Proadrenomedullin N-terminal 20 peptide (PAMP) inhibits food intake and gastric emptying in mice

We found that proadrenomedullin N-terminal 20 peptide (PAMP) decreased dose-dependently (3-30 nmol/mouse) food intake after intra-third cerebroventricular administration in fasted ddY mice. Gastric emptying also was delayed after central injection of PAMP. In our previous study, PAMP was demonstrated to elicit hyperglycemia via bombesin (BN) receptor. Then, we examined whether the effects of PAMP on feeding and gastric emptying were induced through BN receptor. Surprisingly, PAMP-induced reductions in feeding and gastric emptying rate were not blocked by a BN antagonist, [D-Phe(6), Leu-NHEt(13), des-Met(14)]-BN (6-14). PAMP suppressed feeding in mice lacking gastrin-releasing peptide receptor or BN receptor subtype-3. These results indicate that centrally administered PAMP inhibits food intake, involving the delayed gastric emptying, not through BN receptors but through selective PAMP receptor.     

Proadrenomedullin N-terminal 20 peptide (PAMP) enhances proliferation of rat zona glomerulosa cells by activating MAPK cascade.

The effect of proadrenomedullin N-terminal 20 peptide (PAMP) on the proliferative activity of rat zona glomerulosa (ZG) cells has been investigated. Dispersed rat ZG cells were cultured in vitro for 24 h and then exposed to PAMP for an additional 24 h, and the proliferation rate was assessed by the 5-bromo-2'-deoxyuridine (BrdU) incorporation technique. PAMP dose-dependently increased the percentage of BrdU-positive cells, with a maximal effective concentration observed at 10(-8) M. The tyrosine kinase (TK) inhibitor, tyrphostin-23, and the p42/p44 MAPK inhibitor, PD-98059, abolished the proliferogenic effect of PAMP, while the protein kinase (PK) A inhibitor, H-89, and the PKC inhibitor, calphostin-C, were ineffective in blocking the response to PAMP. PAMP (10(-8) M) enhanced TK and MAPK activity of dispersed rat ZG cells. The stimulatory action of PAMP on TK activity was annulled by tyrphostin-23, while that on MAPK activity was abolished by either tyrphostin-23 or PD-98059. Taken together, these data indicate that PAMP enhances proliferation of cultured rat ZG cells, through the TK-dependent activation of p42/p44 MAPK cascade.     

Proadrenomedullin NH(2)-terminal 20 peptide (PAMP) and adrenomedullin bind to teratocarcinoma cells

Proadrenomedullin NH(2-)terminal 20 peptide (PAMP) and adrenomedullin (ADM) bind to teratocarcinoma cells. The effects of PAMP and ADM on teratocarcinoma cells were investigated. (125)I-PAMP bound to PA1 cells with moderate affinity (K(d) = 110 nM) to a single class of sites (B(max) = 110 000/cell). Specific (125)I-PAMP binding was inhibited by PAMP (IC(50) of 100 nM) but not ADM, calcitonin gene-related peptide (CGRP), or amylin. Specific (125)I-ADM binding was inhibited with high affinity by ADM, CGRP, and CGRP(8-37) (IC(50) values of 10, 10, and 15 nM respectively) but not PAMP or amylin. ADM elevated cAMP (ED(50) value of 100 nM), whereas PAMP had no effect on basal cAMP but inhibited the increase in cAMP caused by 10 nM ADM. Also, the increase in cAMP caused by ADM was inhibited CGRP(8-37), suggesting that ADM is binding to CGRP receptors. ADM (100 nM) stimulated transiently c-fos mRNA, whereas PAMP (1000 nM) had little effect; however, PAMP inhibited the increase in c-fos mRNA caused by ADM. ADM stimulated [(3)H]thymidine uptake into PA1 cells, whereas PAMP inhibited the increase in thymidine uptake caused by ADM. These results indicate that ADM and PAMP are both biologically active in teratocarcinoma cells.     

