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One of the major limits of chemotherapy is depending on the ability of the cancer cells to elude and adapt to different drugs. Recently, we demonstrated how the activation of the M2 muscarinic receptor could impair neuroblastoma cell proliferation. In the present paper, we investigate the possible effects mediated by the preferential M2 receptor agonist arecaidine propargyl ester (APE) on drug resistance in two neuroblastoma cell lines, SK-N-BE and SK-N-BE(2C), a sub-clone presenting drug resistance. In both cell lines, we compare the expression of the M2 receptor and the effects mediated by the M2 agonist APE on cell cycle, demonstrating a decreased percentage of cells in S phase and an accumulation of SK-N-BE cells in G1 phase, while the APE treatment of SK-N-BE(2C) cells induced a block in G2/M phase. The withdrawal of the M2 agonist from the medium shows that only the SK-N-BE(2C) cells are able to rescue cell proliferation. Further, we demonstrate that the co-treatment of low doses of APE with doxorubicin or cisplatin significantly counteracts cell proliferation when compared with the single treatment. Analysis of the expression of ATP-binding cassette (ABC) efflux pumps demonstrates the ability of the M2 agonist to downregulate their expression and that this negative modulation may be dependent on N-MYC decreased expression induced by the M2 agonist. Our data demonstrate that the combined effect of low doses of conventional drugs and the M2 agonist may represent a new promising therapeutic approach in neuroblastoma treatment, in light of its significant impact on drug resistance and the possible reduction in the side effects caused by high doses of chemotherapy drugs.

Triple negative tumors are more aggressive than other breast cancer subtypes and there is a lack of specific therapeutic targets on them. Since muscarinic receptors have been linked to tumor progression, we investigated the effect of metronomic therapy employing a traditional anti-cancer drug, paclitaxel plus muscarinic agonists at low doses on this type of tumor. We observed that MDA-MB231 tumor cells express muscarinic receptors, while they are absent in the non-tumorigenic MCF-10A cell line, which was used as control. The addition of carbachol or arecaidine propargyl ester, a non-selective or a selective subtype 2 muscarinic receptor agonist respectively, plus paclitaxel reduces cell viability involving a down-regulation in the expression of ATP "binding cassette" G2 drug transporter and epidermal growth factor receptor. We also detected an inhibition of tumor cell migration and anti-angiogenic effects produced by those drug combinations in vitro and in vivo (in NUDE mice) respectively. Our findings provide substantial evidence about subtype 2 muscarinic receptors as therapeutic targets for the treatment of triple negative tumors., Competing Interests: The authors have declared that no competing interests exist.

Background/purpose: Oral submucous fibrosis (OSF) represents a precancerous lesion of oral mucosa that may progress into oral cancer and its major etiological factor is areca nut chewing. Carboxyl-terminus of Hsp70-interacting protein (CHIP) functions as an ubiquitin E3 ligase and is associated with fibrosis diseases. In the current study, we sought to investigate whether CHIP participated in the areca nut-mediated OSF development., Methods: The mRNA expression of CHIP in arecoline-stimulated buccal mucosal fibroblasts (BMFs) and OSF tissues was determined by qRT-PCR. Collagen gel contraction, migration and invasion assays were carried out to evaluate the myofibroblast activation. The protein expression levels of α-SMA and transglutaminase 2 (TGM2) were assessed by Western blot., Results: The expression level of CHIP was reduced in BMFs following arecoline treatment in a dose-dependent manner, which was consistent with the observation of lower CHIP expression in OSF specimen compared to the normal counterparts. Ectopic expression of CHIP mitigated the myofibroblast activities, including elevated collagen gel contractility and cell motility. In addition, we showed that overexpression of CHIP downregulated the α-SMA and TGM-2 expression, which may lead to less fibrosis alteration., Conclusion: CHIP may not only function as a key regulator of protein quality control but also a critical deciding factor to oral fibrogenesis. Our findings suggested that CHIP possesses the anti-fibrotic effect, which may be mediated by TGM2 regulation. Restoration of CHIP could be a therapeutic direction to help OSF patients., Competing Interests: Declaration of competing interest None declared., (Copyright © 2019. Published by Elsevier B.V.)

Background: Ataxia telangiectasia mutated (ATM) regulates DNA repair and cell cycle. The present study analyzed arecoline-induced ATM expression during oral cancer progression., Methods: In vitro studies were performed using oral squamous cell carcinoma (OSCC) cell lines treated with arecoline to analyze cell response and ATM regulation. in vivo studies were performed using immunohistochemistry to detect ATM expression in normal, oral potentially malignant disorder (OPMD), and OSCC tissues., Results: Low-dose arecoline induced cell proliferation, ATM promoter activity, and DNA repair. High-dose arecoline induced cell cycle arrest, apoptosis, and DNA damage. ATM was overexpressed in OPMD tissues but was downregulated in OSCC tissues. ATM expression level was associated with the risk of developing dysplasia, buccal-OSCC, and with OSCC survival rate., Conclusion: High ATM expression helps DNA repair mechanisms to maintain the cells in the OPMD stage, but low ATM expression causes DNA damage accumulation to increase cell malignancy., (© 2019 Wiley Periodicals, Inc.)

Millions of people consume betel nut for increased capacity to work and for stress reduction. The nut contains arecoline, which has multiple side effects on endocrine functions. Objective of the work is to investigate pineal-testicular responses to noise and after arecoline treatment in noise in rats. Noise exposure (100 dB, 6 h daily, 10 days) caused pineal stimulation ultrastructurally and at indoleamines level. Leydig cell dysfunction with fall of testosterone level and suppression of sex accessories were noticed. In contrast, pineal activity was inhibited and reproductive functions were stimulated after arecoline administration, confirmed from reversed changes to those of noise. Arecoline treatment in noise exposure showed same results as in noise both in pineal and in reproductive functions. It is concluded that noise causes testicular dysfunction probably by gonadotropin suppression induced by pineal melatonin in noise. Furthermore, arecoline cannot prevent it in noise in rats.

Arecoline, a major alkaloid in areca nuts, is involved in the pathogenesis of oral diseases. Mammalian taste buds are the structural unit for detecting taste stimuli in the oral cavity. The effects of arecoline on taste bud morphology are poorly understood. Arecoline was injected intraperitoneally (IP) into C57BL/6 mice twice daily for 1-4 weeks. After arecoline treatment, the vallate papillae were processed for electron microscopy and immunohistochemistry analysis of taste receptor proteins (T1R2, T1R3, T1R1, and T2R) and taste associated proteins (α-gustducin, PLCβ2, and SNAP25). Body weight, food intake and water consumption were recorded. A 2-bottle preference test was also performed. The results demonstrated that 1) arecoline treatment didn't change the number and size of the taste buds or taste bud cells, 2) electron microscopy revealed the change of organelles and the accumulation of autophagosomes in type II cells, 3) immunohistochemistry demonstrated a decrease of taste receptor T1R2- and T1R3-expressing cells, 4) the body weight and food intake were markedly reduced, and 5) the sweet preference behavior was reduced. We concluded that the long-term injection of arecoline alters the morphology of type II taste bud cells, retards the growth of mice, and affects discrimination competencies for sweet tastants., (© The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Oral squamous cell carcinoma (OSCC) is the sixth most prevalent malignancy worldwide and the third most common cancer in developing nation. Most OSCC patients relapse within months after receiving treatment. Therefore, searching the biomarkers of recurrence is urgently required to improve OSCC patient survival., Methods: We set out to explore whether expression of ZEB1 could be triggered in oral epithelial cells (SG and FaDu) by arecoline in vitro. Control and ZEB1-knockdown arecoline-stimulated SG and FaDu were subjected to migration/invasiveness/anchorage-independent growth assay. Primary and recurrent OSCC tissues from areca quid chewers were analyzed using real-time RT-PCR analysis for ZEB1 expression., Results: Arecoline led to dose-dependent elevation of ZEB1 expression in SG and FaDu cells. Downregulation of ZEB1 by lentiviral infection significantly reversed arecoline-induced oncogenicity including migration ability, cell invasiveness, and anchorage-independent growth in SG and FaDu cells. Clinically, the level of ZEB1 expression was higher in recurrent OSCC tumor samples but lower in primary lesions., Conclusions: Targeting ZEB1 might offer a new strategy for the treatment of OSCC patients. ZEB1 can serve as a progression and relapse marker in OSCC patients., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

