agonist at the MC5 receptor, its stimulation of LH release is more likely to be mediated via a specific MCH receptor that has common properties with the MC5 receptor.

ACTH1-17 is a more potent agonist at the human MC1 receptor than alpha-MSH.

The melanocortin receptor MC1 is expressed on melanocytes and is an important control point for melanogenesis and other responses. Alpha-MSH, which is considered to be the major ligand at the human melanocortin (MC)1 receptor (hMC1R), is produced from proopiomelanocortin (POMC) in the pituitary and in the skin by melanocytes and keratinocytes. Other POMC peptides are also produced in the skin and their concentrations exceed those of alpha-MSH by several fold. One of the most abundant is ACTH1-17. We have shown that adrenocorticotrophic hormone (ACTH)1-17 is more potent than alpha-MSH in stimulating melanogenesis in human melanocytes and unlike alpha-MSH produces a biphasic dose response curve. In this study we have examined the ability of ACTH1-17 to function as a ligand at the hMC1R. Competitive binding assays with [125I]Nle4 DPhe7 alpha-MSH as labelled ligand were carried out in HEK 293 cells transfected with the hMC1R. ACTH1-17 showed high affinity for the hMC1R with a Ki value of 0.21 +/- 0.03 nM which was slightly higher than that of 0.13 +/- 0.005 nM for alpha-MSH. ACTH1-17 was, however, more potent than alpha-MSH in increasing cAMP and IP3 production in the transfected cells. Our results demonstrate that ACTH1-17 is a potent agonist at the hMC1R. It is therefore possible that ACTH1-17, which is found in the skin in greater concentrations than alpha-MSH, has an important role in the regulation of human melanocytes and other cell types that express the hMC1R.

Binding of human IgM from a rheumatoid factor to IgG of 12 animal species.

The binding of IgM from a rheumatoid factor (RF-IgM) to IgG from 12 animal species was analyzed by an ELISA system. The RF-IgM bound various animal IgG with dissimilar affinities. The binding of RF-IgM to animal IgG was inhibited by addition of protein A, which binds some animal IgG by recognizing the junctional site on CH2-CH3 domains in the Fc region. As previously reported, no significant correlation was observed between the binding of RF-IgM to IgG and the content of galactose-free oligosaccharides, which is increased in IgG of rheumatoid arthritis patients or autoimmune mice. We suggest that the crucial epitope of IgG for RF-IgM binding is not the oligosaccharide structure generated specifically in IgG of autoimmune diseases but that RF-IgM may recognize a certain protein conformation of a region in IgG near the binding site of protein A.

Enterotoxicity and immunological properties of two mutant forms of Escherichia coli STIp with lysine or arginine substituted for the asparagine residue at position 11.

Two variants of Escherichia coli heat-stable enterotoxin Ip, in which the amino acid residue at position 11 was substituted with lysine or arginine, were purified to near homogeneity from the culture supernatants of toxin-producing mutant strains. Neither the purified heat-stable enterotoxin Ip(Lys-11) nor the purified heat-stable enterotoxin Ip(Arg-11) showed a positive response in the suckling mouse assay or in the mouse intestinal loop assay. Furthermore, live bacteria producing these mutant heat-stable Ip enterotoxins did not cause fluid accumulation in mouse intestinal loops, in contrast to bacteria producing native heat-stable enterotoxin Ip. Nevertheless, antisera raised against both heat-stable enterotoxin Ip(Lys-11) and heat-stable enterotoxin Ip(Arg-11) neutralized the enterotoxic activity of native heat-stable enterotoxin Ip. These results demonstrate that heat-stable enterotoxin Ip(Lys-11) and heat-stable enterotoxin Ip(Arg-11) lose enterotoxicity but retain epitopes which are common to native heat-stable enterotoxin Ip.

Reduction of enterotoxic activity of Escherichia coli heat-stable enterotoxin by substitution for an asparagine residue.

The Escherichia coli heat-stable enterotoxins (STs) are small peptide toxins consisting of 18 (STp) or 19 (STh) amino acids. STp and STh share biologically active sequences which reside in the C-terminal 13 amino acid residues, but the role of each amino acid in the active sequences is not clear. We substituted in vivo Asp, Tyr, His, Gln, Lys, and Arg for the Asn residue at position 11 of STp by oligonucleotide-directed site-specific mutagenesis and examined the biological activities of the resulting mutants. All mutant STs reacted with both monoclonal and polyclonal antibodies, demonstrating that the amino acid substitutions at position 11 did not cause a significant change in the conformation of STp. However, the substitutions invariably caused a significant decrease in enterotoxic activities. The most remarkable decrease was observed with Asn-11----Lys-11 and Asn-11----Arg-11 mutations; that is, enterotoxic activity could not be detected in the culture supernatant of either of these mutant strains. These results indicate that Asn-11 of STp plays an essential role in the enterotoxic activity. The amide group and the length of side chain of Asn-11 seem to be especially important for enterotoxic activity.

Substitutions of cysteine residues of Escherichia coli heat-stable enterotoxin by oligonucleotide-directed mutagenesis.

The Escherichia coli 18-amino-acid, heat-stable enterotoxin STp has six cysteine residues linked intramolecularly by three disulfide bonds. These disulfide bonds are important for toxic activity, but the precise role of each bond is not clear. We substituted cysteine residues of STp in vivo by oligonucleotide-directed site-specific mutagenesis to dissociate each disulfide bond and examined the biological activities of the resulting mutants. The Cys-6----Ala and Cys-17----Ala mutations caused a complete loss of toxic activity. The Cys-5----Ala, Cys-10----Ser, and Gly-16, Cys-17----Cys-16, Gly-17 mutations caused a large decrease in toxic activity. These results mean that all three disulfide bonds formed at fixed positions are required for full expression of the biological activity of STp. However, a weak but significant toxicity still remained after three mutations, Cys-5----Ala, Cys-10----Ser, and Gly-16, Cys-17----Cys-16, Gly-17. This indicates that STp has some flexibilities in its conformation to exert toxic activity and that the role of each disulfide bond exerting toxic activity is not quite the same.

Cytotoxicity and calcium-dependent antigen of Yersinia.

The relationship between invasiveness and calcium dependency was examined in various strains of Yersinia enterocolitica and Y. pseudotuberculosis by using established cell lines. Infection with calcium-dependent bacteria resulted in the formation of microvilli and the adherence of bacteria on the cell surface, and the adherent bacteria were ingested 1.5 hr after infection. Morphological changes in the cells became visible 2 to 3 hr after infection, and intracellular multiplication of the ingested bacteria was noted. When the cells were incubated with bacteria at 37 C for 1.5 hr and then at 25 C, however, the morphological changes in the infected cells were not observed. No isogenic strains that had lost calcium dependency for growth at 37 C were able to elicit the morphological changes in the cells, though they possessed the ability to adhere to and penetrate the cells. The antigen(s) supposedly related to cytotoxicity of the calcium-dependent Yersinia was sought by using antibodies prepared against calcium-dependent bacteria and then absorbed with calcium-independent bacteria and with calcium-independent bacterial cytosol. Double diffusion tests between the antisera and bacterial cytosol extracts revealed the presence of an antigen which was a cytoplasmic substance common to all calcium-dependent but not calcium-independent strains of Y. enterocolitica and Y. pseudotuberculosis.