Novel actions of proadrenomedullin N-terminal 20 peptide (PAMP).

Poadrenomedullin N-terminal 20 peptide (PAMP) is a hypotensive peptide derived from the precursor of adrenomedullin. We identified novel actions of proadrenomedullin N-terminal 20 peptide (PAMP) on blood glucose, food intake and gastric emptying after exogenous administration. PAMP elevated blood glucose levels after central injection in fasted mice. PAMP had affinity for bombesin (BN) receptor and the hyperglycemic effect of PAMP was blocked by a BN antagonist, indicating that the elevation of blood glucose after central administration of PAMP was mediated by BN receptor. Centrally administered PAMP inhibited food intake and gastric emptying in fasted conscious mice. However, studies using a BN antagonist and BN receptor knockout mice suggested that the inhibitory effects of PAMP on feeding and gastric emptying were mediated not via BN receptor but via another receptor specific for PAMP. In the present review, we summarize these effects of PAMP and report other novel actions of PAMP on body temperature and oxygen consumption. In addition, the mechanism underlying the cardiovascular functions of PAMP is discussed.     

Adrenomedullin and proadrenomedullin N-terminal 20 peptide (PAMP) in adrenal chromaffin cells.

Adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP) are peptides having multiple physiological functions and are most abundantly expressed in the adrenal medulla. In addition to PAMP, PAMP12, a 12 amino acid peptide with sequence identity to PAMP between amino acids 9-20, has also been shown to be expressed in the adrenal medulla. AM, PAMP and PAMP12 are released along with catecholamines by regulated exocytosis upon stimulation of adrenal chromaffin cells. PAMP and PAMP12 regulate catecholamine release and synthesis by interfering with nicotinic cholinergic receptors in these chromaffin cells. AM may also cause gradual release of catecholamine from these cells. AM, PAMP and PAMP12 are endogenous peptides that modulate chromaffin cell function via different mechanisms.     

Injection of the insulin receptor alpha subunit increases blood glucose levels in mice

Using the expression vector of the truncated human insulin receptor (hIR), we have constructed a stable Chinese hamster ovary (CHO) cell line which secretes the His-tagged alpha subunit (insulin-binding domain) of hIR into medium. To examine characteristics of the His-tagged hIRalpha, we purified the protein secreted from the CHO cells. The His-tagged hIRalpha was glycosylated and processed a dimer. The molecule bound insulin with an affinity similar to that of the intact hIR. The His-tagged full length of hIR was autophosphorylated by insulin stimulation in CHO cells. Injection of the purified His-tagged hIRalpha into veins of mice increased in the concentration of blood glucose within 30 min. The intraperitoneal glucose tolerance test (ipGTT) done after injection of the purified His-tagged hIRalpha showed evidence of a marked hyperglycemia. These findings provide direct evidence that the presence of hIRalpha in the blood stream inhibits insulin actions by binding with plasma insulin.     

A review of the biological properties and clinical implications of adrenomedullin and proadrenomedullin N-terminal 20 peptide (PAMP), hypotensive and vasodilating peptides.

Adrenomedullin (AM), identified from pheochromocytoma and having 52 amino acids, elicits a long-lasting vasodilatation and diuresis. AM is mainly mediated by the intracellular adenylate cyclase coupled with cyclic adenosine monophosphate (cAMP) and nitric oxide (NO) -cyclic guanosine monophosphate (cGMP) pathway through its specific receptor. The calcitonin receptor-like receptor (CLCR) and receptor-activity modifying protein (RAMP) 2 or RAMP3 models have been proposed as the candidate receptor. AM is produced mainly in cardiovascular tissues in response to stimuli such as shear stress and stretch, hormonal factors and cytokines. Recently established AM knockout mice lines revealed that AM is essential for development of vitelline vessels of embryo. Plasma AM levels elevate in cardiovascular diseases such as heart failure, hypertension and septic shock, where AM may play protective roles through its characteristic biological activities. Human AM gene delivery improves hypertension, renal function, cardiac hypertrophy and nephrosclerosis in the hypertensive rats. AM decreases cardiac preload and afterload and improves cardiac contractility and diuresis in patients with heart failure and hypertension. Advances in gene engineering and receptor studies may contribute to further understandings of biological implication and therapeutic availability of AM.     