A subchronic toxicity test was conducted in rats on the basis of a previous acute toxicity test to evaluate the safety of arecoline hydrobromide (Ah), to systematically study its pharmacological effects and to provide experimental support for a safe clinical dose. Eighty rats were randomly divided into four groups: a high-dose group (1000 mg/kg), medium-dose group (200 mg/kg), low-dose group (100mg/kg) and blank control group. The doses were administered daily via gastric lavage for 14 consecutive days. There were no significant differences in the low-dose Ah group compared to the control group (P>0.05) with regard to body weight, organ coefficients, hematological parameters and histopathological changes. The high-dose of Ah influenced some of these parameters, which requires further study. The results of this study indicated that a long-term, continuous high dose of Ah was toxic. However, it is safe to use Ah according to the clinically recommended dosing parameters. The level of Ah at which no adverse effects were observed was 100 mg/kg/day under the present study conditions.

Betel-quid use is associated with the risk of liver cirrhosis and hepatocellular carcinoma. The aim of the present work was to evaluate the impact of arecoline on human hepatic cytochrome P450 (CYP) enzymes in vitro and rat hepatic CYP enzymes, as well as the hepatic oxidative stress and liver injury of rats in vivo. The in vitro results indicated that arecoline hydrobromide (AH) has no significant effect on the activities of CYP2B, 2C9, 3A4, 1A2, 2E1 and 2D6 in human liver microsome (HLM). However, oral administration of AH at 4 and 20 mg/kg/d for seven consecutive days significantly increased the activities of rat hepatic CYP2B, 2E1, 2D, 3A, 2C and 1A2. In addition, AH at 100 mg/kg/d significantly increased the levels of ALT, AST and MDA, decreased the levels of SOD, CAT, GSH-Px and GSH, in rat liver. The in vivo induction of AH on rat hepatic CYP isoforms suggested that the high risk of metabolic interaction should be existed when the substrate drugs of the six kinds of CYP isoforms was administered in betel-quid use human. Furthermore, the in vivo results also suggested that AH-induced hepatoxicity should be associated with the induction of AH on rat hepatic CYP2E1 and 2B.

The peripheral application of the M2 cholinergic agonist arecaidine on sensory nerve endings shows anti-nociceptive properties. In this work, we analyze in vitro, the mechanisms downstream M2 receptor activation causing the analgesic effects, and in vivo the effects produced by M2 agonist arecaidine administration on nociceptive responses in a murine model of nerve growth factor (NGF)-induced pain. Cultured DRG neurons treated with arecaidine showed a decreased level of VR1 and SP transcripts. Conversely, we found an increased expression of VR1 and SP transcripts in DRG from M2/M4(-/-) mice compared to WT and M1(-/-) mice, confirming the inhibitory effect in particular of M2 receptors on SP and VR1 expression. Patch-clamp experiments in the whole-cell configuration showed that arecaidine treatment caused a reduction of the fraction of capsaicin-responsive cells, without altering the mean capsaicin-activated current in responsive cells. We also demonstrated that arecaidine prevents PKCϵ translocation to the plasma membrane after inflammatory agent stimulation, mainly in medium-small sensory neurons. Finally, in mice, we have observed that intraperitoneal injection of arecaidine reduces VR1 expression blocking hyperalgesia and allodynia caused by NGF intraplantar administration. In conclusion, our data demonstrate that in vivo M2 receptor activation induces desensitization to mechanical and heat stimuli by a down-regulation of VR1 expression and by the inhibition of PKCϵ activity hindering its translocation to the plasma membrane, as suggested by in vitro experiments., (© 2013 Wiley Periodicals, Inc.)

Oral submucous fibrosis (OSF) is considered as a pre-cancerous condition of the oral mucosa and is highly associated with habitual areca quid chewing. Arecoline is the major alkaloid in areca quid and is thought to be involved in the pathogenesis of OSF. Our previous studies have demonstrated that arecoline could induce epithelial-mesenchymal transition (EMT)-related factors in primary human buccal mucosal fibroblasts (BMFs). Therefore, we investigated the expression of zinc finger E-box binding homeobox 1 (ZEB1), which is a well-known transcriptional factor in EMT, in OSF tissues and its role in arecoline-induced myofibroblast transdifferentiation from BMFs. The expression of ZEB1, as well as the myofibroblast marker α-smooth muscle actin (α-SMA), was significantly increased in OSF tissues, respectively. With immunofluorescence analysis, arecoline induced the formation of α-SMA-positive stress fibres in BMFs expressing nuclear ZEB1. Arecoline also induced collagen contraction of BMFs in vitro. By chromatin immunoprecipitation, the binding of ZEB1 to the α-SMA promoter in BMFs was increased by arecoline. The promoter activity of α-SMA in BMFs was also induced by arecoline, while knockdown of ZEB1 abolished arecoline-induced α-SMA promoter activity and collagen contraction of BMFs. Long-term exposure of BMFs to arecoline induced the expression of fibrogenic genes and ZEB1. Silencing of ZEB1 in fibrotic BMFs from an OSF patient also suppressed the expression of α-SMA and myofibroblast activity. Inhibition of insulin-like growth factor receptor-1 could suppress arecoline-induced ZEB1 activation in BMFs. Our data suggest that ZEB1 may participate in the pathogenesis of areca quid-associated OSF by activating the α-SMA promoter and inducing myofibroblast transdifferentiation from BMFs., (© 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

The response surface methodology (RSM) including polynomial equations has been used to design an optimal patch formulation with appropriate adhesion and flux. The patch formulations were composed of different polymers, including Eudragit RS 100 (ERS), Eudragit RL 100 (ERL) and polyvinylpyrrolidone K30 (PVP), plasticizers (PEG 400), and drug. In addition, using terpenes as enhancers could increase the flux of the drug. Menthol showed the highest enhancement effect on the flux of arecoline.

