Intravenous Administration of Arecoline Induces Biphasic Modulations in Blood Pressure in Anaesthetized Rats

1School of Educational Sciences and Guangdong Provincial Key Laboratory of Development and Education for Special Needs Children, Lingnan Normal University, 29 Cunjing Road, Zhanjiang 524048, Guangdong Province, China. 2Department of Oncology, Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Hongshan Road Shizi Street 100, Nanjing, Jiangsu 210028, P.R. China. Article Information Received 01 September 2020 Revised 15 October 2020 Accepted 05 November 2020 Available online 06 January 2021


INTRODUCTION
A recoline (molecular formula shown in Fig. 1) is a major pharmacological alkaloid of areca (betel) nut, a chewable fruit endemic to South and Southeast Asia that is reported to produce effects of anti-depression, antifatigue, attention-focusing, and relaxation (Volgin et al., 2019). Areca nut also acts as a traditional herbal medicine widely used for vermifuge and as a digestant in tropical and subtropical countries (Peng et al., 2015). Arecoline has several cholinomimetic effects on the parasympathetic nervous system (Dasgupta et al., 2017(Dasgupta et al., , 2018. Because of the cholinergic features, arecoline is a therapeutic treatment for patients with Alzheimer's dementia (AD), and ameliorates the symptoms of psychosis and schizophrenia (Bales et al., 2009;Christie et al., 1981;Dasgupta et al., 2006;Pomara and Sidtis, 2010;Xu et al., 2019).

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Corresponding author: peilingzhouvip@163.com, robertluoyi@126.com 0030-9923/2021/0001-0001 $ 9.00/0 Copyright 2021 Zoological Society of Pakistan Notably, cholinergic actions exert significant effects on cardiovascular activities, but with complicated phenotypes. For example, administration of acetylcholine into specific brain regions induced the blood pressure (BP) depressor response (Shafei et al., 2013;Zhang et al., 2016; O n l i n e F i r s t A r t i c l e Zhu et al., 2015), activation of the muscarinic or nicotinic acetylcholine receptors in the rostral or caudal ventrolateral medulla led to BP pressor or depressor responses (Aberger et al., 2001;Kumar et al., 2009). Studies regarding arecoline on BP modulations have also been published. For example, intraperitoneal (i.p.) injection of arecoline produced cardiodepression in rats and dogs (Beil et al., 1986;Dahl et al., 1994), and a meta-analysis demonstrated that chewing areca nut increased the risk of cardiovascular disorders (Peng et al., 2015;Zhang et al., 2010). This study was designed to determine the effects of arecoline on systemic BP modulations, as well as the modulatory characteristics on such effects to provide basic data for physiopharmacological investigations, and to provide perspectives of the cardiovascular concerns associated with arecoline use in clinical practice.

Animals
Young adult male Sprague-Dawley rats (2 months, 240 ± 20 g; n = 30) were obtained from Jinan Pengyue Experimental Animal Breeding Co. LTD (Jinan, China) and used in this study. The animals were housed in a temperature-controlled (25 ± 1°C) environment with a 12/12 h light/dark-cycle with ad libitum food and water. All animal experiments were conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23, revised in 1996), and were approved by the Academic and Ethics Committee of Lingnan Normal University (LNU20191018). All efforts were made to minimize the number of animals used, as well as their suffering.

Surgical procedures
The BP recording methods followed those detailed in our previous reports Zhu et al. 2015). Briefly, rats were anesthetized with urethane (1.4 g/kg body weight i.p. injection; Sinopharm Chemical Reagent Co. Ltd., Shanghai, China) and processed for cervical surgery using tracheal intubation. The carotid artery pressure was recorded using a catheter (BL-2020; Taimeng Sci-Tec Co., Ltd., Chengdu, China) that was connected to a signal collecting and processing apparatus (BL-420F, Taimeng Sci-Tech Co., Ltd.) via a BP transducer (PT-100, Taimeng Sci-Tech Co., Ltd.).

