Adrenomedullin (ADM), acting through ADM(22-52)-sensitive receptors, is involved in the endotoxin-induced hypotension in rats

The possible involvement of adrenomedullin (ADM) in the endotoxin-induced hypotension has been investigated in the rat. Lipopolysaccharide (LPS, 500 &kg intraperitoneum) caused a severe decrease in the blood pressure (BP), reaching maximum 2-3 h after the injection and subsiding after 12 h. The putative ADM-receptor antagonist ADM(22-52) (3 nmoHkg) counteracted LPS-induced BP lowering at 1 and 2 h, and reversed it at 3 and 6 h CGRP(S-37), a selective antagonist of the CGRPl receptors, was ineffective. Both ADM(22-52) and CGRP(%37) did not evoke significant changes in the basal BP. Our findings provide strong support to the view ADM overproduction plays a major role in the LPS-induced decrease in BP, and suggest a potentially important therapeutic effect of the blockade of ADM(22-52)-sensitive receptors during endotoxic shock

Human adrenomedullin gene silencing by short interfering RNAs: A preliminary study

Adrenomedullin (AM) is a regulatory peptide widely expressed, along its receptors, in cells and tissues, of which it controls many basic and specific functions acting in an autocrine-paracrine manner. However, the unequivocal demonstration of the physiological relevance of the regulatory role of AM would require the study of cells where endogenous AM system had been suppressed. Hence, we developed a protocol to silence the human AM gene by transfection with short interfering RNAs (siRNAs). Eight possible AM-siRNA sequences were designed: six siRNAs were synthesized in our laboratory and two were provided by Ambion. As positive control the suppression of the glyceraldehyde-3-phosphate dehydrogenase (GADPH) gene was tested using the Ambion Silencer GADPH siRNA kit. Cultured human embryonal kidney cell line HEK-293 and human umbilical vein endothelial cells (HUVECs) were transfected using either the Qiagen or the Ambion transfection reagent, and transfection visualization, carried out using Cy3-labeled siRNA and examining red fluorescence within the cells, showed that the former reagent was the most efficient. AM-gene silencing was determined in HUVECs by measuring AM mRNA levels in transfected and control cells by real-time polymerase chain reaction. Only Ambion siRNAs were effective, and the best AM-gene silencing (about 80%) was observed 48 or 72 h after transfection with 3 or 6 microg of siRNAs. The conclusion is drawn that siRNA technology can be useful in the investigations on AM functions, but that the complete suppression of the AM-gene transcription is very difficult to obtain

Reciprocal regulation of endothelin-1 and nitric oxide: relevance in the physiology and pathology of the cardiovascular system.

The endothelium plays a crucial role in the regulation of cardiovascular structure and function by releasing several mediators in response to biochemical and physical stimuli. These mediators are grouped into two classes: (1) endothelium-derived constricting factors (EDCFs) and (2) endothelium-derived relaxing factors (EDRFs), the roles of which are considered to be detrimental and beneficial, respectively. Endothelin-1 (ET-1) and nitric oxide (NO) are the prototypes of EDCFs and EDRFs, respectively, and their effects on the cardiovascular system have been studied in depth. Numerous conditions characterized by an impaired availability of NO have been found to be associated with enhanced synthesis of ET-1, and vice versa, thereby suggesting that these two factors have a reciprocal regulation. Experimental studies have provided evidence that ET-1 may exert a bidirectional effect by either enhancing NO production via ETB receptors located in endothelial cells or blunting it via ETA receptors prevalently located in the vascular smooth muscle cells. Conversely, NO was found to inhibit ET-1 synthesis in different cell types. In vitro and in vivo studies have started to unravel the molecular mechanisms involved in this complex interaction. It has been clarified that several factors affect in opposite directions the transcription of preproET-1 and NO-synthase genes, nuclear factor-KB and peroxisome proliferator-activated receptors playing a key role in these regulatory mechanisms. ET-1 and NO interplay seems to have a great relevance in the physiological regulation of vascular tone and blood pressure, as well as in vascular remodeling. Moreover, an imbalance between ET-1 and NO systems may underly the mechanisms involved in the pathogenesis of systemic and pulmonary hypertension and atherosclerosis

Endogenous ligands of PACAP/VIP receptors in the autocrine-paracrine regulation of the adrenal gland

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are the main endogenous ligands of a class of G protein-coupled receptors (Rs). Three subtypes of PACAP/VIP Rs have been identified and named PAC(1)-Rs, VPAC(1)-Rs, and VPAC(2)-Rs. The PAC(1)-R almost exclusively binds PACAP, while the other two subtypes bind with about equal efficiency VIP and PACAP. VIP, PACAP, and their receptors are widely distributed in the body tissues, including the adrenal gland. VIP and PACAP are synthesized in adrenomedullary chromaffin cells, and are released in the adrenal cortex and medulla by VIPergic and PACAPergic nerve fibers. PAC(1)-Rs are almost exclusively present in the adrenal medulla, while VPAC(1)-Rs and VPAC(2)-Rs are expressed in both the adrenal cortex and medulla. Evidence indicates that VIP and PACAP, acting via VPAC(1)-Rs and VPAC(2)-Rs coupled to adenylate cyclase (AC)- and phospholipase C (PLC)dependent cascades, stimulate aldosterone secretion from zona glomerulosa (ZG) cells. There is also proof that they can also enhance aldosterone secretion indirectly, by eliciting the release from medullary chromaffin cells of catecholamines and adrenocorticotropic hormone (ACTH), which in turn may act on the cortical cells in a paracrine manner. The involvement of VIP and PACAP in the regulation of glucocorticoid secretion from inner adrenocortical cells is doubtful and surely of minor relevance. VIP and PACAP stimulate the synthesis and release of adrenomedullary catecholamines, and all three subtypes of PACAP/VIP Rs mediate this effect, PAC(1)-Rs being coupled to AC, VPAC(1)-Rs to both AC and PLC, and VPAC2-Rs only to PLC. A privotal role in the catecholamine secretagogue action of VIP and PACAP is played by Ca(2+). VIP and PACAP may also modulate the growth of the adrenal cortex and medulla. The concentrations attained by VIP and PACAP in the blood rule out the possibility that they act as true circulating hormones. Conversely, their adrenal content is consistent with a local autocrine-paracrine mechanism of action