Background: O(6) -methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that can protect cells from carcinogenic effects of alkylating agents by removing adducts from the O(6) position of guanine. Evidences indicated that areca quid chewing may increase the risk of oral squamous cell carcinoma (OSCC). This study was to investigate the role of MGMT expression in OSCCs and the normal oral tissues., Methods: Thirty-two OSCCs from areca quid chewers and ten normal oral tissue biopsy samples without areca quid chewing were analyzed by the immunohistochemistry for MGMT. Primary human oral keratinocytes (HOKs) were challenged with arecoline, the major alkaloid of areca nut, by Western blot. Nicotine, an important component of cigarette smoke, was added to find the possible regulatory mechanisms., Results: Significant association was observed between low MGMT expression and advanced clinical stage of OSCCs and lymph node metastasis (P = 0.03). MGMT expression was significantly higher in patients only chewing areca quid than patients both chewing areca quid and smoking (P = 0.028). Arecoline was found to elevate MGMT expression in a dose- and time-dependent manner. The addition of nicotine was found to enhance arecoline-induced MGMT expression., Conclusion: Our results indicate that MGMT could be used clinically as a predictive marker for tumor processing, the potential for lymph node metastasis as well as advanced clinical stage. MGMT expression was significantly upregulated by arecoline in HOKs. Nicotine has a synergistic effect of arecoline-induced MGMT expression. The cigarette smoking may act synergistically in the pathogenesis of OSCC in areca quid chewers via the upregulation of MGMT., (© 2012 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Arecoline, the most abundant areca alkaloid, has been reported to decrease interleukin-6 (IL-6) levels in epithelial cancer cells. Since IL-6 overexpression contributes to the tumorigenic potency of basal cell carcinoma (BCC), this study was designed to investigate whether arecoline altered IL-6 expression and its downstream regulation of apoptosis and the cell cycle in cultured BCC-1/KMC cells. BCC-1/KMC cells and a human keratinocyte cell line, HaCaT, were treated with arecoline at concentrations ranging from 10 to 100μg/ml, then IL-6 production and expression of apoptosis- and cell cycle progress-related factors were examined. After 24h exposure, arecoline inhibited BCC-1/KMC cell growth and decreased IL-6 production in terms of mRNA expression and protein secretion, but had no effect on HaCaT cells. Analysis of DNA fragmentation and chromatin condensation showed that arecoline induced apoptosis of BCC-1/KMC cells in a dose-dependent manner, activated caspase-3, and decreased expression of the anti-apoptotic protein Bcl-2. In addition, arecoline induced progressive and sustained accumulation of BCC-1/KMC cells in G2/M phase as a result of reducing checkpoint Cdc2 activity by decreasing Cdc25C phosphatase levels and increasing p53 levels. Furthermore, subcutaneous injection of arecoline led to decreased BCC-1/KMC tumor growth in BALB/c mice by inducing apoptosis. This study demonstrates that arecoline has potential for preventing BCC tumorigenesis by reducing levels of the tumor cell survival factor IL-6, increasing levels of the tumor suppressor factor p53, and eliciting cell cycle arrest, followed by apoptosis., (Crown Copyright © 2011. Published by Elsevier Inc. All rights reserved.)

Background: The beneficial effects of magnesium (Mg) salts on central manifestations of Mg deficiency are well known. Mg replacement therapy can be effective to prevent some of the serious depression-like and anxiety-related behaviour sequelae of Mg deficiency. However, few experimental studies have been undertaken on Mg-deficiency-induced behavioural changes. Even fewer studies have been carried out on acute behavioural responses to clonidine, D-amphetamine, arecoline, nicotine, apomorphine, and L-5-hydroxytryptophan (HTP), which might characterize possible neuromediator changes in Mg deficiency. The effects of correcting Mg deficiency by magnesium chloride (MgCl₂ · 6H₂O) and the combination of this salt with vitamin B₆, on the behavioural manifestations of Mg deficiency have never been described as well., Objective: The aims of this study were: to estimate effect of MgCl₂ · 6H₂O alone and in combination with vitamin B6 on acute behavioural responses to agonists or blockers of the main neurotransmitter systems in CNS, psychomotor activity and emotional status of rats fed with Mg-deficient diet for 49 days. In our study open field test has shown that in Mg-deficient rats locomotor activity and vertical activity, number of visiting and residence time in central squares were decreased significantly. In the elevated plus maze test, the number of visiting open arms and residence time of rats were significantly less as compared with the control group. In the forced swimming test, time immobile was significantly increased by 44.29% and time of swimming was decreased by 52.79% compared to control., Results: In our study Mg-deficient rats were more sensitive to d-amphetamine-induced motor stereotypes. Mg deficiency antagonized 5-hydroxytryptophan-induced head-twitch response and arecoline-induced tremor. Supplement of MgCl₂ · 6H₂O with vitamin B₆ administered to a Mg-deficient rat increased the Mg level in plasma and erythrocytes. Furthermore, this increase was in relation to vitamin B₆ given to the animal. Mg supplementation alone and in combination with pyridoxine normalized acute behavioural responses to d-amphetamine, 5-hydroxytryptophan, and arecoline in Mg deficient rats with a return to pre-deficient levels observed in the Mg sufficient group., Discussion: Combination of Mg salts and pyridoxine hydrochloride can be effective at treating some behavior form of primary Mg deficiency.

Ca(2+)-independent phospholipase A(2)β (iPLA(2)β) selectively hydrolyzes docosahexaenoic acid (DHA, 22:6n-3) in vitro from phospholipid. Mutations in the PLA2G6 gene encoding this enzyme occur in patients with idiopathic neurodegeneration plus brain iron accumulation and dystonia-parkinsonism without iron accumulation, whereas mice lacking PLA2G6 show neurological dysfunction and neuropathology after 13 months. We hypothesized that brain DHA metabolism and signaling would be reduced in 4-month-old iPLA(2)β-deficient mice without overt neuropathology. Saline or the cholinergic muscarinic M(1,3,5) receptor agonist arecoline (30 mg/kg) was administered to unanesthetized iPLA(2)β(-/-), iPLA(2)β(+/-), and iPLA(2)β(+/+) mice, and [1-(14)C]DHA was infused intravenously. DHA incorporation coefficients k* and rates J(in), representing DHA metabolism, were determined using quantitative autoradiography in 81 brain regions. iPLA(2)β(-/-) or iPLA(2)β(+/-) compared with iPLA(2)β(+/+) mice showed widespread and significant baseline reductions in k* and J(in) for DHA. Arecoline increased both parameters in brain regions of iPLA(2)β(+/+) mice but quantitatively less so in iPLA(2)β(-/-) and iPLA(2)β(+/-) mice. Consistent with iPLA(2)β's reported ability to selectively hydrolyze DHA from phospholipid in vitro, iPLA(2)β deficiency reduces brain DHA metabolism and signaling in vivo at baseline and following M(1,3,5) receptor activation. Positron emission tomography might be used to image disturbed brain DHA metabolism in patients with PLA2G6 mutations.

Background: Epidemiological evidence of co-use of alcohol and areca nuts suggests a potential central interaction between arecoline, a major alkaloid of areca and a muscarinic receptor agonist, and ethanol. Moreover, the central cholinergic system plays an important role in the depressant action of ethanol and barbiturates. The purpose of this study was to investigate the effects of arecoline on pentobarbital- and ethanol-induced hypnosis in mice., Methods: Male ICR mice were tested for locomotor activity following acute systemic administration of ethanol alone, arecoline alone, or ethanol plus arecoline. For the loss of the righting reflex (LORR) induced by pentobarbital and ethanol, sleep latency and sleeping duration were evaluated in mice treated with arecoline alone or the combination of arecoline and scopolamine or methscopolamine., Results: Ethanol (1.0 to 3.0 g/kg, i.p.) reduced locomotor activity significantly and a declining trend was observed after treatment with arecoline (0.25 to 1.0 mg/kg, i.p.), but there were no synergistic effects of ethanol and arecoline on locomotor activity. The experiments on LORR demonstrated that arecoline (0.125 to 1.0 mg/kg, s.c.) shortened the duration of sleeping induced by ethanol (4.0 g/kg, i.p.), but not pentobarbital (45 mg/kg, i.p.). In addition, alterations of sleep latency were not obvious in both pentobarbital- and ethanol-induced LORR. Statistical analyses revealed that scopolamine (centrally acting), but not methscopolamine (peripherally acting), could antagonize the effect of arecoline on the duration of ethanol-induced LORR in mice., Conclusions: These results suggest that central muscarinic receptor is a pharmacological target for the action of arecoline to modulate ethanol-induced hypnosis.