BP measurements
Once the BP was stable, saline (0.9% NaCl) and arecoline (at 0.06, 0.2, and 0.6 mg/kg/0.2ml) were injected separately (~10 s) into the vein. Repeated injections were administered at ≥60 min intervals, according to the persistent period from our preliminary experiments in cardiovascular recordings and prior reports on behavioral tests and metabolic examinations (Dasgupta et al., 2018;Sarbani et al., 2006), to avoid mutual interference between arecoline administrations. In general, each animal received 3-5 different drug injections, and the BPs that failed to return to basal values (deviated by >15% of the basal levels) were excluded.
The drug effects on BP regulation were considered substance-specific if they were reversible and reproducible. The mean arterial pressure (MAP), maximum decreased MAP (MDMAP), maximum increased MAP (MIMAP), the latency for arecoline-induced BP changes, the BP reaction time for Period 1 (the duration from the onset of BP decrease to recovery), BP reaction time for Period 2 (the duration from the onset of BP increase to recovery), and the area under the curve (AUC; the changes in MAP relative to the reaction time, as calculated using Graph Pad Prism 5.0 software; Graph Pad Software Inc., San Diego, CA) in response to the drug stimulations were analyzed. As no alterations in BP were observed upon saline treatment, the MAPs in Period 1 and Period 2 for the saline treatment group were calculated from two 60-s BP sequences beginning at 10 s and 100 s, respectively. The pre-test MAPs were calculated form a 60-s BP sequence prior to injections.

Statistical analysis
All data are presented as the mean ± standard error. A one-way analysis of variance followed by a Fisher's least significant difference post-hoc test was conducted for statistical analyses. P values <0.05 were considered significant.

DISCUSSION
Arecoline is the main pharmacological alkaloid from areca nut (Dasgupta et al., 2017), and is a therapeutic drug for various ailments, and in particular, for parasitic diseases, digestive disorders, depression, and potentially for AD and schizophrenia treatments (Bales et al., 2009;Chandra et al., 2008;Pomara and Sidtis, 2010). Arecoline primarily exhibits parasympathomimetic features and cardiovascular actions (Beil et al., 1986;Chiou and Kuo, 2008).
The present study showed that intravenous injection of arecoline induced systemic BP modulations with dose-dependent effects. Such effects were reversible and reproducible (Fig. 2), indicating that arecoline exerts substance-specific roles on the regulation of BP. Our current findings demonstrate that arecoline evokes biphasic BP regulations in rats, with an initial downregulation and a subsequent upregulation. However, some reports demonstrated that arecoline only reduced BP in dogs and humans (Beil et al., 1986;Dahl et al., 1994), or a sole BP elevation in rats (Barnes and Roberts, 1991). The discrepancies between the current and previous studies may be due to differences in arecoline doses (a low dose of arecoline may exhibit a primarily depressor response) or sampling times (arecoline induces a transitory BP depression following a longer BP elevation phase; Figs. 3 and 6). Remarkably, the latency of arecolineinduced BP changes via intravenous injection is within 10 seconds in the current study (Fig. 3A), while the onset of cardiovascular activities following areca nut chewing was ~2 minutes (Chu, 2002); thus indicating that different routes of arecoline administration may modulate blood pressure modulation differently.
As arecoline can cross the blood brain barrier (Soncrant et al., 1989), arecoline-mediated BP modulations could be exerted through the central (the brain) and peripheral (mainly the cardiovascular) nervous systems. The downregulation in PB may be due to arecoline activation of the vagal neural circuit, which relaxes the aorta endothelium and improves vasorelaxation (Liu et al., 2016); all such factors correlate with BP depressor responses. Conversely, the upregulation may be due to sympathoadrenal responses (Chu, 2002) since arecoline can activate the hypothalamic-pituitary-adrenal (HPA) axis, which stimulates adrenocorticotropic hormones and corticosterone release (Calogero et al., 1989); thus, leading to a pressor response as proven in many reports (Núñez et al., 2008;Scoggins et al., 1983). Such hypotheses are also supported by evidence that chewing areca nut promotes cadiovascular activities that are associated with sympathoadrenal activation (Chu, 1993(Chu, , 2002. However, determinations of the exact mechanisms require further investigations.
In summary, this study found that arecoline induced biphasic BP modulations, including an initial downregulation followed by upregulation. Nevertheless, further investigations are required to determine the exact mechanisms controlling such fluctuations in BP.