Similar sequence-free amplification of human glyceraldehyde-3- phosphate dehydrogenase for real time RT-PCR applications

One of the major applications of real time polymerase chain reaction (PCR) is relative quantification, where the expression of a target gene is determined as a ratio to a stably expressed reference gene, the so-called housekeeping gene. Glyceraldehyde-3-phosphate dehydrogenase (GAPD) is a glycolytic enzyme, which is active in all mammalian tissues and is frequently used as housekeeping gene in expression studies. The functional locus maps to human chromosome 12p13, but several GAPD-related sequences, including processed pseudogenes, GenBank homologous sequences and computationally predicted sequences are present along the human genome. Due to the high level of GAPD-related sequences it is very important to avoid genomic DNA amplification when GAPD is used as endogenous control in mRNA quantification. We have outlined a GAPD couple of primers that avoid any genomic DNA amplification for real time reverse transcription PCR applications by SYBR-Green Dye. These new designed primers are an useful and chip alternative to probe technologies, and can carry out specific and reproducible data in mRNA expression studies

Adrenomedullin stimulates proliferation and inhibits apoptosis of immature rat thymocytes cultured in vitro

Adrenomedullin (AM) is a hypotensive peptide, which derives from the proteolytic cleavage of pro(p)AM, and acts through two subtypes of receptors, named L1-receptor (L1-R) and calcitonin receptor-like receptor (CRLR). CRLR functions as either a calcitonin gene-related peptide (CGRP) receptor or a selective AM receptor depending on which member of a family of receptor-activity-modifying proteins (RAMPs) is expressed: RAMP1 generates CGRP receptors, while RAMP2 and RAMP3 produce AM receptors. Reverse transcription (RT)-polymerase chain reaction (PCR) consistently allowed the detection of pAM and peptidyl-glycine alpha-amidating monooxygenase (the enzyme converting immature AM to the mature peptide) mRNAs in the thymus cortex of immature (10-day-old) rats. Accordingly, radioimmune assay (RIA) measured low but sizeable AM concentrations in this tissue. RT-PCR also demonstrated the presence of the specific mRNAs of L1-R, CRLR and RAMPs. AM (from 10(-9) to 10(-7)M) increased proliferation index and lowered apoptotic index of cultured immature rat thymocytes, and the effects were annulled by the AM receptor antagonist AM(22-52). In conclusion, our study demonstrated that (1) immature rat thymus cortex expresses AM and the AM receptors L1-R and CRLR/RAMP; and (2) AM, acting via AM(22-52)-sensitive receptors, exerts a potent growth promoting effect on immature rat thymus, by enhancing proliferation and lowering apoptotic death of thymocytes. Taken together, these findings could suggest that AM may play a role in the development of immunity

Proadrenomedullin-derived peptides as autocrine-paracrine regulators of cell growth

Proadrenomedullin (pADM)-derived peptides, adrenomedullin (ADM) and pADM N-terminal 20 peptide (PAMP), are hypotensive peptides, which are expressed, along with their receptors, in several tissues and organs, the function of which they regulate by acting in an autocrine-paracrine manner. Apart from their involvement in the regulation of blood pressure and fluid and electrolyte homeostasis, pADM-derived peptides appear to play a role in the modulation of cell and tissue growth. Evidence has been provided that ADM: 1) favors the remodeling of cardiovascular system under pathological conditions, by exerting an antiapoptotic effect on endothelial cells and an antiproliferogenic and antimigratory action on vascular smooth-muscle cells during neointimal hyperplasia, and by decreasing proliferation and protein synthesis of cardiac myocytes and fibroblasts. These last two effects are mediated by calcitonin gene-related peptide type 1 (CGRP1) receptors coupled to the adenylate cyclase (AC)/protein kinase (PK) A-dependent cascade; 2) inhibits proliferation and enhances apoptosis of kidney mesangial cells, through the modulation of mitogen-activated PK (MAPK) cascades; 3) stimulates proliferation of adrenal zona glomerulosa cells, acting via CGRP1 receptor coupled to the tyrosine kinase-dependent MAPK cascade, thereby possibly being involved in the maintenance and stimulation of adrenal growth; 4) enhances proliferation of skin and mucosa epithelial cells and fibroblasts, by activating CGRP1 receptor coupled to the AC/PKA signaling pathway; and 5) enhances proliferation of several tumor-cell lines through the activation of the AC/PKA cascade, which suggests a potential role for ADM as promoter of neoplastic growth. The growth effects of PAMP have been far less investigated: findings indicate that this peptide, like ADM, enhances adrenal zona glomerulosa-cell proliferation, and, in contrast with ADM, depresses DNA synthesis in some cancer-cell lines. Both pADM-derived peptides are thought to be involved in embryogenesis, such a contention being based on the demonstration of high pADM-gene expression during the crucial phases of organ growth and differentiation