Background: Betel nut and tobacco chewing is a common practice in south-east Asia. In India, betel nut is commonly chewed in the form of pan, with or without tobacco. Numerous studies have shown the carcinogenic potential of betel nut and tobacco. Betel nut and tobacco are also known to have deleterious effects on the oral tissues., Purpose: The aim of our study was to evaluate and compare the periodontal effects of pan chewing with or without the use of tobacco as an ingredient., Materials and Methods: The periodontal status of 300 subjects (150 subjects were pan chewers with tobacco and 150 subjects were pan chewers without tobacco) was evaluated using the community periodontal index (CPI). The subjects were selected by the stratified random sampling method. The oral hygiene status of the subjects was evaluated using the simplified oral hygiene index., Results: CPI code-4, with a probing depth of 6 mm or more, was seen in 30% of pan chewers with tobacco compared with 7.3% of pan chewers without tobacco. It was found that pan chewers with tobacco had 4.7 times more risk of having pockets than pan chewers without tobacco. The higher codes of loss of attachment were seen in pan chewers with tobacco compared with pan chewers without tobacco. It was found that pan chewers with tobacco had 7 times more risk of having loss of attachment when compared with the pan chewers without tobacco., Conclusions: The results show higher incidence of periodontal diseases in pan chewers who use tobacco compared with pan chewers who do not use tobacco. Based on the results, it was concluded that, although betel nut has deleterious effects on the periodontium, the addition of tobacco leads to a synergistic effect between betel nut and tobacco on the periodontal tissues.

Arecoline is an alkaloid of betel nut of Areca catechu. Betel nut is chewed by millions of people in the world and it causes oral and hepatic cancers in human. It has therapeutic value for the treatment of Alzheimer and schizophrenia. Arecoline has immunosuppressive, mutagenic and genotoxic effects in laboratory animals. It also affects endocrine functions. The objective of this study was to investigate the effects of arecoline on pineal-testicular axis in rats. Since pineal activity is different between day and night, the current study is undertaken in both the photophase and scotophase. The findings were evaluated by ultrastructural and hormonal studies of pineal and testicular Leydig cells, with quantitations of fructose and sialic acid of sex accessories. Arecoline treatment (10 mg/kg body weight daily for 10 days) caused suppression of pineal activity at ultrastructural level by showing dilatation of the cisternae of the rough endoplasmic reticulum (RER), large autophagosome-like bodies with swollen mitochondrial cristae, numerous lysosomes, degenerated synaptic ribbons and reduced number of synaptic-like microvesicles. Moreover, pineal and serum N-acetylserotonin and melatonin levels were decreased with increased serotonin levels in both the gland and serum. In contrast, testicular Leydig cell activity was stimulated with abundance of smooth endoplasmic reticulum (SER), electron-dense core vesicles and vacuolated secretory vesicles, and increased testosterone level in the arecoline recipients. Consequently, the testosterone target, like prostate, was ultrastructurally stimulated with abundance of RER and accumulation of secretory vesicles. Fructose and sialic acid concentrations were also significantly increased respectively in the coagulating gland and seminal vesicle. These results were more significant in the scotophase than the photophase. The findings suggest that arecoline inhibits pineal activity, but stimulates testicular function (testosterone level) and its target organs presumably via muscarinic cholinergic receptor in rats.

The alkaloid arecoline is a main constituent of areca nuts that are chewed by approximately 600 million persons worldwide. A principal metabolite of arecoline is arecoline 1-oxide whose metabolism has been poorly studied. To redress this, synthetic (+/-)-arecoline 1-oxide was administered to mice (20mg/kg p.o.) and a metabolomic study performed on 0-12h urine using ultra-performance liquid chromatography-coupled time-of-flight mass spectrometry (UPLC-TOFMS) with multivariate data analysis. A total of 16 mass/retention time pairs yielded 13 metabolites of (+/-)-arecoline 1-oxide, most of them novel. Identity of metabolites was confirmed by tandem mass spectrometry. The principal pathways of metabolism of (+/-)-arecoline 1-oxide were mercapturic acid formation, with catabolism to mercaptan and methylmercaptan metabolites, apparent CC double-bond reduction, carboxylic acid reduction to the aldehyde (a novel pathway in mammals), N-oxide reduction, and de-esterification. Relative percentages of metabolites were determined directly from the metabolomic data. Approximately, 50% of the urinary metabolites corresponded to unchanged (+/-)-arecoline 1-oxide, 25% to other N-oxide metabolites, while approximately, 30% corresponded to mercapturic acids or their metabolites. Many metabolites, principally mercapturic acids and their derivatives, were excreted as diastereomers that could be resolved by UPLC-TOFMS. Arecoline was converted to arecoline 1-oxide in vitro by human flavin-containing monooxygenases FMO1 (K(M): 13.6+/-4.9muM; V(MAX): 0.114+/-0.01nmolmin(-1)microg(-1) protein) and FMO3 (K(M): 44.5+/-8.0microM; V(MAX): 0.014+/-0.001nmolmin(-1)microg(-1) protein), but not by FMO5 or any of 11 human cytochromes P450. This report underscores the power of metabolomics in drug metabolite mining.

The areca alkaloids comprise arecoline, arecaidine, guvacoline, and guvacine. Approximately 600 million users of areca nut products, for example, betel quid chewers, are exposed to these alkaloids, principally arecoline and arecaidine. Metabolism of arecoline (20 mg/kg p.o. and i.p.) and arecaidine (20 mg/kg p.o. and i.p.) was investigated in the mouse using a metabolomic approach employing ultra-performance liquid chromatography-time-of-flight mass spectrometric analysis of urines. Eleven metabolites of arecoline were identified, including arecaidine, arecoline N-oxide, arecaidine N-oxide, N-methylnipecotic acid, N-methylnipecotylglycine, arecaidinylglycine, arecaidinylglycerol, arecaidine mercapturic acid, arecoline mercapturic acid, and arecoline N-oxide mercapturic acid, together with nine unidentified metabolites. Arecaidine shared six of these metabolites with arecoline. Unchanged arecoline comprised 0.3-0.4%, arecaidine 7.1-13.1%, arecoline N-oxide 7.4-19.0%, and N-methylnipecotic acid 13.5-30.3% of the dose excreted in 0-12 h urine after arecoline administration. Unchanged arecaidine comprised 15.1-23.0%, and N-methylnipecotic acid 14.8%-37.7% of the dose excreted in 0-12 h urine after arecaidine administration. The major metabolite of both arecoline and arecaidine, N-methylnipecotic acid, is a novel metabolite arising from carbon-carbon double-bond reduction. Another unusual metabolite found was the monoacylglyceride of arecaidine. What role, if any, that is played by these uncommon metabolites in the toxicology of arecoline and arecaidine is not known. However, the enhanced understanding of the metabolic transformation of arecoline and arecaidine should contribute to further research into the clinical toxicology of the areca alkaloids.

