Serum levels of insulin-like growth factors (IGFs) and their binding proteins (IGFBPs), -1, -2, -3, in oral cancer.

INTRODUCTION: Insulin-like growth factor (IGF) possesses mitogenic properties promoting cellular proliferation and inhibiting cellular apoptosis. Reported evidence suggests that cancer (non-oral) is associated with high circulating levels of IGF-1 and low levels of IGFBP-3. AIMS: This study measured circulating levels of IFG-1 and IGFBPs -1, -2 and -3 in oral cancer patients. METHODOLOGY: Blood was collected from surgical patients and controls. Samples were assayed for IGF-1 and IGFBPs -1, -2, -3, using commercially available enzyme-linked immunosorbant assays (ELISA; R&D Systems Europe, Oxon, UK). RESULTS: Twenty-seven oral cancer and 31 age- and sex-matched patients were recruited. Mean IGF-1 and IGFBP-3 levels were significantly lower in cancer patients than controls (85.3 ng/ml and 2008 ng/ml versus 191 ng/ml and 2935 ng/ml, P<0.001). In contrast, levels of IGFBPs 1 and 2 were significantly higher in cancer patients than in controls. No significant association was demonstrated with tumour size or nodal metastases. DISCUSSION: This study has shown that in contrast to other cancers, circulating levels of IGF-1 and IGFBP-3 may both be lowered in patients with head and neck cancer.

Differentiation of the HaCaT keratinocyte cell line: modulation by adrenomedullin.

BACKGROUND: Adrenomedullin (AM) is a multifunctional peptide produced by a wide variety of cells, including keratinocytes. We, and others, have demonstrated that AM has a role as a growth regulatory factor of the skin and contributes as an antimicrobial agent in the integument's protective barrier. It is not known whether AM has a role in differentiating keratinocytes. OBJECTIVES: To study the role of AM in keratinocyte differentiation, modulating the effects of calcium and in addition, to assess whether differentiated keratinocytes are still capable of initiating an inflammatory response. METHODS: HaCaT cells were differentiated using CaCl2. Expression of transglutaminase type 1 (TG1) and E2F1 genes was used to monitor differentiation. AM secretion was measured by enzyme-linked immunosorbent assay (ELISA). NF-kappaB activity and interleukin (IL)-6 secretion in the cells were assessed after exposure to calcium and AM by electrophoretic mobility shift assay and ELISA, respectively. RESULTS: Secretion of AM by the keratinocyte cell line HaCaT was found to be increased during 1 mmol L(-1) CaCl2-induced cell differentiation but not 0.1 mmol L(-1) CaCl2. All treatments showed low levels of the cell proliferation marker, E2F1. Over time, cells incubated in the presence of 0.1 mmol L(-1) or 1 mmol L(-1) of CaCl2 showed an increase in TG1 expression, a marker of early differentiation. The addition of AM showed a decrease in TG1 expression when combined with 0.1 mmol L(-1) CaCl2, but not with 1 mmol L(-1) CaCl2. In addition, cells kept in 0.1 mmol L(-1) CaCl2 showed translocation of NF-kappaB after 48 h and 72 h of incubation, which was abolished when AM was added to the cells. Treatment with 1 mmol L(-1) CaCl2 led to earlier translocation of NF-kappaB at 24 h after treatment and addition of AM did not abolish the effect of 1 mmol L(-1) CaCl2 on NF-kappaB activation. Cells incubated in 0.1 mmol L(-1) CaCl2 showed increased secretion of IL-6 over time, consistent with NF-kappaB activation. The addition of AM to cells incubated with 0.1 mmol L(-1) CaCl2 showed a rapid decrease in IL-6 secretion after only 6 h. However, 1 mmol L(-1) CaCl2 did not induce secretion of IL-6 and the addition of AM did not affect the result. CONCLUSIONS: Our data indicate that AM can reverse calcium-induced differentiation when 0.1 mmol L(-1) CaCl2 is used but not 1 mmol L(-1) CaCl2. Cells differentiated with 0.1 mmol L(-1) CaCl2 are still capable of generating an inflammatory response, showing signs of late NF-kappaB activation and IL-6 secretion that can be inhibited by AM. However, cells differentiated with 1 mmol L(-1) CaCl2 lose their ability to secrete IL-6 but not AM, which could be acting as an antimicrobial peptide.

