Supriya Kapas

Adrenomedullin and mucosal defence: interaction between host and microorganism

Many surface epithelial cells express adrenomedullin (AM) and it is postulated that it may have an important protective role. This peptide has many properties in common with other cationic antimicrobial peptides including the human beta-defensins. Antimicrobial activity against members of the human skin, oral, respiratory tract and gastric microflora has been demonstrated. Both pathogenic and commensal strains of bacteria are sensitive; Gram-positive and Gram-negative bacteria being equally susceptible. No activity against the yeast Candida albicans was observed. Minimum inhibitory and minimum bacteriocidal concentrations range from 7.75 x 10(-4) to 12.5 and 0.003 to >25.0 microg ml(-1), respectively. On exposure of oral, skin and gastric epithelial cells to whole cells and culture supernatants from bacteria isolated from these sites an increase in AM peptide and gene expression has been observed. No upregulation was detected with C. albicans. In cultured cells and an animal infection model increased AM peptide and gene expression has been demonstrated using immunohistochemical and in situ hybridization techniques. These collective findings suggest that AM represents a new category of antimicrobial peptide, which contributes to the mucosal host defence system.

ADRENOMEDULLIN HAS MITOGENIC EFFECTS ON HUMAN ORAL KERATINOCYTES : INVOLVEMENT OF CYCLIC AMP

The effects of the novel vasoactive regulatory peptide, adrenomedullin, on human oral keratinocytes was investigated. Adrenomedullin, acting via its specific receptor, stimulated a dose‐dependent increase in DNA synthesis, and, in addition, stimulated further changes in the cell cycle resulting in the proliferation of keratinocytes. When cells were incubated in the presence of increasing concentrations of adrenomedullin, there was a rapid and dose‐dependent rise in intracellular cyclic AMP levels. Stimulation of mitogenesis and cell proliferation in these cells were mimicked by the cell permeable cAMP analogue, di‐butyryl cAMP. Adrenomedullin‐stimulated mitogenesis was attenuated by the adenylyl cyclase inhibitor SQ22,536, but was unaffected by inhibitors of PKC, tyrosine kinase or the CGRP receptor antagonist, CGRP(8–37). These data identify adrenomedullin as a new mitogenic regulatory peptide of keratinocytes acting via the cAMP cascade.

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.

Mechanisms of adrenomedullin antimicrobial action

The mechanism of antimicrobial action of the multifunctional peptide adrenomedullin (AM) against Escherichia coli and Staphylococcus aureus was investigated. AM (52 residues) and AM fragments (1-12, 1-21, 13-52, 16-21, 16-52, 22-52, 26-52 and 34-52 residues) were tested for activity. Carboxy-terminal fragments were shown to be up to 250-fold more active than the parent molecule. Minimum inhibitory concentration values of the most active fragments (13-52 and 16-52) and the parent molecule were 4.9 x 10(-2) and 12.5 microg/ml, respectively, with E. coli. Ultrastructural analyses of AM treated cells demonstrated marked cell wall disruption with E. coli within 0.5 h. Abnormal septum formation with no apparent peripheral cell wall disruption was observed with S. aureus after 2 h. Outer membrane permeabilisation assays with E. coli confirmed that the C-terminal fragments were significantly (P < 0.05) more active. It is suggested that postsecretory processing may generate multiple AM congeners that have enhanced antimicrobial activities against a range of potential targets.

The relationship between adrenal vascular events and steroid secretion: the role of mast cells and endothelin.

The actions of ACTH on the adrenal cortex are known to be 2-fold. In addition to increased steroidogenesis, ACTH also causes marked vasodilation, reflected by an increased rate of blood flow through the gland. Our studies, using the in situ isolated perfused rat adrenal preparation, have shown that zona fasciculata function and corticosterone secretion are closely related to vascular events, with an increase in perfusion medium flow rate causing an increase in corticosterone secretion, in the absence of any known stimulant. These observations give rise to two important questions: how does ACTH stimulate blood flow; and how does increased blood (or perfusion medium) flow stimulate steroidogenesis? Addressing the first question, we have recently identified mast cells in the adrenal capsule, and shown that Compound 48/80, a mast cell degranulator, mimics the actions of ACTH on adrenal blood flow and corticosterone secretion. We have also demonstrated an inhibition of the adrenal vascular response to ACTH in the presence of disodium cromoglycate, which prevents mast cell degranulation. We conclude, therefore, that ACTH stimulates adrenal blood flow by its actions on mast cells in the adrenal capsule. Addressing the second question, we looked at the role of endothelin in the rat adrenal cortex. Endothelin 1, 2 and 3 caused significant stimulation of steroid secretion by collagenase dispersed cells from both the zona glomerulosa and the zona fasciculata. A sensitive response was seen, with significant stimulation at an endothelin concentration of 10(-13) mol/l or lower. Endothelin secretion by the in situ isolated perfused rat adrenal gland was measured using the Amersham assay kit. Administration of ACTH (300 fmol) caused an increase in the rate of immunoreactive endothelin secretion, from an average of 28.7 +/- 2.6 to 52.6 +/- 6 fmol/10 min (P less than 0.01, n = 5). An increase in immunoreactive endothelin secretion was also seen in response to histamine, an adrenal vasodilator, which stimulates corticosterone secretion in the intact gland, but has no effect on collagenase-dispersed cells. From these data we conclude that endothelin may mediate the effects of vasodilation on corticosterone secretion, and this mechanism may explain some of the differences in response characteristics between the intact gland and dispersed cells.