Background: Areca quid chewers have a higher prevalence of periodontal disease than non-chewers. Little is known about the influence of areca quid on the immune system. This study was to determine the possible effects of the areca nut on phagocytic activity of human neutrophils., Methods: Aqueous extracts of ripe areca nut without husk (rANE), fresh and tender areca nut with husk (tANE), a major alkaloid (arecoline), and a phenolic component ([+]-catechin) of areca nut were examined for their effects on cellular viability using trypan blue exclusion assay. The possible effects on the phagocytic activity of neutrophils against a periodontal pathogen, Actinobacillus actinomycetemcomitans ATCC 33384, were determined using flow cytometry and confocal laser scanning microscopy., Results: At the concentrations tested, rANE, tANE, arecoline, and (+)-catechin did not significantly affect viability of neutrophils. However, rANE, tANE, arecoline, and (+)-catechin inhibited the phagocytic activity of neutrophils in a dose-dependent manner. Approximately 50% of the relative phagocytic activity of neutrophils was affected when 50 microg/ml of rANE, 400 microg/ml of tANE, 20,000 microg/ml of arecoline, or 2,500 microg/ml of (+)- catechin was used. Decreased levels of internalized fluorescent bacteria were also demonstrated. However, arecoline or (+)-catechin alone could not be used to explain the inhibitory effects observed for rANE and tANE., Conclusions: Components of areca nut reduced the uptake of A. actinomycetemcomitans ATCC 33384 by human neutrophils. The inhibition of areca nut on phagocytosis of neutrophils may be one possible mechanism by which the areca nut compromises the periodontal health of areca quid chewers.

This study investigated the effects of arecoline, an active ingredient of the areca nut, on the tone of human umbilical arteries and veins and on the eNOS expression and cell proliferation of human umbilical vein endothelial cells (HUVECs). We found that arecoline relaxes the human umbilical artery and vein rings in a concentration-dependent manner; the higher the concentration of arecoline, the greater the relaxation of the rings. However, the relaxation decreases after the endothelium was removed or pretreated with L-NAME, a nitric oxide synthase inhibitor. Moreover, arecoline increases in a dose-dependent way the cGMP levels of human umbilical arteries and veins. In HUVECs, arecoline also increases the eNOS expression. Therefore, the relaxant effects of arecoline on the umbilical artery and vein rings were endothelium-dependent through the NO-cGMP systems. In addition, arecoline at higher doses (100-1000 microM) inhibits endothelial cell proliferation; the exposure toarecoline (100-1000 microM) for 24 and 48 h induces G2/M cell cycle arrest of HUVECs. Our results indicate that arecoline would decrease vascular tone, in part mediated by NO. Higher doses of arecoline inhibit endothelial cell growth, which suggest that long-term use or high doses of areca nut might induce endothelial dysfunction and associated diseases.

Background: Oral submucous fibrosis (OSF) is associated with the betel quid chewing habit, and 86% of betel quid chewers in Taiwan are also smokers. Arecoline and safrole are major principles in the composition of betel quid, and nicotine is the main toxic ingredient of cigarettes., Methods: To explore the pathogenesis of OSF, flow cytometry was used to compare collagen phagocytosis by fibroblasts from the normal and the OSF region of the same 15 OSF patients., Results: The results indicated that heterogeneity of fibroblasts existed because collagen phagocytosis by fibroblasts from the normal region was higher than from the OSF region in the same patient. The percentage of phagocytic cells was significantly inhibited by 10, 25 and 50 microg/ml arecoline, safrole and nicotine in normal fibroblast cultures, respectively, and the percentage of phagocytic cells was significantly reduced by 25, 25 and 50 microg/ml arecoline, safrole and nicotine in OSF fibroblast cultures, respectively. Collagen phagocytosis by fibroblasts exhibited prominent dose-dependent inhibition as the concentration of arecoline, safrole, and nicotine increased. Besides, nicotine had a synergistic effect on arecoline- or safrole-inhibited collagen phagocytosis., Conclusions: The present study concludes that even in the same person, the collagen phagocytosis by fibroblasts is different between normal and OSF region. The deficiency in collagen phagocytosis by fibroblasts of the lesion might participate in the pathogenesis of OSF. Arecoline, safrole and nicotine, which are released in saliva during BQ chewing plus cigarette smoking, inhibit collagen phagocytosis by fibroblasts in a dose-dependent manner and may induce OSF formation in Taiwan's patients.

A number of drugs undergo extensive first-pass metabolism after oral administration, necessitating large doses for effective therapeutic responses in the body. Buccal administration of drugs is becoming more popular because the drugs diffuse into the systemic circulation directly, circumventing the first-pass metabolism. Lower concentrations thus need to be administered and side effects may be minimized. In this study, one of the classic models for human buccal permeability, i.e. the porcine buccal mucosal model, is compared with the more recent human vaginal model and both these are in turn further compared to porcine mouth floor mucosa. To determine the permeability of the different markers (arecoline, 17beta-estradiol, water and vasopressin), a continuous flow-through perfusion system was used (20 degrees C, 24h). Mean steady state flux values were compared statistically using a t-test at a significance level of 5%. Porcine buccal mucosa showed a consistently lower permeability towards all the markers than the other mucosae tested. Porcine mouth floor mucosa was found to be more permeable than porcine buccal mucosa. From these studies we concluded that human vaginal and porcine mouth floor mucosae were superior models for human buccal mucosa than porcine buccal mucosa, using in vitro permeability studies with various chemical markers.

Studies were performed to determine if the reported 'proconvulsant' action of lithium in rats given cholinergic drugs is related to receptor-initiated phospholipase A2 signaling via arachidonic acid. Regional brain incorporation coefficients k* of intravenously injected [1-14C]arachidonic acid, which represent this signaling, were measured by quantitative autoradiography in unanesthetized rats at baseline and following administration of subconvulsant doses of the cholinergic muscarinic agonist, arecoline. In rats fed LiCl for 6 weeks to produce a therapeutically relevant brain lithium concentration, the mean baseline values of k* in brain auditory and visual areas were significantly greater than in rats fed control diet. Arecoline at doses of 2 and 5 mg/kg intraperitoneally increased k* in widespread brain areas in rats fed the control diet as well as the LiCl diet. However, the arecoline-induced increments often were significantly greater in the LiCl-fed than in the control diet-fed rats. Lithium's elevation of baseline k* in auditory and visual regions may correspond to its ability in humans to increase auditory and visual evoked responses. Additionally, its augmentation of the k* responses to arecoline may underlie its reported 'proconvulsant' action with cholinergic drugs, as arachidonic acid and its eicosanoid metabolites can increase neuronal excitability and seizure propagation.

Background, Aims: The habit of betel nut chewing impinges on the daily lives of approximately 200 million people. Betel quid chewers have a higher prevalence of periodontal diseases than non-chewers. This study examined the pathobiological effects of arecoline, a major component of the betel nut alkaloids, on human periodontal ligament fibroblasts (PDLF) in vitro., Method: Cell viability, proliferation, protein synthesis, and cellular thiol levels were used to investigate the effects of human PDLF exposed to arecoline levels of 0 to 200 microg/ml. In addition, nicotine was added to test how it modulated the effects of arecoline., Results: Arecoline significantly inhibited cell proliferation in a dose-dependent manner. At concentrations of 10 and 30 microg/ml, arecoline suppressed the growth of PDLF by 20% and 50% (p < 0.05), respectively. Arecoline also decreased protein synthesis in a dose-dependent manner during a 24-h culture period. A 100 microg/ ml concentration level of arecoline significantly inhibited protein synthesis to only 50% of that in the untreated control (p < 0.05). Moreover, arecoline significantly depleted intracellular thiols in a dose-dependent manner. At concentrations of 25 microg/ml and 100 microg/ml, arecoline depleted about 18% and 56% of thiols (p < 0.05), respectively. This suggests that arecoline itself might augment the destruction of periodontium associated with betel nut use. Furthermore, the addition of nicotine acted with a synergistic effect on the arecoline-induced cytotoxicity. At a concentration of 60 microg/ml, arecoline suppressed the growth of PDLF by about 33% and 5 mM nicotine enhanced the arecoline-induced cytotoxic response to cause about 66% cell death., Conclusion: During thiol depletion, arecoline may render human PDLF more vulnerable to reactive agents within cigarettes. Taken together, people who combine habits of betel nut chewing with cigarette smoking could be more susceptible to periodontium damage than betel nut chewing alone.

