In silico optimization of analogs derived pro-adrenomedullin peptide to evaluate antimicrobial potential.

Diverse computational approaches have been widely used to assist in designing antimicrobial peptides with enhanced activities. This tactic has also been used to address the need for new treatment alternatives to combat resistant bacterial infections. Herein, we have designed eight variants from a natural peptide, pro-adrenomedullin N-terminal 20 peptide (PAMP), using an in silico pattern insertion approach, the Joker algorithm. All the variants show an α-helical conformation, but with differences in the helix percentages according to circular dichroism (CD) results. We found that the C-terminal portion of PAMP may be relevant for its antimicrobial activities, as revealed by the molecular dynamics, CD, and antibacterial results. The analogs showed variable antibacterial potential, but most were not cytotoxic. Nevertheless, PAMP2 exhibited the most potent activities against human and animal-isolated bacteria, showing cytotoxicity only at a substantially higher concentration than its minimal inhibitory concentration (MIC). Our results suggest that the enhanced activity in the profile of PAMP2 may be related to their particular physicochemical properties, along with the adoption of an amphipathic α-helical arrangement with the conserved C-terminus portion. Finally, the peptides designed in this study can constitute scaffolds for the design of improved sequences.

Centrally acting adrenomedullin in the long-term potentiation of sympathetic vasoconstrictor activity induced by intermittent hypoxia in rats.

NEW FINDINGS: What is the central question of this study? Adrenomedullin in the rostral ventrolateral medulla (RVLM) increases sympathetic activity; given that adrenomedullin is released during hypoxia, what are the effects of its agonism and antagonism in the RVLM after chronic intermitent hypoxia (CIH) exposure? What is the main finding and its importance? CIH exposure sensitizes adrenomedullin-dependent mechanisms in the RVLM, supporting its role as a sympathoexcitatory neuromodulator. A novel mechanism was identified for the generation of sympathetic overdrive and hypertension associated with hypoxia, providing potential guidance on new therapeutic approaches for controlling sympathetic hyperactivity in diseases such as sleep apnoea and neurogenic hypertension. ABSTRACT: Adrenomedullin in the rostral ventrolateral medulla (RVLM) has been shown to increase sympathetic activity whereas the antagonism of its receptors inhibited this autonomic activity lowering blood pressure in conditions of hypertension. Given that hypoxia is a stimulant for releasing adrenomedullin, we hypothesized that the presence of this peptide in the RVLM associated with chronic intermittent hypoxia (CIH) would cause sympathetic overdrive. Juvenile male rats (50-55 g) submitted to CIH (6% oxygen every 9 min, 8 h day-1 for 10 days) were studied in an arterially perfused in situ preparation where sympathetic activity was recorded. In control rats (n = 6), exogenously applied adrenomedullin in the RVLM raised baseline sympathetic activity when combined with episodic activation of peripheral chemoreceptors (KCN 0.05%, 5 times every 5 min). This sympathoexcitatory response was markedly amplified in rats previously exposed to CIH (n = 6). The antagonism of adrenomedullin receptors in the RVLM caused a significant reduction in sympathetic activity in the CIH group (n = 7), but not in controls (n = 8). The transient reflex-evoked sympathoexcitatory response to peripheral chemoreceptor stimulation was not affected by either adrenomedullin or adrenomedullin receptor antagonism in the RVLM of control and CIH rats. Our findings indicate that CIH sensitizes the sympathoexcitatory networks within the RVLM to adrenomedullin, supporting its role as an excitatory neuromodulator when intermittent hypoxia is present. These data reveal novel state-dependent mechanistic insights into the generation of sympathetic overdrive and provide potential guidance on possible unique approaches for controlling sympathetic discharge in diseases such as sleep apnoea and neurogenic hypertension.