Investigation of bombesin peptide as a targeting ligand for the gastrin releasing peptide (GRP) receptor

Gastrin releasing peptide (GRP) receptor (GRPR), a bombesin family receptor, is overexpressed in many cancers including breast, prostate, pancreatic and lung. The targeting of therapeutics to GRPR can be achieved using the full-length (14 amino acid) GRP analogue Bombesin (BBN) or the truncated BBN(6–14) sequence, both of which bind GRPR with high affinity and specificity. In this study, we have investigated the level of GRPR expression in various cancerous (Caco-2, HeLa, LNCap, MDA-MB-231, and PC-3) and non-cancerous (WPMY-1) cell lines using a western blotting approach. Such information is currently lacking in the literature, and is therefore of importance for the in vitro assessment of GRPR targeted therapeutics. Of the cell lines assessed, the PC-3 (prostate cancer) and Caco-2 (colon cancer) cell lines demonstrated the highest and lowest levels of GRPR expression respectively. Using this information, we further investigated the cellular uptake of carboxyfluorescein-labelled BBN and BBN(6–14) peptides by flow cytometry and confocal microscopy using cell lines that express GRPR (Caco-2, HeLa, PC-3). The uptake of each of these peptides was similar, suggesting that the shorter BBN(6–14) peptide is sufficient for GRPR targeting. Further, the uptake of these peptides could be inhibited by competition with unlabelled BBN peptides, suggesting their cellular uptake is GRPR-mediated, while the level of BBN uptake (as measured by flow cytometry) was found to be directly proportional to the level of GRPR expression. Overall, the information obtained from these studies provides useful information for the in vitro assessment of GRPR targeted therapeutics.     

Proadrenomedullin N-Terminal 20 Peptide (PAMP), an Endogenous Anticholinergic Peptide: Its Exocytotic Secretion and Inhibition of Catecholamine Secretion in Adrenal Medulla

Abstract: In cultured bovine adrenal medullary cells, stimulation of nicotinic receptors by carbachol evoked the Ca²⁺-dependent exocytotic cosecretion of proadrenomedullin N-terminal 20 peptide (PAMP) (EC₅₀ = 50.1 µ M ) and catecholamines (EC₅₀ = 63.0 µ M ), with the molar ratio of PAMP/catecholamines secreted being equal to the ratio in the cells. Addition of PAMP[1–20]NH₂ inhibited carbachol-induced ²²Na⁺ influx via nicotinic receptors (IC₅₀ = 2.5 µ M ) in a noncompetitive manner and thereby reduced carbachol-induced ⁴⁵Ca²⁺ influx via voltage-dependent Ca²⁺ channels (IC₅₀ = 1.0 µ M ) and catecholamine secretion (IC₅₀ = 1.6 µ M ). It did not alter high K⁺-induced ⁴⁵Ca²⁺ influx via voltage-dependent Ca²⁺ channels or veratridine-induced ²²Na⁺ influx via voltage-dependent Na⁺ channels. PAMP seems to be a novel antinicotinic peptide cosecreted with catecholamines by a Ca²⁺-dependent exocytosis in response to nicotinic receptor stimulation.     