Betel nut chewing, like cigarette smoking, is a popular oral habit which impinges on the daily lives of a population of approximately 200 million. People who chew betel nuts have a higher prevalence of periodontal diseases than those who do not. Many of the undesirable effects of betel nuts have been attributed to arecoline, a major component of the particular alkaloid in betel nuts. In this in vitro study, we have focused on the effects of arecoline and the role it could play in periodontal breakdown via its direct effects on human gingival fibroblasts. Human gingival fibroblasts were derived from three healthy individuals undergoing crown-lengthening procedures. We found that arecoline is cytotoxic to human gingival fibroblasts at a concentration higher than 50 micrograms/ml by depleting intracellular thiols and inhibiting mitochondrial activity (P < 0.05). In addition, the cells displayed a marked arrest at G2/M phase in a dose-dependent manner. Repeated and long-term exposure to arecoline could impair the gingival fibroblast functions. As they are cytotoxic, the use of betel nut products in conjunction with periodontal therapy may interfere with optimal healing and/or lead to further periodontal breakdown.

The carcinogenic potentiality of the major alkaloid of betel nut, arecoline (ARC), is well established. This study was undertaken to determine the differences in genotoxic effects of ARC when given by two different routes (oral administration (OA) and intraperitoneal injection (IP)) in mouse bone marrow cells (BMC) since ARC carcinogenicity was observed only when ARC was given orally. The data indicate that ARC-OA induced a higher frequency of cancers, a greater delay in the cell cycle and greater sister chromatid exchanges than ARC-IP. The presence of N-acetyl cysteine along with ARC-OA significantly reduced the effect of ARC.

Arecoline (ARC), an alkaloid of the betel nut (Areca catechu), is a major ingredient of betel quid. The carcinogenic potentiality as well as its cell transformation ability has already been reported. Reduced glutathione (GSH), a major non-protein thiol substance plays an important role in protection of cells against the toxic effect of exogenous compounds. In order to understand the role of factors which affect ARC sensitivity, we have made an attempt to establish a relationship between ARC-induced DNA damage and the endogenous GSH status of the cells. ARC was administered to untreated and buthionine sulfoximine (BSO) (a GSH-depleting agent)-treated mice. Exogenous GSH was also added to ARC-administered mice. Cells were fixed at 20 h and both chromosome aberrations (CAs) and sister chromatid exchanges (SCEs) were scored. Both CAs and SCEs were significantly induced by ARC and the frequency of both these parameters were increased further when ARC was given to BSO-treated mice. However, GSH reduced the frequency of CAs induced by ARC but failed to do so for SCEs. The data indicate that ARC-induced DNA damage is influenced by endogenous GSH level. The failure of GSH to reduce the frequency of SCEs indicates that the mechanism of induction of CAs and SCEs by ARC are different.

Betel quid chewing has been linked to oral submucous fibrosis and oral cancer. Cytotoxicity and genotoxicity assays were used to investigate the pathobiological effects of arecoline on cultured human buccal fibroblasts. Arecoline increased double-stranded polynucleic acid at the concentration of 0.1 to 10 micrograms/ml in a concentration-dependent manner. At a concentration higher than 50 micrograms/ml, arecoline was cytotoxic to cultured fibroblasts and the cytotoxicity was dose-dependent. No genotoxicity for arecoline was found even at a concentration of 400 micrograms/ml. On the other hand, 600 micrograms/ml glutathione (GSH) and 200 micrograms/ml glycyrrhizin could prevent the arecoline-induced cytotoxicity. These results indicate that arecoline is a cytotoxic agent and no genotoxicity was found to human buccal fibroblasts. Furthermore, increasing consumption of GSH- and glycyrrhizin-rich foods may reduce the oral diseases associated with betel quid chewing.

This study investigates the effects of different muscarinic receptor agonists on extracellular glutamate and aspartate concentrations in the rat neostriatum. In vivo intracerebral perfusions were undertaken in the conscious rat using a concentric push-pull cannulae system. Amino acid concentrations in samples were determined by HPLC with fluorometric detection. The intrastriatal perfusion of arecoline, a M1-M2 muscarinic receptor agonist, produced a significant decrease in extracellular [ASP] (45% of decrease) but not in extracellular [GLU]. These effects were blocked by scopolamine, a M1-M2 muscarinic receptor antagonist. McN-A-343, a M1 muscarinic receptor agonist, but not the M2 muscarinic receptor agonist, oxotremorine, produced a significant decrease in extracellular [ASP] (40% of decrease) but not in extracellular [GLU]. The effects of McN-A-343 on extracellular [ASP] were blocked by pirenzepine, a M1 muscarinic receptor antagonist. These results suggest that the decrease in extracellular [ASP] could be mediated, at least in part, by M1 muscarinic receptor activation in the rat neostriatum.

Bilateral kainic acid lesions of the ventro-medial (VM) thalamic nucleus of rats which greatly reduced the catalepsy produced by haloperidol (2 mg/kg i.p.) not only did not reduce, but even enhanced, the cataleptogenic effect of eserine (1 mg/kg i.p.) and arecoline (30 mg/kg i.p.). This finding is in accord with former conclusions that catalepsy produced by cholinergic drugs does not depend on striatal mechanisms. In rats with kainic acid lesions of the VM thalamic nucleus, and similarly in intact, non-lesioned rats, systemic administration of eserine and arecoline potentiated the catalepsy produced by microinjections of carbachol (2 microg) into the pontine reticular formation (PRF). Atropine microinjected bilaterally into the PRF attenuated the cataleptogenic effect of eserine and arecoline i.p. We suggest that the PRF is a site at which systemically given cholinergic drugs act to produce catalepsy.

Objective: To study the pharmacokinetics and pharmacodynamics of intravenously administered arecoline in subjects with Alzheimer's disease., Methods: Plasma arecoline concentrations were measured during and after high-dose (i.e., 5 mg intravenously over 30 minutes) and up to 2 weeks of continuous multiple-dose steady-state intravenous infusions of arecoline in 15 subjects with mild to moderate Alzheimer's disease. During multiple-dose infusions, the dose of arecoline was escalated from 0.5 to 40 mg/day. Psychometric tests were administered at baseline and every other dose to determine an "optimal dose" for each subject. This dose then was administered for 1 week using a randomized, placebo-controlled, double blind, crossover design. Plasma drug concentrations were measured by GC-MS., Results: The optimal dose of arecoline varied fourfold across subjects (4 mg/day, n = 6; 16 mg/day, n = 3) with mean plasma half-lives of 0.95 +/- 0.54 and 9.3 +/- 4.5 (SD) minutes. Clearance and volume of distribution were 13.6 +/- 5.8 L/min and 205 +/- 170 (SD) L, respectively. At the dose that optimized memory, the mean plasma level was 0.31 +/- 0.14 (SD) ng/ml, and it predicted the optimal dose in all subjects., Conclusions: Because optimal dose variation is due to differing plasma kinetics, the plasma arecoline level measured at a single infusion rate can be used to choose the optimal dose for memory enhancement in patients with Alzheimer's disease.