The role of neuropeptides in the regulation of adrenal vascular tone: effects of vasoactive intestinal polypeptide, substance P, neuropeptide Y, neurotensin, Met-enkephalin, and Leu-enkephalin on perfusion medium flow rate in the intact perfused rat adrenal

There is evidence that adrenal blood flow may be regulated in part by neuropeptides released from the capsular region of the adrenal gland in response to splanchnic nerve stimulation. The present study investigated the effects of various neuropeptides on the rate of perfusion medium flow through an intact in situ perfused rat adrenal preparation. Vasoactive intestinal polypeptide (VIP) had the greatest effect, causing a 136% increase in flow at the highest dose used (10 nmol in a 200 microliters bolus). Of the other peptides tested Met-enkephalin caused a 50% increase in flow, and the others (Leu-enkephalin, neurotensin and substance P) had only a minor effect, increasing perfusion medium flow rate by no more than around 35%. Neuropeptide Y, in contrast, caused a significant decrease in perfusion medium flow rate: the maximum effect was a 30% decrease with a dose of 1 nmol in a 200 microliters bolus. The significance of this observation awaits elucidation. It is clear from the actions of the neuropeptides tested that they may have a significant role in the regulation of adrenal blood flow. In view of the findings of other authors: that VIP is released in response to splanchnic nerve stimulation, and that it is specifically localised in the capsular region of the adrenal, it seems most likely that VIP is the major peptide involved in mediating the increased adrenal blood flow following splanchnic nerve stimulation.

The role of neuropeptides in the regulation of adrenal zona fasciculata/reticularis function : effects of vasoactive intestinal polypeptide, substance P, neuropeptide Y, Met- and Leu-enkephalin and neurotensin on corticosterone secretion in the intact perfused rat adrenal gland in situ

There is much evidence to suggest that glucocorticoid secretion may be influenced by the splanchnic innervation to the adrenal gland, and that this effect may be mediated by neuropeptides. The present studies investigated the effects of several neuropeptides on corticosterone secretion by the intact perfused rat adrenal gland in situ. Both vasoactive intestinal polypeptide and Met-enkephalin caused a dose-dependent increase in corticosterone secretion, with a maximum response of 450% and 370% increment in corticosterone respectively. Of the other peptides tested, Leu-enkephalin, substance P and neurotensin all stimulated corticosterone secretion, with a maximum response of around 160% increase in each case. Neuropeptide Y on the other hand, had only a minor effect, which was only apparent over a small dose range. These results support the theory that adrenal neuropeptides may have a role in the regulation of glucocorticoid secretion.

Actions of Neuropeptide Y on the Rat Adrenal Cortex1

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 β1-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 medi...

Effect of the endothelins on aldosterone secretion by rat zona glomerulosa cells In vitro

Endothelins are thought to be involved in the local regulation of blood flow and tissue function. These experiments were carried out to investigate the possible role of endothelins in the control of aldosterone secretion by the rat adrenal. Suspensions of zona glomerulosa cells were prepared by collagenase digestion of capsular tissue, and incubated in the presence of increasing concentrations of endothelin. Aldosterone was measured by RIA. All three peptides caused a dose-dependent increase in the secretion rate of aldosterone by zona glomerulosa cells. The minimum concentration of peptide required to give a significant response was 10(-14) mol/l for endothelins 2 and 3 and 10(-13) mol/l for endothelin 1. At a concentration of 10(-7) mol/l endothelin 2 elicited a 20-fold increase over basal aldosterone secretion, while both endothelins 1 and 3 elicited a 30-fold increase (P less than 0.001 in all cases). These results show that the endothelins are potent stimulators of aldosterone secretion, and suggest that these peptides may have a role in the control of zona glomerulosa function.

Adrenomedullin Expression by Gastric Epithelial Cells in Response to Infection

ABSTRACT Many surface epithelial cells express adrenomedullin, a multifunctional peptide found in a wide number of body and cell systems. Recently, we and others have proposed that adrenomedullin has an important novel role in host defense. This peptide has many properties in common with other cationic antimicrobial peptides, including the human β-defensins. Upon exposure of human gastric epithelial cells to viable cells of invasive or noninvasive strains of Helicobacter pylori, Escherichia coli, Salmonella enterica, or Streptococcus bovis, a significant increase in adrenomedullin secretion from these cells was demonstrated. Adrenomedullin gene expression was also increased in response to these microorganisms. Similar observations were noted when these cells were incubated with proinflammatory cytokines such as interleukin 1α (IL-1α), IL-6, tumor necrosis factor alpha and lipopolysaccharide. In cultured cells and an animal infection model, increased adrenomedullin peptide and gene expression was demonstrated when exposed to E. coli or Mycobacterium paratuberculosis, respectively. The data suggest there is a strong association between epithelial infection, inflammation, and adrenomedullin expression, which may have clinical relevance. The regulation of adrenomedullin expression may have therapeutic applications, such as improving or enhancing mucosal immunity.