Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) act as neurotransmitters in numerous biological responses. We previously reported that the replacement of Lys by Arg, and Met by Leu in VIP (IK312532; [Arg 15, 20, 21, Leu 17]-VIP) resulted in a significant improvement in metabolic stability and biological activity. In the present study, we investigated the effect of VIP and its related peptides including long-acting VIP derivative (IK312532) and PACAP27 on the cytotoxicity of cigarette smoke extract (CSE), a causative factor of chronic obstructive pulmonary disease (COPD), in rat alveolar L2 cells. RT-PCR displayed the dominant expression of mRNA for the VIP-specific VPAC2 receptor in L2 cells, and VIP and the related peptides showed the specific binding activity and potent stimulation of adenylate cyclase. CSE at a concentration of 0.1% or higher induced significant apoptotic death of L2 cells. Interestingly, the addition of neuropeptides at a concentration of 10−11 M or higher in L2 cells with CSE (0.25%) resulted in significant attenuation of cell death with the deactivation of CSE-evoked caspase-3 activity. IK312532 was much stable against the enzymatic digestion compared to VIP, and the protective effect of IK312532 was 1.6-fold higher than that of VIP. Taken together with our previous report showing that IK312532 has long-acting relaxant activity in the lung, IK312532 may be a potential candidate for drug treatment of asthma and COPD.
Currently, the incidence of chronic obstructive pulmonary disease (COPD) is increasing, and is expected to be the fourth largest cause of death in the world by 2010 [1]. This disease is characterized by a chronic, slowly progressive airway disorder resulting from a combination of pulmonary emphysema and irreversible reduction in the caliber of the small airways of the lung. The toxicity of cigarette smoke is associated with the occurrence of COPD in developing countries, and in vitro studies have shown that cigarette smoke induces apoptotic death of some alveolar cells including human alveolar A549 cells [2]. The chemical composition of cigarette smoke is complex. Therefore, it is difficult to predict which compound individually, or in combination, may be involved in the toxicity of cigarette smoke. Recently, oxidative damage to cellular components was found to be involved in the mechanism of cigarette smoke toxicity, and to cause DNA damage and cell death [3].
In our previous investigation, we confirmed that vasoactive intestinal peptide (VIP) [4] and pituitary adenylate cyclase-activating polypeptide (PACAP) [5] have a potent neuroprotective effect against the several oxidative stresses induced by NMDA-type glutamate receptor agonist, prion protein, and β-amyloid in rat pheochromocytoma PC12 cells [6], [7], [8]. VIP and PACAP are similar neuropeptides belonging to the glucagon/secretin family, and act as a neurotransmitter in the central and peripheral nervous systems [9]. In vivo studies as well as our investigation also showed the potent neuroprotective effect of VIP and PACAP on oxidative stress-induced toxicity in some tissues and neuronal cells [10], [11], [12], whereas the effect of these neuropeptides on the toxicity of cigarette smoke has not been fully elucidated.
In this study, we have used a rat alveolar epithelial cell line (L2 cells) to study the ability of cigarette smoke to injure epithelial cells. The aims of this investigation were to demonstrate the protective effect of VIP and PACAP on the toxicity of cigarette smoke and their ability to deactivate the cigarette smoke-evoked caspase-3 in L2 cell in vitro. Furthermore, we now report on extended studies exploring the relative potency and efficacy of IK312532 ([R 15, 20, 21, L 17]-VIP-GRR), a novel long-acting VIP derivative, compared to VIP.
Peptides (Table 1) were synthesized by a solid-phase strategy employing optimal side-chain protection as reported previously [13]. The reference cigarette (2R4F) was obtained from the Smoking and Health Institute of the University of Kentucky (Lexington, KY). WST-8 (4-[3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2 H-5-tetrazolio]-1, 3-benzene disulfonate sodium salt) was purchased from Dojindo (Kumamoto, Japan). Dibutyryl-cAMP (db-cAMP) was purchased from Sigma (St. Louis, MO). [125 I]VIP (81.4
To characterize PACAP/VIP receptors expressed in L2 cells, RT-PCR was performed with primers which are specific for the three distinct PACAP/VIP receptors; the PACAP-specific PAC1 receptor, and the two VIP/PACAP receptors, VPAC1 and VPAC2. A PCR product of the VPAC2 receptor mRNA was obtained from L2 cells, whereas PAC1 and VPAC2 receptors were negligible (Fig. 1). VPAC2 receptors in L2 cells were identified and characterized by a radioligand binding assay using [125 I]VIP, and the estimated
For the first time, we demonstrated that CSE induced the delayed cell death in L2 cells in a concentration-dependent manner, and that neuropeptides including VIP, PACAP27, and long-acting VIP derivative IK312532 significantly attenuated CSE cytotoxicity through deactivation of CSE-evoked caspase-3.
Recently, it has been reported that numerous chemical components of cigarette smoke, including acrolein [22], nicotine [23], benzo[a]pyrene [24], and N-nitrosamines [25], appear to play an important