Nine patients with possible or probable dementia of the Alzheimer type were tested on nine cognitive tests prior to (two times) and during continuous intravenous administration of five different doses of the muscarinic cholinergic agonist arecoline (1, 4, 16, 28, and 40 mg/day). The present analysis examined whether improvement on cognitive testing for each patient during arecoline treatment was most likely to occur at the same dose for all tests or whether different test scores improved at different doses of arecoline. Results indicated there were significant differences among tests in the dose at which most patients showed improved cognitive performance. These differences may have therapeutic significance, as verbal ability tended to improve at low doses of arecoline, whereas attention and visuospatial ability tended to improve at higher doses of arecoline.

Because betel quid (BQ) chewing has been linked to a higher prevalence of periodontal diseases, the pathobiological effects of arecoline, a main alkaloid found in areca nut, were investigated in cultured human gingival fibroblasts. At concentrations higher than 0.4 mM, arecoline inhibits cell attachment, cell spreading and cell migration in a dose-dependent manner. These inhibitory effects were associated with intracellular depletion of glutathione (GSH). At concentrations of 0.4 mM and 1 mM, arecoline depleted about 26% and 45% of GSH after 2 h incubation. Exposure of cells to arecoline at concentrations lower than 0.4 mM for 2 h showed no significant decrease in either cell viability or intracellular GSH. However, incubation of cells for 24 h in 1 mM are colined decreased the cell numbers to only 35% of those in the untreated control. Arecoline also decreased cell growth and collagen synthesis in a dose-dependent manner. Because of repeated and long-term exposure to arecoline, BQ chewers could be more susceptible to periodontal damage and less responsive to new attachment procedures.

The aim of this study was to investigate the effect on the growth of salivary and selected oral microorganisms of areca nut, aqueous extracts of the nut, its major alkaloid arecoline and the components tannic acid and catechin of its tannin fraction. The antibacterial properties of the above were tested on Streptococcus mutans, Streptococcus salivarius, Candida albicans and Fusobacterium nucleatum and, as a control, Staphylococcus aureus. This was followed by investigating its effect on salivary organisms cultured from the saliva after chewing boiled areca nut. Extracts inhibited the growth of the selected organisms in a concentration dependent manner, baked and boiled nuts being significantly more potent than raw nut. Growth of C. albicans was the least affected by the nut extracts. Tannic acid was strongly antibacterial but not catechin or arecoline. No antibacterial effect could be demonstrated on salivary organisms after chewing the nut for 5 minutes but exposure of saliva to the cud for 1 hour caused a significant depression of bacterial growth. It is concluded that the hydrolysable tannins in the tannin fraction, which include tannic acid, are responsible for the antibacterial properties of the nut and that prolonged intraoral exposure to the nut can suppress bacteria in the mouth.

This study describes the receptor-binding characteristics, antimuscarinic and antinicotinic activities of 2 beta-(2'-phenyl-2'-cyclopentyl-2'-hydroxy-ethoxy)tropane (beta-PCT) and its four optical isomers. Both arecoline-induced tremor and nicotine-induced convulsion in mice were antagonized by beta-PCT and its optical isomers. These compounds were less potent than atropine in their antimuscarinic potencies, but more potent than atropine in their antinicotinic activities. The isomer with the 1S-2 beta-2'R configuration was about one order of magnitude more potent than the isomer with the 1R-2 beta-2'S configuration in their antimuscarinic activity, but the antinicotinic potencies of these compounds did not differ significantly. The order of potencies of beta-PCT and its optical isomers to displace the specific binding of [3H]quinuclidinyl benzilate to muscarinic receptors was similar to that of their antimuscarinic potencies. The binding of [3H]nicotine to central nicotinic receptors was not inhibited by these compounds. The findings indicate that beta-PCT and its optical isomers are useful affinity ligands to examine the biochemical and functional characteristics of brain cholinergic receptors.

The purpose of the present study was to determine the minimal arecaidine concentrations showing a synergistic effect on DMBA-induced hamster cheek pouch carcinogenesis. One hundred and twelve male adult Syrian golden hamsters were divided into 16 groups, each containing seven animals. After eight weeks of DMBA initiation and then four weeks of arecaidine promotion, 100% tumor incidence was found with arecaidine concentrations of 400 micrograms/ml and 500 micrograms/ml; average tumor numbers were 1.86 +/- 0.63 and 1.86 +/- 0.93 respectively (P < 0.05). After four weeks of DMBA and a subsequent eight weeks of arecaidine painting, all hamsters developed visible tumors with arecaidine concentrations of 900 micrograms/ml and 1000 micrograms/ml; average tumor numbers were 1.86 +/- 0.82 and 2.14 +/- 1.09 respectively (P < 0.05). The tumor dimensions varied little and differences were not statistically significant. Without DMBA pretreatment, regardless of the high arecaidine concentrations (1000 micrograms/ml, 2000 micrograms/ml and 3000 micrograms/ml) applied, no visible tumor growth was observed; only hyperkeratosis and inflammation could be discerned histologically. Thus, the minimal concentrations of arecaidine displaying a synergistic effect in the DMBA-induced hamster cheek pouch of carcinogenesis were found to be 400 micrograms/ ml applied for four weeks after eight weeks of DMBA application, and 900 micrograms/ml applied for eight weeks after four weeks of DMBA painting. These findings may be useful for other studies concerning the tumorgenicity of arecaidine.

Denny and co-workers (Navazesh et al., 1992) recently reported decreased concentrations of MG1 and MG2 mucins in resting and stimulated whole human saliva with age. The current study was therefore conducted to examine whether there is a corresponding attenuation with age in stimulus secretion coupling regulating mucous cell exocrine secretion. We utilized an in vitro model system, isolated rat sublingual acini, to evaluate the regulation of mucous cell exocrine secretion. Rat sublingual glands are similar to human sublingual and minor mucous glands, both histologically and in terms of their pattern of innervation, which is predominantly parasympathetic. Mucin secretion is thus activated primarily by muscarinic cholinergic agonist and to a lesser extent by vasoactive intestinal peptide (VIP), which is co-localized with acetylcholine in parasympathetic nerve terminals. We isolated sublingual mucous acini from five-month-old and 24-month-old rats and compared the concentration responses for mucin secretion induced by VIP and the muscarinic agonist, arecaidine propargyl ester (APE). Concentration-response curves for VIP were nearly identical for mucous acini from the five-month-old and 24-month-old animals. Values for basal secretion, maximal secretion, and EC50 (approximately equal to 200 nmol/L VIP) were statistically equivalent between both age groups. Concentration-response curves for APE were also very similar between age groups, with no statistically significant difference in basal secretion or EC50 values (approximately equal to 50 nmol/L APE). Maximal secretion was slightly less but statistically different for 24-month-old vs. five-month-old animals, 158% vs. 169% above basal secretion, respectively. Collectively, we found no substantial age-related changes in the secretory responsiveness of salivary mucous cells.

