Calcium channels blockers toxins attenuate abdominal hyperalgesia and inflammatory response associated with the cerulein-induced acute pancreatitis in rats
Vanice Paula Ricardo Carvalho 1, Juliana Figueira da Silva 1, Marcelo Araújo Buzelin 1, Cláudio Antônio da Silva Júnior 1, Duana Carvalho Dos Santos 1, Danuza Montijo Diniz 1, Nancy Scardua Binda 2, Márcia Helena Borges 3, André Luiz Senna Guimarães 4, Elizete Maria Rita Pereira 1, Marcus Vinicius Gomez 5
Abstract
Agents that modulate the activity of high-voltage gated calcium channels (HVCCs) exhibit experimentally and clinically significant effect by relieving visceral pain. Among these agents, the toxins Phα1β and ω-conotoxin MVIIA effectively reduce chronic pain in rodent models. The molecular mechanisms underlying the chronic pain associated with acute pancreatitis (AP) are poorly understood. Hypercalcemia is a risk factor; the role of cytosolic calcium is considered to be a modulator of pancreatitis. Blockade of Ca2+ signals may be useful as a prophylactic treatment of pancreatitis.
We explored the pathophysiological roles of three peptide toxins: Phα1β and its recombinant form CTK 01512-2—blockers of TRPA1 receptor and HVCCs and ω-conotoxin MVIIA, a specific blocker of N-type calcium channels in cerulein-induced AP. Cerulein injection elicits AP in rats, evidenced by an increase in hyperalgesic pain, inflammatory infiltration, amylase and lipase secretion, and reactive oxygen species, TNF-α, and p65 NF-κB levels. These effects of cerulein-induced AP were abolished by Phα1β and its recombinant form CTK 01512-2, whereas ω-conotoxin MVIIA had no effect on the induced increase in pancreatic enzyme secretion. Our results demonstrate that Phα1β and CTK 01512-2 toxins—antagonists of HVCCs and TRPA1 receptor presented an effective response profile, in the control of nociception and inflammatory process in the AP model in rats, without causing changes in spontaneous locomotion of the rats.
Introduction
Acute pancreatitis (AP) is an inflammatory disease of the pancreas. The cause of pancreatitis is related to biliary disease or excessive alcohol consumption (Kim et al., 2015a,b). Clinically, AP symptoms consist of abdominal pain, nausea, and hyperamylasemia (Kim et al., 2015a,b); some cases of AP become severe, extremely painful, and include multisystem organ failure accompanied by a high mortality rate (Stumpf et al., 2016). Other features of AP include oedema, hemorrhage, acinar cell necrosis, and severe pancreatic inflammation (Zhao et al., 2013). The pathogenic mechanism of AP includes autodigestion and acute inflammation of the pancreas by activation of pancreatic proteases and acute inflammatory cells (Kim et al., 2015a,b). Several experimental animal models of AP have been developed, with AP induced by cerulein being one of the most commonly used versions (Lerch and Gorelick, 2013).
The venom of the armed spider Phoneutria nigriventer contains peptide toxins that are potent and selective ion channel blockers with a great pharmaceutical potential (Gomez et al., 2002). The most studied peptide called PhTx3 consists of six peptides isoforms (PhTx3-1 to 3–6). The peptide PhTx3-6, patented as Phα1β, blocks high-voltage-gated calcium channels (HVCCs) types N, R, P/Q and L, in this order of potency (Vieira et al., 2005) and is also a specific inhibitor of the TRPA1 channel (Tonello et al., 2017).
Phα1β has been shown to have an antinociceptive effect in several rodent pain models, including visceral pain (Diniz et al., 2014); postsurgical, inflammatory, and neuropathic pain (Souza et al., 2008; De Souza et al., 2013 and 2014); and cancer pain (Rigo et al., 2013). The recombinant form of Phα1β, CTK 01512-2, shows similar activity as the native peptide (Rigo et al., 2017; Tonello et al., 2017). Ward et al. (1995) suggested that an abnormally elevated Ca2+ may be a trigger for AP. Cerulein-induced pancreatitis is associated with its ability to initiate Ca2+ signals. Because HVCC-blocking agents appear to prevent the abnormal increase in Ca2+ concentration associated with AP, we decided to test their effect on cerulein-induced pancreatitis.
Section snippets
Materials
The native Phα1β toxin was purified from the PhTx3 fraction of P. nigriventer venom as described by Cordeiro et al. (1993). The recombinant Phα1β toxin (CTK 01512-2) was synthesized by Giotto Biotech (www.giottobiotech.com). The native and recombinant toxins have the same sequence of 55 amino acids (ACIPRGEICTDDCECCGCDNQCYCPPGSS LGIFKCSCAHANKYFCNRKKEKCKKA). Phα1β is a reversible blocker of HVCCs (Vieira et al., 2005); the recombinant CTK 01512-2 reproduces the analgesic effect of the native Acute pancreatitis induced by five or seven doses of cerulein (5.0 μg/kg, i.p).
Acute pancreatitis induction by five or seven intraperitoneal injections of cerulein (5 μg/kg, i.p) caused similar abdominal mechanical hyperalgesia during 30–120 min after the last injection of cerulein, Fig. 2. There were no significant differences in the nociceptive behavior measured by abdominal hyperalgesia when the AP model was induced by five or seven doses of cerulein with 5.0 μg/kg i.p. Therefore, we used five intraperitoneal injections of cerulein 5.0 μg/kg i.p to induce the AP.
Discussion
The present study showed that ω-conotoxin MVIIA, Phα1β, and recombinant CTK 01512-2 toxins attenuated pancreas inflammation and hyperalgesia response associated with cerulein-induced AP in rats. Cerulein-induced AP is the most common and accepted model of AP and is associated with studying nociception in experimental rats (Stumpf et al., 2016). Intraperitoneally administered cerulein—a synthetic agonist of cholecystokinin—increases pancreatic enzyme secretion causing autolysis of pancreatic.
Conclusion
Our results demonstrated that Phα1β and CTK 01512-2 toxins—inhibitors of HVCCs and antagonists of the TRPA1 receptor—elicited an effective response profile in the control of inflammation and nociception processes in the cerulein-induced AP model in rats, without causing changes in spontaneous locomotion of the rats. Thus suggesting the potential use of these FI-6934 neuropeptides as analgesic drugs for the treatment of AP pain.
Ethical statement
The authors warrant that the above manuscript is an original work. It has neither been published before nor has it been submitted for publication elsewhere. The authors do not have any competing financial interests. All the authors have contributed significantly to the execution, analyses, and writing of the study, which is now being reported.