Pharmacological characterization of the relaxant effect induced by adrenomedullin in rat cavernosal smooth muscle

The aim of the present study was to determine the mechanisms underlying the relaxant effect of adrenomedullin (AM) in rat cavernosal smooth muscle (CSM) and the expression of AM system components in this tissue. Functional assays using standard muscle bath procedures were performed in CSM isolated from male Wistar rats. Protein and mRNA levels of pre-pro-AM, calcitonin receptor-like receptor (CRLR), and Subtypes 1, 2 and 3 of the receptor activity-modifying protein (RAMP) family were assessed by Western immunoblotting and quantitative real-time polymerase chain reaction, respectively. Nitrate and 6-keto-prostaglandin F1α (6-keto-PGF1α; a stable product of prostacyclin) levels were determined using commercially available kits. Protein and mRNA of AM, CRLR, and RAMP 1, -2, and -3 were detected in rat CSM. Immunohistochemical assays demonstrated that AM and CRLR were expressed in rat CSM. AM relaxed CSM strips in a concentration-dependent manner. AM22-52, a selective antagonist for AM receptors, reduced the relaxation induced by AM. Conversely, CGRP8-37, a selective antagonist for calcitonin gene-related peptide receptors, did not affect AM-induced relaxation. Preincubation of CSM strips with NG-nitro-L-arginine-methyl-ester (L-NAME, nitric oxide synthase inhibitor), 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, quanylyl cyclase inhibitor), Rp-8-Br-PET-cGMPS (cGMP-dependent protein kinase inhibitor), SC560 [5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethyl pyrazole, selective cyclooxygenase-1 inhibitor], and 4-aminopyridine (voltage-dependent K+ channel blocker) reduced AM-induced relaxation. On the other hand, 7-nitroindazole (selective neuronal nitric oxide synthase inhibitor), wortmannin (phosphatidylinositol 3-kinase inhibitor), H89 (protein kinase A inhibitor), SQ22536 [9-(tetrahydro-2-furanyl)-9H-purin-6-amine, adenylate cyclase inhibitor], glibenclamide (selective blocker of ATP-sensitive K+ channels), and apamin (Ca2+-activated channel blocker) did not affect AM-induced relaxation. AM increased nitrate levels and 6-keto-PGF1α in rat CSM. The major new contribution of this research is that it demonstrated expression of AM and its receptor in rat CSM. Moreover, we provided evidence that AM-induced relaxation in this tissue is mediated by AM receptors by a mechanism that involves the nitric oxide-cGMP pathway, a vasodilator prostanoid, and the opening of voltage-dependent K+ channels.

Pharmacological characterization of the relaxant effect induced by adrenomedullin in rat cavernosal smooth muscle.

The aim of the present study was to determine the mechanisms underlying the relaxant effect of adrenomedullin (AM) in rat cavernosal smooth muscle (CSM) and the expression of AM system components in this tissue. Functional assays using standard muscle bath procedures were performed in CSM isolated from male Wistar rats. Protein and mRNA levels of pre-pro-AM, calcitonin receptor-like receptor (CRLR), and Subtypes 1, 2 and 3 of the receptor activity-modifying protein (RAMP) family were assessed by Western immunoblotting and quantitative real-time polymerase chain reaction, respectively. Nitrate and 6-keto-prostaglandin F(1α) (6-keto-PGF(1α); a stable product of prostacyclin) levels were determined using commercially available kits. Protein and mRNA of AM, CRLR, and RAMP 1, -2, and -3 were detected in rat CSM. Immunohistochemical assays demonstrated that AM and CRLR were expressed in rat CSM. AM relaxed CSM strips in a concentration-dependent manner. AM(22-52), a selective antagonist for AM receptors, reduced the relaxation induced by AM. Conversely, CGRP(8-37), a selective antagonist for calcitonin gene-related peptide receptors, did not affect AM-induced relaxation. Preincubation of CSM strips with N(G)-nitro-L-arginine-methyl-ester (L-NAME, nitric oxide synthase inhibitor), 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, quanylyl cyclase inhibitor), Rp-8-Br-PET-cGMPS (cGMP-dependent protein kinase inhibitor), SC560 [5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethyl pyrazole, selective cyclooxygenase-1 inhibitor], and 4-aminopyridine (voltage-dependent K(+) channel blocker) reduced AM-induced relaxation. On the other hand, 7-nitroindazole (selective neuronal nitric oxide synthase inhibitor), wortmannin (phosphatidylinositol 3-kinase inhibitor), H89 (protein kinase A inhibitor), SQ22536 [9-(tetrahydro-2-furanyl)-9H-purin-6-amine, adenylate cyclase inhibitor], glibenclamide (selective blocker of ATP-sensitive K(+) channels), and apamin (Ca(2+)-activated channel blocker) did not affect AM-induced relaxation. AM increased nitrate levels and 6-keto-PGF1α in rat CSM. The major new contribution of this research is that it demonstrated expression of AM and its receptor in rat CSM. Moreover, we provided evidence that AM-induced relaxation in this tissue is mediated by AM receptors by a mechanism that involves the nitric oxide-cGMP pathway, a vasodilator prostanoid, and the opening of voltage-dependent K(+) channels.