A human oral keratinocyte cell line responds to human heat shock protein 60 through activation of ERK1/2 MAP kinases and up- regulation of IL-1beta.

Heat shock proteins (HSP) are released by cells in response to stress signals. It is hypothesized that pathogenic bacteria stimulate the cells in the periodontium to up-regulate the expression of HSP60, which would stimulate macrophages, and possibly other cells, to produce proinflammatory cytokines. We sought to determine whether oral keratinocytes responded to recombinant human HSP60 and to identify the signalling pathways involved. In addition, whether oral keratinocytes are a source of endogenous HSP60 was also investigated. RT-PCR revealed that rhHSP60 induced expression of the IL-1beta gene in the Human Oral Keratinocyte (HOK-16B) cell line and it was highest at the lowest concentration used (0.1 microg/ml). These responses were mediated via activation of p44/42 MAP-kinases and to a lesser extend the MAP-kinase SAP/JNK. Similar data was obtained from analysis of intracellular signalling pathways in HOK-16B cells by rhHSP70 and LPS (from both E. coli and the oral pathogen Porphyromonas gingivalis). However, there was little activation of p38 by rhHSP60. Blocking of the p44/42 pathway decreased HSP60-induced IL-1beta gene expression and protein secretion. In addition, we discovered that self-HSP60 proteins were constitutively secreted by HOK-16B cells. Secretion of self-HSP60 was up-regulated in cells treated with LPS from P. gingivalis, but down-regulated with LPS from E. coli. To summarize, oral keratinocytes respond to exogenous HSP60 by triggering expression of the inflammatory cytokine IL-1beta through activation of p44/42 MAP kinase. Oral keratinocytes are also a source for self-HSP60 and the secretion of this protein may be differentially modified by LPS from different bacterial species. These results highlight the importance of oral keratinocytes and HSPs in the development of an immune response against bacterial infection.

Expression patterns of integrins on pleomorphic adenoma and adenoid cystic carcinoma: study on specimens and in vitro investigation of the effects of extracellular matrix on the expression of these adhesion molecules.

BACKGROUND: Pleomorphic adenoma (PA) and adenoid cystic carcinoma (ACC) are neoplasms of distinct behaviour, showing similar origin, cell components and marked presence of extracellular matrix (ECM). Interactions between cells and ECM are important in the biology of tumours, being partially mediated by integrins. This study investigated these interactions on PA and ACC using paraffin-embedded tissue and an in vitro model of these conditions. METHODS: Expression of integrins in paraffin-embedded samples was assessed by immunohistochemistry. Cells from PA and ACC were characterized using immunofluorescence, and integrin patterns of expression were investigated on cells cultivated on different ECM proteins. RESULTS: Luminal cells of both PA and ACC were more intensely positive for integrins than myoepithelial cells. In vitro studies revealed that PA cells expressed more integrins than ACC cells regardless the ECM protein present. CONCLUSIONS: This study revealed particular patterns of integrin expression in both specimens and in vitro models of PA and ACC. This might prove useful for a better understanding of the biology of these lesions.

Expression of adrenomedullin and its receptors in human salivary tissue.

Adrenomedullin is a multifunctional peptide produced by a wide range of different cells and tissues. This study was designed to investigate whether adrenomedullin is present in human saliva and in salivary glands. It was expected that saliva may contain high concentrations of adrenomedullin, which has antimicrobial activity in vitro, which may have functional implications in the oral cavity. Saliva from the submandibular and parotid glands contained higher concentrations of adrenomedullin than did the circulation, but lower concentrations than in whole saliva. This suggests that oral epithelium may contribute the majority of the adrenomedullin peptide found in saliva. Specific adrenomedullin receptors were found in cell lines from the submandibular (HSG) and parotid (HSY) salivary glands. These findings suggest a paracrine/autocrine role for adrenomedullin in these tissues; however, the concentration of adrenomedullin in saliva was insufficient to suggest a significant antimicrobial action in the healthy oral cavity.

Salivary gland tumours: immunoexpression of integrins beta 1, beta 3 and beta 4.