Improved blood glucose disposal and altered insulin secretion patterns in adenosine A(1) receptor knockout mice

Type 2 diabetes mellitus (T2DM) is characterized by the inability of the pancreatic β-cells to secrete enough insulin to meet the demands of the body. Therefore, research of potential therapeutic approaches to treat T2DM has focused on increasing insulin output from β-cells or improving systemic sensitivity to circulating insulin. In this study, we examined the role of the A(1) receptor in glucose homeostasis with the use of A(1) receptor knockout mice (A(1)R(-/-)). A(1)R(-/-) mice exhibited superior glucose tolerance compared with wild-type controls. However, glucose-stimulated insulin release, insulin sensitivity, weight gain, and food intake were comparable between the two genotypes. Following a glucose challenge, plasma glucagon levels in wild-type controls decreased, but this was not observed in A(1)R(-/-) mice. In addition, pancreas perfusion with oscillatory glucose levels of 10-min intervals produced a regular pattern of pulsatile insulin release with a 10-min cycling period in wild-type controls and 5 min in A(1)R(-/-) mice. When the mice were fed a high-fat diet (HFD), both genotypes exhibited impaired glucose tolerance and insulin resistance. Increased insulin release was observed in HFD-fed mice in both genotypes, but increased glucagon release was observed only in HFD-fed A(1)R(-/-) mice. In addition, the regular patterns of insulin release following oscillatory glucose perfusion were abolished in HFD-fed mice in both genotypes. In conclusion, A(1) receptors in the pancreas are involved in regulating the temporal patterns of insulin release, which could have implications in the development of glucose intolerance seen in T2DM.     

Solubilization of the receptor for the neuropeptide gastrin-releasing peptide (bombesin) with functional ligand binding properties

The receptor for the neuropeptide gastrin-releasing peptide, the mammalian homologue of bombesin, was solubilized from rat brain and Swiss 3T3 cells by using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) and the cholesteryl hemisuccinate ester (CHS). Only the combination of the detergent CHAPS and the cholesteryl ester CHS in a glycerol-containing buffer satisfactorily preserved the binding activity upon solubilization. Specific binding activity was only solubilized from cell lines and tissue preparations known to express the GRP receptor. The dissociation constant (Kd) for the receptor solubilized from rat brain and Swiss 3T3 cells was 0.6 nM, similar to the value of 0.8 nM calculated for the membrane-bound receptor. Binding was saturable and reached equilibrium after approximately 2 h at 4 degrees C. The identity of the solubilized receptor with the membrane-bound one was further confirmed by the concordance of the relative binding affinities of various established bombesin analogues.     

Bioactivity of adrenomedullin and proadrenomedullin N-terminal 20 peptide in man.

Although the biological effects of adrenomedullin (AM) and PAMP have been reported extensively in animal studies and from in-vitro experiments, relatively little information is available on responses to the hormone administered to man. This review summarizes data from the few studies carried out in man. In healthy volunteers, i.v. infusion of AM reduces arterial pressure, probably at a lower rate of administration than is required to elicit other responses. AM stimulates heart rate, cardiac output, plasma levels of cAMP, prolactin, norepinephrine and renin whilst inhibiting any concomitant response in plasma aldosterone. Little or no increase in urine volume or sodium excretion has been observed. Patients with essential hypertension differ only in showing a greater fall in arterial pressure and in the development of facial flushing and headache. In patients with heart failure or chronic renal failure, i.v. AM has similar effects to those seen in normal subjects but also induces a diuresis and natriuresis, depending on the dose administered. Infusion of AM into the brachial artery results in a dose-related increase in forearm and skin blood flow, more prominent and more dependent on endogenous nitric oxide in healthy volunteers than in patients with cardiac failure. When infused into a dorsal hand vein, AM partially reversed the venoconstrictor action of norepinephrine. Although much more information is required to clarify the role of AM under physiological and pathophysiological circumstances, it is clear that it has prominent hemodynamic and neurohormonal effects, though generally lesser urinary effects when administered short-term in doses sufficient to raise its levels in plasma to those seen in a number of clinical disorders. The only study of PAMP in man showed that its skeletal muscle vasodilator potency, when infused into the brachial artery of healthy volunteers, was less than one hundredth that of AM, and it was without effect on skin blood flow.     