Pharmacological characterisation of the mechanisms underlying the relaxant effect of adrenomedullin in the rat carotid artery.

OBJECTIVES: We investigated the mechanisms underlying the relaxant effect of adrenomedullin (AM) in the rat carotid artery and verified the expression of AM system components in this tissue. METHODS: The carotid artery was isolated from male Wistar rats and immunohistochemical, Western immunoblotting, real-time polymerase chain reaction and functional assays were conducted. KEY FINDINGS: Protein and mRNA expression of AM, calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMP)1, 2, 3 were detected in carotid segments from male Wistar rats. Immunohistochemical assays showed that AM and CRLR receptors are expressed in the endothelium and smooth muscle cells. Functional assays showed that AM concentration dependently relaxed carotid rings with intact endothelium. Endothelial removal reduced, but not abolished, the relaxation induced by AM. AM22-52 (selective antagonist for AM receptors) and calcitonin gene-related peptide (CGRP)8-37 (selective CGRP receptor antagonist) reduced AM-induced relaxation in endothelium-intact rings. Pre-incubation of endothelium-intact rings with N-nitro-L-arginine methyl ester, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one or Rp-8-Bromo-?-phenyl-1,N2-ethenoguanosine 3',5'cyclic monophosphorothioate reduced AM-induced relaxation. Inhibition of cyclooxygenase-1 and protein kinase A (PKA) reduced AM-induced relaxation. The relaxation induced by AM was attenuated by the K(+) channel blockers apamin and glibenclamide. AM increased nitrate levels and 6-keto-prostaglandin F1α (stable product of prostacyclin) in the rat carotid. In endothelium-denuded rings, AM22-52 , glibenclamide and PKA inhibition by H89 reduced AM-induced relaxation. CONCLUSIONS: The novelty of this work is that it first demonstrated functionally that AM-induced relaxation is mediated by AM and CGRP receptors located on the endothelium and AM receptors located on smooth muscle of rat carotid arteries. AM-induced relaxation involves the nitric oxide-cGMP pathway, a vasodilator prostanoid, the opening of K(+) channels and the activation of PKA.

Cardiovascular effects of the intracerebroventricular injection of adrenomedullin: roles of the peripheral vasopressin and central cholinergic systems

Our objective was to investigate in conscious Sprague-Dawley (6-8 weeks, 250-300 g) female rats (N = 7 in each group) the effects of intracerebroventricularly (icv) injected adrenomedullin (ADM) on blood pressure and heart rate (HR), and to determine if ADM and calcitonin gene-related peptide (CGRP) receptors, peripheral V1 receptors or the central cholinergic system play roles in these cardiovascular effects. Blood pressure and HR were observed before and for 30 min following drug injections. The following results were obtained: 1) icv ADM (750 ng/10 µL) caused an increase in both blood pressure and HR (DMAP = 11.8 ± 2.3 mmHg and ΔHR = 39.7 ± 4.8 bpm). 2) Pretreatment with a CGRP receptor antagonist (CGRP8-37) and ADM receptor antagonist (ADM22-52) blocked the effect of central ADM on blood pressure and HR. 3) The nicotinic receptor antagonist mecamylamine (25 µg/10 µL, icv) and the muscarinic receptor antagonist atropine (5 µg/10 µL, icv) prevented the stimulating effect of ADM on blood pressure. The effect of ADM on HR was blocked only by atropine (5 µg/10 µL, icv). 4) The V1 receptor antagonist [β-mercapto-β-β-cyclopentamethylenepropionyl¹, O-me-Tyr²,Arg8]-vasopressin (V2255; 10 µg/kg), that was applied intravenously, prevented the effect of ADM on blood pressure and HR. This is the first study reporting the role of specific ADM and CGRP receptors, especially the role of nicotinic and muscarinic central cholinergic receptors and the role of peripheral V1 receptors in the increasing effects of icv ADM on blood pressure and HR.