BACKGROUND: Integrins, heterodimeric transmembranic adhesion molecules composed of alpha and beta subunits, have been implicated in normal and neoplastic biological processes. We investigated the patterns of expression of integrins subunits beta 1, beta 3 and beta 4 in neoplasms derived from the terminal segment of salivary glands in order to understand their expression patterns in the different structures of these tumours. METHODS: Immunohistochemistry using 'catalized signal amplification' (CSA) detection system was applied to paraffin-embedded specimens of polymorphous low-grade adenocarcinoma, adenoid cystic carcinoma and acinic cell carcinoma. Normal salivary glands were used as controls. RESULTS: Immunoexpression of integrins showed that neoplastic structures of the tumours studied tend to mimic the patterns seen in the normal structures of salivary glands from which they are derived. Anaplastic cells were negative for all integrins. CONCLUSIONS: This study revealed detailed topography of integrins in malignant tumours derived from intercalated acinar segment of salivary gland and this might be useful in the diagnosis of these lesions, especially of fine-needle aspiration (FNA) products or small specimens derived form incisional biopsies.

Adrenomedullin: expression and possible role in human skin and hair growth.

BACKGROUND: Adrenomedullin (AM) is a regulatory peptide that is synthesized and secreted by a wide number of cells and tissues. AM is a potent vasodilator, but also exerts other functions, such as regulating cell growth and antimicrobial defence. Two receptors, L1 and calcitonin receptor-like receptor (CRLR), which are able to bind AM, have been cloned and characterized. OBJECTIVES: To investigate expression of AM protein and its receptors in human skin and during different stages of the human hair cycle and, moreover, because of the suggested antimicrobial function of AM in skin, to investigate AM immunoreactivity (IR) in inflammatory acne lesions compared with healthy pilosebaceous follicles. METHODS: We used immunohistochemistry to determine the distribution of AM and its receptors in human skin and during different stages of the human hair cycle. AM IR in inflammatory acne lesions was investigated to evaluate the antimicrobial function of the protein, and hair follicle cultures were performed to examine the role of AM in differentiation and proliferation of hair follicle keratinocytes. RESULTS: Strong IR for AM and its receptors was present in the suprabasal epidermis, in the melanocytes of the epidermis, and in sweat and sebaceous glands. In the hair follicle, AM protein was strongly expressed in the basal and suprabasal layers of the hair bulb and the proximal outer root sheath (ORS). In the distal ORS, AM expression was increasingly suprabasal, especially in proximity to the bulge region where the basal cell layer was free of IR. IR for the CRLR revealed a similar expression pattern to that seen for AM. In contrast, L1 IR showed a suprabasal pattern of IR throughout the ORS. Similar expression of AM and its receptors was observed in catagen and early anagen follicles. AM expression was not markedly upregulated in acne lesions, suggesting a minor role for this antimicrobial peptide in acne. Despite its well-documented mitogenic effects, particularly in oral and skin keratinocytes, AM had no significant effect on hair follicle growth in vitro. CONCLUSIONS: AM and its receptors are expressed in human hair follicles, and both AM and its receptors are colocalized in the same compartments and cell types of the skin. This finding is consistent with the proposed autocrine/paracrine mechanism in the physiology of AM.

Regulation of rat adrenal vasoactive intestinal peptide content: effects of adrenocorticotropic hormone treatment and changes in dietary sodium intake.

Vasoactive intestinal peptide (VIP) is well established as a paracrine regulator of adrenal function. It is present in nerves supplying the adrenal cortex, although previous studies have found that the amount of VIP in the outer zones of the rat adrenal is not affected by ligating the splanchnic nerve supplying the adrenal gland. The present studies were designed to investigate the mechanisms involved in regulating the VIP content of the rat adrenal gland. This study examined the effects of changes in electrolyte balance and adrenocorticotropic hormone (ACTH) administration on the adrenal content of VIP as measured by radioimmunoassay. Rats on a low sodium diet had a significantly increased capsular/zona glomerulosa immunoreactive VIP (irVIP) level, while rats on a high sodium diet had suppressed levels relative to controls. Changes in dietary sodium did not affect inner zone/medullary VIP content. Administration of ACTH caused a decrease in irVIP levels in the capsular/zona glomerulosa portion of the adrenal gland but had no effect on the inner zone/medulla. Analysis of mRNA encoding VIP revealed a large increase in expression of VIP in the sodium-deplete group compared with the control, with no change in VIP expression in the sodium-loaded group. ACTH treatment was found to significantly decrease VIP mRNA levels in the capsular portion. Neither ACTH treatment nor changes in sodium intake affected inner zones/medullary VIP message. These data suggest that VIP in the capsule and zona glomerulosa region of the adrenal cortex is regulated in response to the physiological status of the animal, with changes in capsular/zona glomerulosa VIP correlating with changes in zona glomerulosa function.