The bombesin/gastrin releasing peptide receptor antagonist RC-3095 blocks apomorphine but not MK-801-induced stereotypy in mice

Bombesin (BN)-like peptides might be involved in the pathogenesis of neuropsychiatric disorders such as schizophrenia. Stereotyped behaviors induced by the dopamine receptor agonist apomorphine or the N-methyl-D-aspartate glutamate receptor antagonist dizocilpine (MK-801) in rodents have been proposed as animal models of schizophrenic psychosis. In the present study we evaluated the effects of the BN/gastrin-releasing peptide receptor (GRP) antagonist (D-Tpi6, Leu13 psi[CH2NH]-Leu14) bombesin (6-14) (RC-3095) on apomorphine and MK-801-induced stereotyped behavior in mice. An intraperitoneal (i.p.) injection of RC-3095 (1.0, 10.0 or 100.0 mg/kg) blocked apomorphine-induced stereotypy. The inhibitory effect of RC-3095 on apomorhine-induced stereotypy was similar to that induced by haloperidol (0.5 mg/kg). RC-3095 did not affect stereotyped behavior induced by MK-801 (0.5 mg/kg). The results provide the first evidence that BN/GRP receptor antagonism blocks stereotyped behavior induced by a dopamine agonist. Together with previous evidence, the present study indicates that the BN/GRP receptor can be considered a drug target in the investigation of potential new agents for treating neuropsychiatric disorders.     

Gastrin-releasing peptide (GRP,mammalian bombesin) in the pathogenesis of lung cancer

Established human lung cancer exhibits a complex pattern of genetic changes as well as several distinct autocrine growth factor loops for regulatory peptides. The best studied example is that of gastrin-releasing peptide (GRP), the mammalian homolog of the amphibian bombesin. It is produced by up to 70% of small cell lung cancers and 10–20% of non-small cell lung cancers. GRP stimulates the growth of normal bronchial epithelium as well as that of small cell lung cancer, and its blockade with the use of antibodies or synthetic antagonists inhibits the growth of these tumors. Study of its molecular biology has revealed a complex pattern of mRNA processing which has lead to the recent isolation of a novel family of peptides termed gastrin-releasing peptide gene-associated peptides (GGAPs), present in normal and malignant human tissues. Additional efforts have been directed at characterizing the GRP receptor as well as its intracellular signaling pathways which have been reported both as G protein phospholipase C coupled events as well as activation of a membrane associated tyrosine kinase. In view of its expression in normal bronchial epithelium and its mitogenic effects on this tissue, GRP appears to play a central role in the early events of pulmonary carcinogenesis.     

Discovery of potent and selective peptide agonists at the GRP-preferring bombesin receptor (BB2).

Analogues of the nonselective bombesin receptor synthetic agonist H-D-Phe-Gln-Trp-Ala-Val-betaAla-His-Phe-Nle-NH2 were prepared and their biological activity assessed at the NMB-preferring/bombesin receptor (NMB-R: BB1), the GRP-preferring/bombesin receptor (GRP-R: BB2) and the orphan receptor bombesin receptor subtype-3 (BRS-3; BB3). Progressive N-terminal deletions identified the minimum C-terminal sequences required for maintaining a significant agonist effect, whilst an alanine scan, targeted changes in stereochemistry and other pertinent substitutions identified key side-chain and stereochemical requirements for activation. Key structural elements required for functional potency at BB1 BB2 and BB3, and for selectivity between these receptor subtypes were established. Synthetic peptides were discovered. which were highly potent agonists at BB2 and extremely selective over both BB1 and BB3.     

Overexpression of proadrenomedullin N-terminal 20 peptide blunts blood pressure rise and attenuates myocardial hypertrophy and fibrosis in hypertensive rats

We developed a transgenic (Tg) rat model that overexpresses human proadrenomedullin N-terminal 20 peptide (PAMP) only and then compared the effects of unilateral nephrectomy followed by a high salt diet for five weeks in Tg and wild-type rats. We found that systolic blood pressure was significantly lower in Tg UNX rats and cardiac hypertrophy and myocardial fibrosis was also attenuated in Tg rats. Evaluation of gene expression showed suppression of cardiac local renin-angiotensin system (RAS) in Tg rat. These results suggest that in addition to reducing blood pressure, PAMP suppresses cardiac hypertrophy through negative regulation of the local cardiac RAS.     