Cardiovascular effects of the intracerebroventricular injection of adrenomedullin: roles of the peripheral vasopressin and central cholinergic systems.

Our objective was to investigate in conscious Sprague-Dawley (6-8 weeks, 250-300 g) female rats (N = 7 in each group) the effects of intracerebroventricularly (icv) injected adrenomedullin (ADM) on blood pressure and heart rate (HR), and to determine if ADM and calcitonin gene-related peptide (CGRP) receptors, peripheral V1 receptors or the central cholinergic system play roles in these cardiovascular effects. Blood pressure and HR were observed before and for 30 min following drug injections. The following results were obtained: 1) icv ADM (750 ng/10 µL) caused an increase in both blood pressure and HR (DMAP = 11.8 ± 2.3 mmHg and ΔHR = 39.7 ± 4.8 bpm). 2) Pretreatment with a CGRP receptor antagonist (CGRP8-37) and ADM receptor antagonist (ADM22-52) blocked the effect of central ADM on blood pressure and HR. 3) The nicotinic receptor antagonist mecamylamine (25 µg/10 µL, icv) and the muscarinic receptor antagonist atropine (5 µg/10 µL, icv) prevented the stimulating effect of ADM on blood pressure. The effect of ADM on HR was blocked only by atropine (5 µg/10 µL, icv). 4) The V1 receptor antagonist [ß-mercapto-ß-ß-cyclopentamethylenepropionyl¹, O-me-Tyr²,Arg8]-vasopressin (V2255; 10 µg/kg), that was applied intravenously, prevented the effect of ADM on blood pressure and HR. This is the first study reporting the role of specific ADM and CGRP receptors, especially the role of nicotinic and muscarinic central cholinergic receptors and the role of peripheral V1 receptors in the increasing effects of icv ADM on blood pressure and HR.

Chronic ethanol consumption reduces adrenomedullin-induced relaxation in the isolated rat aorta.

Adrenomedullin (AM) is a peptide that displays cardiovascular protective activity. We investigated the effects of chronic ethanol consumption on vascular reactivity to AM and the expression of AM system components in the rat aorta. Male Wistar rats were treated with ethanol (20% vol/vol) for 6 weeks. Vascular reactivity experiments were performed in the isolated rat aorta. Metalloproteinase-2 (MMP-2) levels were determined by gelatin zymography. Nitrite and nitrate generation was measured by chemiluminescence. Protein and mRNA levels of pre-pro-AM, calcitonin receptor-like receptor (CRLR) and RAMP1, 2, and 3 (receptor-activity-modifying proteins) were assessed by western blot and quantitative real-time polymerase chain reaction, respectively. Ethanol intake reduced AM-induced relaxation in endothelium-intact rat aortas, whereas calcitonin gene-related peptide-, acetylcholine-, and sodium nitroprusside-induced relaxation were not affected by ethanol intake. N(G)-nitro-l-arginine-methyl-ester (l-NAME), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, and tetraethylammonium reduced AM-induced relaxation in aortic rings from both control and ethanol-treated rats. Ethanol consumption did not alter basal levels of nitrate and nitrite, nor did it affect the expression of MMP-2 in the rat aorta. Ethanol consumption increased mRNA levels of pre-pro-AM and RAMP1. Protein levels of AM, CRLR, and RAMP1, 2, and 3 were not affected by ethanol consumption. The major findings of the present study are that ethanol consumption reduces the vascular relaxation induced by AM and changes the mRNA expression of the components of the AM system in the vasculature. This response could be one of the mechanisms by which ethanol predisposes individuals to vascular dysfunction and hypertension.