Regulation of adrenomedullin secretion in cultured human skin and oral keratinocytes.

Adrenomedullin, a potent vasoactive peptide, is actively secreted from primary cultures of human oral and skin keratinocytes, but nothing is known of the regulation of its release. This study describes the effects of a range of substances on adrenomedullin production from cultures of oral and skin keratinocytes. We have established that keratinocytes do not store adrenomedullin but secrete it constitutively. Cytokines interleukin-1alpha and -1beta, tumor necrosis factor-alpha and -beta, and the bacterial product, lipopolysaccharide, significantly stimulate adrenomedullin secretion from oral but not skin keratinocytes. Both transforming growth factor-beta1 and interferon-gamma are potent suppressors of adrenomedullin secretion from both cell types, as are forskolin, di-butyryl cyclic adenosine monophosphate, and adrenocorticotropin. The peptides thrombin and endothelin-1 increase adrenomedullin production, particularly from skin keratinocytes. These findings indicate that there are differences in the regulation of adrenomedullin production between oral and skin keratinocytes and that oral keratinocytes are particularly responsive to the action of inflammatory cytokines. This raises the possibility that adrenomedullin may serve a different functions in oral mucosa and skin.

Adrenomedullin expression in pathogen-challenged oral epithelial cells.

Adrenomedullin, a multifunctional peptide, is expressed by many surface epithelial cells and, previously, we have demonstrated that adrenomedullin has antimicrobial activity. The oral cavity contains an epithelium that is permanently colonized by microflora, yet infections in a host are rare. We exposed oral keratinocytes to whole, live cells from four microorganisms commonly isolated from the oral cavity, Porphyromonas gingivalis, Streptococcus mutans, Candida albicans and Eikenella corrodens. There was upregulation of protein and gene expression in these cells in response to bacterial suspensions, but not with the yeast, Candida albicans. We propose there is a potential role for microbial products in enhancing mucosal defense mechanisms and that adrenomedullin participates in the prevention of local infection, thus contributing to host defense mechanisms.

Autocrine role of adrenomedullin in the human adrenal cortex.

Previous studies from our laboratory have reported that adrenomedullin is synthesised in rat zona glomerulosa cells. In the present studies, it was found that the human adrenocortical cell line H295R expresses the gene encoding adrenomedullin, and that immunoreactive adrenomedullin is released into the culture medium. Furthermore, it was found that secretion of adrenomedullin is regulated by angiotensin II and forskolin. Studies on the actions of adrenomedullin and calcitonin gene-related peptide (CGRP) revealed a stimulatory effect of adrenomedullin, but not of CGRP, on aldosterone and cortisol secretion. These data suggest that adrenomedullin is not acting by a CGRP receptor-mediated mechanism in the H295R cell line. Adrenomedullin was also found to increase cAMP production, suggesting that in the adrenal, as in other cell types, cAMP is a second messenger for adrenomedullin action. However, the effects of adrenomedullin were not fully mimicked by forskolin, possibly suggesting a role for an additional second messenger. The presence of mRNA encoding both the putative adrenomedullin receptors, L1 and calcitonin receptorlike receptor/receptor-associated modulatory protein 2 (CRLR/RAMP-2), was demonstrated in H295R cells, but RAMP-1 was not detected, suggesting that these cells do not express the CGRPI receptor CRLR/RAMP-1. Taken together, these data have demonstrated that adrenomedullin is synthesised and secreted by H295R cells. The observed rate of adrenomedullin synthesis suggests that this peptide exerts a paracrine/autocrine effect in this adrenocortical cell line, probably acting through a specific adrenomedullin receptor, to stimulate steroidogenesis and increase aldosterone synthase expression.

Aldosterone secretion by the rat adrenal cortex is stimulated by the activation of protease-activated receptor 1.