Relationship between blood glucose levels and hepatic Fto mRNA expression in mice

Common variants in the fat mass and obesity associated (FTO) gene are associated with obesity and type 2 diabetes. Fto-deficient mice develop hepatic insulin resistance, leading to the hypothesis that hepatic Fto plays a role in the regulation of glucose metabolism and that hepatic Fto expression is regulated by metabolic states. We found that hepatic Fto mRNA levels were increased by fasting in mice. Intraperitoneal glucose injection reduced hepatic Fto mRNA levels without significant changes in body weight in fasted mice. The inverse correlation between Fto mRNA and glucose remained significant after adjusting for body weight. There were positive correlations between hepatic Fto mRNA expression and gluconeogenic gene expression. These data support the hypothesis that hepatic Fto expression changes in response to metabolic states and glucose reduces hepatic Fto mRNA expression independently of body weight. Hepatic Fto may participate in the feedback regulation of glucose metabolism via gluconeogenesis.     

Decrease of TSH levels and epithelium/colloid ratio in rat thyroid glands following administration of proadrenomedullin N-terminal peptide (12-20).

Adrenomedullin (AM) exerts a potent and long-lasting hypotensive effect and is considered to be an important hormone in blood pressure control. AM is a 52-amino-acid peptide synthesized as part of a 185-amino-acid preprohormone that also contains 20-amino-acid residues in the N-terminus, which has similar biological activity. This sequence is named a proadrenomedullin N-terminal 20 peptide (PAMP). Also, proadrenomedullin N-terminal peptide (PAMP)(12-20) exerts vasodepressor response, however this response is 3-fold less potent than the effect evoked by full-sequence peptide. Both AM and PAMP controls secretory activity of the pituitary gland and adrenal cortex, however, their action on the other endocrine glands is not recognized. Therefore, the aim of the study was to examine whether PAMP(12-20) is able to affect the structure and function of the rat thyroid gland. In adult female rats, subcutaneous PAMP(12-20) administration (1 or 4 nmol/rat/day for 6 days, autopsy 60 min after the last injection) had no effect on the weight of the thyroid gland. Peptide administration however, resulted in a dose-dependent increase in the volume of thyroid colloid, and lowered epithelium/colloid ratio in the gland (3.76 +/- 0.49, 2.66 +/- 0.27, 2.38 +/- 0.26, means +/- SE, n = 6, control, 1 and 4nmol PAMP/rat, respectively). PAMP administration changed neither the length of thyroid capillaries per unit area of surface nor their diameter. Lower dose of PAMP(12-20) significantly lowered blood TSH concentration (p < 0.01) while total and free T3 and T4 concentrations remained unchanged. Collectively, these findings suggest that PAMP(12-20) exerts a mild inhibitory effect on secretory activity of the rat thyroid gland.     

Spider silk fibroin enhances insulin secretion and reduces blood glucose levels in diabetic mice

Fibroin silk proteins make up the cocoons of silkworms and spider webs and are rich in glycine and alanine residues. Recent studies have shown that silk fibroin hydrolysate from silkworms improves blood glucose and lipid metabolism. In the present study, we investigated the anti-diabetic effects of spider silk fibroin protein in type 2 diabetic mice. Recombinant AvMaSp-R, which consists of the 240 amino acid repetitive domain of major ampullate silk protein (AvMaSp) from the spider Araneus ventricosus, was produced in baculovirus-infected insect cells. We tested the effects of oral AvMaSp-R administration on serum insulin and blood glucose levels in diabetic mice and found that AvMaSp-R increases serum insulin levels and reduces blood glucose levels in diabetic mice. Consequently, our results are the first to provide evidence that silk fibroin protein from spiders enhances insulin secretion, which leads to reduced blood glucose levels in type 2 diabetic mice.