1. The characteristics of muscarinic receptors mediating relaxation and/or contraction in the rat iris dilator muscle were examined. 2. Relaxation was induced in a dilator muscle by application of acetylcholine (ACh) at low doses (3 microM or less) and contraction was induced by high doses. Methacholine and carbachol also showed biphasic effects similar to those of ACh; in contrast, bethanechol, arecoline, pilocarpine and McN-A-343 induced mainly relaxation but no substantial contraction. 3. After parasympathetic denervation by ciliary ganglionectomy, the relaxant response to muscarinic agonists disappeared upon nerve stimulation. Application of McN-A-343 and pilocarpine induced only small contractions in denervated dilator muscles, indicating that these are partial agonists for contraction. 4. pA2 values of pirenzepine, methoctramine, AF-DX 116, himbacine, and 4-DAMP for antagonism to pilocarpine-induced relaxation in normal dilator muscles and those for antagonism to ACh-induced contraction in denervated dilator muscles were determined. The pA2 values for antagonism to relaxation of all these antagonists were most similar to those for M3-type muscarinic receptors. 5. Although pA2 values for contraction of these antagonists, except for methoctramine, were very close to those for relaxation, contraction was not significantly antagonized by methoctramine. Contraction might be mediated by M3-like receptors which have a very low affinity for methoctramine. 6. In conclusion, ACh-induced biphasic responses in rat iris dilator muscles were clearly distinguished from each other by specific muscarinic agonists and parasympathetic denervation, whereas muscarinic receptors could not be subclassified according to the pA2 values of 5 specific antagonists only.

We have reported that arecoline, a muscarinic receptor agonist replicably enhanced verbal memory in five of nine subjects with Alzheimer's disease (AD). To investigate the mechanism of cognitive improvement, circulating hormone measurements were made during high-dose acute and low-dose chronic intravenous (i.v.) arecoline administration to AD patients. Acute hormone responses were measured during, and for 6 h after, infusion of arecoline 5 mg i.v. over 30 min. Chronic responses were measured in cognitive responders during continuous i.v. infusion of arecoline escalating over 2 weeks (0.5-40 mg/day) and then during a 1 week infusion of the dose optimizing cognition (4-16 mg/day). Acute arecoline administered to 14 subjects produced unpleasant side-effects (e.g. nausea, vomiting), mean adrenocorticotrophic hormone (p = .0006), cortisol (p = .0001) and beta-endorphin (p = .0001) levels were elevated. During chronic arecoline treatment, no side-effects occurred and plasma cortisol, adrenocorticotrophic hormone and beta-endorphin levels were unchanged in nine subjects overall and in five cognitive responders. Thus, high-dose arecoline activates the hypothalamic-pituitary-adrenal (HPA) axis and may increase other anterior pituitary hormone levels, likely representing a 'stress response', but cognition-enhancing, low doses of arecoline do not produce a glucocorticoid response. Hence, arecoline-induced memory improvement is not due to the induction of 'stress' nor to the elevation of peripheral corticosteroid levels.

Children with major depressive disorder often fail to exhibit electroencephalographic (EEG) sleep abnormalities similar to those reported in depressed adults. It was hypothesized that a cholinergic rapid eye movement (REM) induction test would contribute to the identification of EEG sleep abnormalities in depressed children. To test this hypothesis, prepubertal children meeting research diagnostic criteria for major depressive disorder (n = 33) and carefully screened healthy control children (n = 15) were enrolled in a 4-day psychobiologic protocol that included 1 night with infusion of arecoline (0.5 mg) during the first non-REM sleep period. Although there had been no significant group differences in baseline sleep measures, results on the arecoline night revealed significantly shorter REM latency in the group of depressed children compared with the control children (mean +/- SD = 105 +/- 51 minutes vs. 140 +/- 46 minutes). The design of the protocol (with an interval break immediately preceding the arecoline night) prevented a direct estimation of arecoline effects within subjects; however, these data provide promising preliminary results regarding cholinergic REM induction tests in childhood depression.

The modifying potential of arecoline alkaloid was studied on hepatic drug metabolizing system enzymes, acid soluble sulfhydryl (-SH) content and microsomal lipid peroxidation. Arecoline was administered intraperitoneally to Swiss albino mice at the dose levels of 10, 20 or 40 mg/kg body wt./day for 10 or 30 days. Significant increase in the levels of glutathione S-transferase (GST), cytochrome b5 (Cyt.b5), cytochrome P-450 (Cyt.P-450) and malondialdehyde (MDA) was observed in the arecoline treated groups. Decreased -SH content was apparent by the administration of 40 mg arecoline for 10 or 30 days. The modulation in biotransformation system enzymes has wide significance in the process of neoplastic development as well as in detecting the further role of biometabolized chemicals.

Arecoline, a major alkaloid of arecanut was examined to explore its modulatory influence on B cell-mediated immune response in a murine model system. The in vivo and in vitro effects were evaluated at sub-toxic concentrations of arecoline. The number of primary antibody forming cells (AFC) and hemagglutinating and hemolysis antibody titers to Sheep Red Blood Cells (SRBC) were evaluated in male mice. Arecoline exposure for a week invoked dose-dependent effect on primary antibody forming cells to SRBC with a maximum reduction at the dosage of 20 mg/kg bw, a moderate reduction at 10 mg/kg bw and no effect at 5 mg/kg bw dose level. HA and HL antibody titers to SRBC were suppressed markedly at arecoline dosage of 20 mg/kg bw and moderately at a dose of 10 mg/kg bw, given daily for 1, 2 or 3 weeks. The inhibitory effect of arecoline was not dependent on the duration of treatment. Like the primary antibody response, the secondary HA and HL antibody titers were also decreased after arecoline exposure. The administration of arecoline dosages 10 and 20 mg/kg bw daily for 4 days following SRBC immunization also, exerted dose-dependent suppression of primary antibody response. Similarly, when treated after 12 h following immunization, significant reduction in response was observed with arecoline dosage of 20 mg/kg bw. While moderate suppression of antibody response was noticed at the dose level of 10 mg/kg bw, there was no alteration in response at a dosage of 5 mg/kg bw.(ABSTRACT TRUNCATED AT 250 WORDS)

Arecoline, a cholinergic agonist, administered at low doses by continuous intravenous infusion for up to 2 weeks, significantly and replicably improved memory in five of nine subjects with mild-moderate Alzheimer's disease. During dose finding, performance on a verbal memory task improved with an inverted U-shaped relation to dose. Six of nine subjects were classified as responders. During blinded, placebo-controlled, individualized optimal dosing for 5 days, verbal memory again improved in five of six responders but not in any non-responder. No adverse drug effects occurred. Arecoline, and possibly other cholinergic agonists, can safely improve memory in Alzheimer's disease at doses much lower than previously studied.

Physostigmine, an acetyl cholinesterase inhibitor, and arecoline, a muscarinic agonist, have been shown to improve Alzheimer presenile dementia in some patients when administered parenterally. Both of these compounds are ineffective orally due to first-pass metabolism. The nasal route was examined as an alternative route of administration for both drugs. Nasal bioavailabilities and dispositions for both drugs were determined in rats. Physostigmine nasal bioavailability was 100% as compared with iv bioavailability, and that of arecoline was 85% when compared with bioavailability following im administration.

The cholinergic rapid eye movement (REM) induction test using arecoline hydrobromide, a cholinergic muscarinic receptor agonist, was studied in patients with affective disorder and in normal controls to determine whether or not depression is associated with enhanced induction of REM sleep by muscarinic agonists. Arecoline induced REM sleep in a dose-dependent fashion in both patients and controls compared with placebo infusions. Compared with normal controls, patients entered REM sleep significantly more rapidly following intravenous administration of 1.0 mg of arecoline hydrobromide than they did following administration of 0.5 mg of arecoline hydrobromide or placebo. These results, as well as those of previous studies, support the hypothesis that patients with affective disorder show a functional supersensitive induction of REM sleep in response to muscarinic receptor agonists and may be consistent with the hypothesis that functional muscarinic receptor "up regulation" is associated with depression.