Stimulation of aldosterone by a serine protease, trypsin, was first reported in 1982, although the mechanism of this effect was unclear. Recently, a family of protease-activated receptors (PARs) has been described and four members of the family characterised and cloned, including the previously recognised thrombin receptor. This study investigated whether PARs mediate the action of trypsin on aldosterone secretion. Using intact rat adrenal capsular tissue, thrombin was found to increase aldosterone secretion, and the effects of trypsin on aldosterone secretion were confirmed. Both trypsin and thrombin were shown to activate phospholipase C, as measured by an increase in inositol triphosphate turnover by adrenal capsular tissue. It was also shown that U73122, a phospholipase C inhibitor, attenuated the aldosterone response to trypsin. These effects were consistent with the activation of a PAR. Northern blot analysis revealed the presence of mRNA encoding PAR-1, but not PARs-2, -3 or -4 in the adrenal capsule/zona glomerulosa. Messenger RNA encoding PAR-1 was increased by dietary sodium depletion, consistent with previous reports of an increased response to trypsin after sodium depletion. These data suggest that the actions of trypsin on aldosterone secretion are mediated by PAR-1.

Cysteine residues are involved in structure and function of melanocortin 1 receptor: Substitution of a cysteine residue in transmembrane segment two converts an agonist to antagonist.

Reduction of disulfide bonds in human melanocortin 1 receptor (hMC1R) with increasing concentrations of DTT (dithiothreitol) resulted in a decrease in the binding of [125I]-ACTH (adrenocorticotropic hormone, L-isomer) in an uniphasic manner and a decrease in [125I]-NDP-MSH ([Nle(4),D-Phe(7)]-alpha-melanocyte stimulating hormone; D-isomer) binding in a biphasic manner. Pretreatment of hMC1R with 10 mM DTT resulted in a 36-fold loss of affinity for alpha-MSH (L-isomer) without affecting the affinity of NDP-MSH (D-isomer). To characterize the role of individual cysteine residues, we employed site-directed mutagenesis to substitute cysteine by glycine at all fourteen positions in hMC1R and analysed wild-type and mutant receptors for ligand binding and cAMP signalling. Single point mutation of four cysteine residues in extracellular loops to glycine (C35G, C267G, C273G, and C275G) resulted in a complete loss of binding for [125I]-NDP-MSH. Moreover, mutants with normal ligand binding, at positions C191G (transmembrane segment 5), C215G (third intracellular loop), and C315G (C-terminal loop) failed to generate cAMP signal in response to both agonists alpha-MSH and NDP-MSH. Mutant at position C78G (with wild-type binding to alpha-MSH as well as NDP-MSH) generated a cAMP signal in response to alpha-MSH (identical to wild-type hMC1R) but interestingly could not be stimulated by NDP-MSH. Moreover, this single amino acid substitution converted NDP-MSH from being an agonist to antagonist at the C78G mutant receptor. These findings demonstrate that (i) alpha-MSH and ACTH (L-isomers) are different from D-isomer NDP-MSH in their sensitivity to DTT for receptor binding, (ii) cysteine residues in N-terminus and extracellular loop three make disulfide bridges and are needed for structural integrity of hMC1R, (iii) cysteine residues in transmembrane segments and intracellular loops are required for receptor-G-protein coupling, (iv) C78 in transmembrane segment two is required for generating a functional response by D-isomer agonist (NDP-MSH) but not by L-isomer agonist (alpha-MSH), and (v) wild-type receptor agonist NDP-MSH is an antagonist at the mutant C78G receptor.

Adrenomedullin and calcitonin gene-related peptide receptors in the rat adrenal cortex.

The actions of calcitonin gene-related peptide (CGRP) and adrenomedullin on steroid hormone secretion from the rat zona glomerulosa are controversial, with reports in the literature of both stimulatory and inhibitory effects. It appears that these results previously obtained may depend on the nature of the receptors expressed by zona glomerulosa cells. The present study was designed to characterize CGRP and adrenomedullin binding in the rat adrenal zona glomerulosa. Specific binding for both peptides was observed, with two CGRP receptor sites found, and a single population of adrenomedullin receptors, but approximately twice the number of adrenomedullin binding sites. Messenger RNA analysis of the candidate genes for CGRP and adrenomedullin receptors revealed an abundance of both CRLR and RAMP1 mRNA, suggesting that these genes encode one of the CGRP receptors in this tissue. Much less RAMP2 expression was observed, however, which suggests that another gene product may account for adrenomedullin binding. There were very low levels of RAMP3 expression, but abundant L1 mRNA present, which may suggest that this rather controversial receptor has a role in the adrenal. The finding of distinct and specific adrenomedullin and CGRP binding in this tissue may account for the different effects these peptides appear to exert on adrenal function.

Adrenomedullin, a multifunctional regulatory peptide.

Since the discovery of adrenomedullin in 1993 several hundred papers have been published regarding the regulation of its secretion and the multiplicity of its actions. It has been shown to be an almost ubiquitous peptide, with the number of tissues and cell types synthesizing adrenomedullin far exceeding those that do not. In Section II of this paper we give a comprehensive review both of tissues and cell lines secreting adrenomedullin and of the mechanisms regulating gene expression. The data on circulating adrenomedullin, obtained with the various assays available, are also reviewed, and the disease states in which plasma adrenomedullin is elevated are listed. In Section III the pharmacology and biochemistry of adrenomedullin binding sites, both specific sites and calcitonin gene-related peptide (CGRP) receptors, are discussed. In particular, the putative adrenomedullin receptor clones and signal transduction pathways are described. In Section IV the various actions of adrenomedullin are discussed: its actions on cellular growth, the cardiovascular system, the central nervous system, and the endocrine system are all considered. Finally, in Section V, we consider some unresolved issues and propose future areas for research.

Adrenomedullin receptor is found exclusively in noradrenaline-secreting cells of the rat adrenal medulla.

Adrenomedullin, originally identified in the adrenal medulla, has binding sites in the adrenal gland; however, its role in the adrenal medulla is unclear. This study was designed to characterise adrenomedullin binding sites in the rat adrenal medulla, using ligand binding studies, immunocytochemistry, and mRNA analysis. A single population of specific adrenomedullin receptors was identified in adrenal medullary homogenates. 125I-Adrenomedullin was displaced only by adrenomedullin1-50 and not by calcitonin gene-related peptide or amylin at concentrations up to 100 nmol/L. The receptor K(D) was 3.64 nmol/L with a receptor density of 570 fmol/mg of protein. Analysis of mRNA revealed that the genes encoding both the putative adrenomedullin receptors, termed calcitonin receptor-like receptor (CRLR) and L1, were expressed in the rat adrenal medulla. Dual-colour indirect-labelled immunofluorescence was used to localise phenylethanolamine N-methyltransferase (PNMT) and the adrenomedullin receptor in the same section. PNMT is the enzyme that converts noradrenaline to adrenaline and is not expressed in noradrenaline-secreting cells. These studies revealed that both CRLR and L1 were expressed only in cells that did not express PNMT, suggesting that adrenomedullin receptors are only found in noradrenaline-secreting cells. Further evidence to support this conclusion was provided by the demonstration of colocalisation of adrenomedullin receptors with dopamine beta-hydroxylase, confirming the presence of the receptors in medullary chromaffin cells. Taken together, these data suggest that adrenomedullin acts through a specific adrenomedullin receptor in the rat adrenal medulla. RT-PCR and northern blot analysis revealed greater abundance of mRNA for L1 than for CRLR, possibly suggesting that L1 may be the major adrenomedullin receptor expressed in this tissue. As it has been reported that adrenomedullin is synthesised predominantly by adrenaline-secreting cells, it appears likely that adrenomedullin is a paracrine regulator in the adrenal medulla.

Actions of neuropeptide Y on the rat adrenal cortex.

Although several studies have demonstrated the presence of neuropeptide Y (NPY) in nerves supplying the mammalian adrenal cortex, its function in this tissue remains unclear, with reports of both stimulatory and inhibitory effects on aldosterone secretion apparently depending on the tissue preparation used. In the present study the effects of NPY on rat adrenal capsular tissue were investigated. NPY significantly stimulated aldosterone secretion in a dose-dependent manner, and this effect was abolished by atenolol, a beta1-adrenergic antagonist. NPY also stimulated the release of catecholamines from intact rat adrenal capsular tissue with the same dose-dependent relationship as the stimulation of aldosterone release. These observations suggest that the actions of NPY may be mediated by the local release of catecholamines from chromaffin cells within adrenal capsular tissue, as we have previously described for vasoactive intestinal peptide. The second part of this study concerned the NPY receptor subtype mediating the actions of NPY on the adrenal cortex. It was found that peptide YY stimulated aldosterone release with a comparable potency to NPY, whereas pancreatic polypeptide (PP) was without effect. The Y1 selective NPY analog Leu31Pro34NPY had a greater effect on aldosterone release than the Y2 selective analog NPY18-36. Studies using the specific Y1 receptor antagonist BIBP 3226 showed significant attenuation of the aldosterone response to NPY, but no effect on the response to added norepinephrine. Binding studies carried out using [125I]NPY revealed the presence of a single population of NPY-binding sites with a Kd of 12.25 nmol/liter and a binding capacity of 623 fmol/mg protein. Competition studies revealed displacement of [125I]NPY specific binding by NPY, peptide YY, and Leu31Pro34NPY, but not by other peptides. Messenger RNA analysis revealed the presence of messenger RNA coding for both the Y1 receptor and the Y4 receptor, but not the other subtypes. Taken together these data suggest that the effects of NPY on the rat adrenal cortex are mediated by the Y1 receptor subtype.

Coexpression of receptors for adrenomedullin, calcitonin gene-related peptide, and amylin in pancreatic beta-cells.

Three receptors have been characterized by their ability to bind adrenomedullin (AM): L1, RDC1, and CRLR. Immunohistochemical analysis and RT-PCR showed that all three receptors are expressed by the insulin-producing cells of the islets of Langerhans. RDC1 and CRLR in the presence of particular modifying proteins can also bind calcitonin gene-related peptide (CGRP). Such data suggest that the inhibitory effect caused by both AM and CGRP on insulin secretion is mediated by a direct interaction with the beta-cell. We also identified receptors for amylin, the third member of the AM peptide family, in mouse insulin-secreting cells. The beta-cells located closer to the periphery of the islets had a stronger immunoreactivity for the AM/ CGRP receptors. This observation could be related to a paracrine mechanism, given the proximity of AM- and CGRP-secreting cells (F and delta-cells, respectively), which are located at the periphery of the islets. Interestingly, the smooth muscle cells in the pancreatic vasculature expressed only RDC1, which is in agreement with physiological data showing that AM functions in the cardiovascular system are mainly mediated through a CGRP1 receptor. These data further implicate AM and the other components of its peptide family as important regulators of insulin release.

An investigation into the antimicrobial effects of adrenomedullin on members of the skin, oral, respiratory tract and gut microflora.

Adrenomedullin, a novel vasoactive peptide, is known to be expressed by many surface epithelial cells and it was postulated that this peptide may have a protective role. The objective of the study was to assess the antimicrobial activity of adrenomedullin against members of the human skin, oral, respiratory tract and gut microflora using disc diffusion and broth microdilution assays. All strains of bacteria screened in an agar diffusion assay were sensitive; gram-positive and gram-negative bacteria were equally susceptible. No activity against the yeast Candida albicans was observed. In a broth microdilution assay, minimum inhibitory and minimum bacteriocidal concentrations ranged from 7.75 x 10(-1) to 12.5 microg ml(-1) and 0.003 to > 25.0 microg ml(-1), respectively. We propose an antimicrobial role for adrenomedullin. participating in the prevention of local infection, thus contributing to host defence systems.

Protease-activated receptor-2 involvement in hypotension in normal and endotoxemic rats in vivo.

BACKGROUND: The protease-activated receptor-2 (PAR-2) is expressed by vascular endothelial cells and upregulated by lipopolysaccharide (LPS) in vitro. PAR-2 is activated by a tethered ligand created after proteolytic cleavage by trypsin or experimentally by a synthetic agonist peptide (PAR-2AP) corresponding to the new amino terminus of the tethered ligand. METHODS AND RESULTS: Intravenous administration of PAR-2AP (0.1, 0.3, and 1 mg/kg) to rats caused a dose-dependent hypotension. A scrambled peptide was without effect. A specific trypsin inhibitor, biotin-SGKR-chloromethylketone, inhibited trypsin-induced hypotension but not that stimulated by PAR-2AP. In animals treated with LPS 20 hours earlier, we found an increased sensitivity to trypsin and PAR-2AP in the hypotensive response. In particular, PAR-2AP caused hypotension at a low concentration of 30 ng/kg. Moreover, PAR-2 was immunolocalized to endothelial and smooth muscle cells in aorta and jugular vein in LPS-treated rats, and increased levels of PAR-2 mRNA were shown by reverse transcription-polymerase chain reaction analysis. CONCLUSIONS: Our findings suggest that PAR-2 is important in the regulation of blood pressure in vivo. A functional upregulation of PAR-2 by LPS was demonstrated by the activity of concentrations of PAR-2AP that were inactive in normal animals. We conclude that PAR-2 may play an important role in the hypotension associated with endotoxic shock and may represent a new therapeutic target.