Ephrin receptor A2 is an epithelial cell receptor for Epstein-Barr virus entry.

Epstein-Barr virus (EBV) is causally associated with nasopharyngeal carcinoma, 10% of gastric carcinoma and various B cell lymphomas 1 . EBV infects both B cells and epithelial cells 2 . Recently, we reported that epidermal growth factor and Neuropilin 1 markedly enhanced EBV entry into nasopharyngeal epithelial cells 3 . However, knowledge of how EBV infects epithelial cells remains incomplete. To understand the mechanisms through which EBV infects epithelial cells, we integrated microarray and RNA interference screen analyses and found that Ephrin receptor A2 (EphA2) is important for EBV entry into the epithelial cells. EphA2 short interfering RNA knockdown or CRISPR-Cas9 knockout markedly reduced EBV epithelial cell infection, which was mostly restored by EphA2 complementary DNA rescue. EphA2 overexpression increased epithelial cell EBV infection. Soluble EphA2 protein, antibodies against EphA2, soluble EphA2 ligand EphrinA1, or the EphA2 inhibitor 2,5-dimethylpyrrolyl benzoic acid efficiently blocked EBV epithelial cell infection. Mechanistically, EphA2 interacted with EBV entry proteins gH/gL and gB to facilitate EBV internalization and fusion. The EphA2 Ephrin-binding domain and fibronectin type III repeats domain were essential for EphA2-mediated EBV infection, while the intracellular domain was dispensable. This is distinct from Kaposi's sarcoma-associated herpesvirus infection through EphA2 4 . Taken together, our results identify EphA2 as a critical player for EBV epithelial cell entry.

GPR55 promotes migration and adhesion of colon cancer cells indicating a role in metastasis.

BACKGROUND AND PURPOSE: Tumour cell migration and adhesion constitute essential features of metastasis. G-protein coupled receptor 55 (GPR55), a lysophospholipid receptor, has been shown to play an important role in carcinogenesis. Here, we investigated the involvement of GPR55 in migration and metastasis of colon cancer cells. EXPERIMENTAL APPROACH: Adhesion and migration assays using the highly metastatic colon cancer cell line HCT116 and an in vivo assay of liver metastasis were performed. The GPR55 antagonist CID16020046, cannabidiol, a putative GPR55 antagonist and GPR55 siRNA were used to block GPR55 activity in HCT116 colon cancer cells. KEY RESULTS: HCT116 cells showed a significant decrease in adhesion to endothelial cells and in migration after blockade with CID16020046 or cannabidiol. The inhibitory effects of CID16020046 or cannabidiol were averted by GPR55 siRNA knock down in cancer cells. The integrity of endothelial cell monolayers was increased after pretreatment of HCT116 cells with the antagonists or after GPR55 siRNA knockdown while pretreatment with lysophosphatidylinositol (LPI), the endogenous ligand of GPR55, decreased integrity of the monolayers. LPI also induced migration in GPR55 overexpressing HCT116 cells that was blocked by GPR55 antagonists. In a mouse model of metastasis, the arrest of HCT116 cancer cells in the liver was reduced after treatment with CID16020046 or cannabidiol. Increased levels of LPI (18:0) were found in colon cancer patients when compared with healthy individuals. CONCLUSIONS AND IMPLICATIONS: GPR55 is involved in the migratory behaviour of colon carcinoma cells and may serve as a pharmacological target for the prevention of metastasis. © 2015 The British Pharmacological Society.

Lack of weight gain after angiotensin AT1 receptor blockade in diet-induced obesity is partly mediated by an angiotensin-(1-7)/Mas-dependent pathway.

BACKGROUND AND PURPOSE: Angiotensin AT1 receptor antagonists induce weight loss; however, the mechanism underlying this phenomenon is unknown. The Mas receptor agonist angiotensin-(1-7) is a metabolite of angiotensin I and of angiotensin II . As an agonist of Mas receptors, angiotensin-(1-7) has beneficial cardiovascular and metabolic effects. EXPERIMENTAL APPROACH: We investigated the anti-obesity effects of transgenically overexpressed angiotensin-(1-7) in rats. We secondly examined whether weight loss due to telmisartan (8 mg·kg(-1) ·d(-1) ) in diet-induced obese Sprague Dawley (SD) rats can be blocked when the animals were co-treated with the Mas receptor antagonist A779 (24 or 72 µg·kg(-1) ·d(-1) ). KEY RESULTS: In contrast to wild-type controls, transgenic rats overexpressing angiotensin-(1-7) had 1.) diminished body weight when they were regularly fed with chow; 2.) were protected from developing obesity although they were fed with cafeteria diet (CD); 3.) showed a reduced energy intake that was mainly related to a lower CD intake; 5.) remained responsive to leptin despite chronic CD feeding; 6.) had a higher, strain-dependent energy expenditure, and 7.) were protected from developing insulin resistance despite CD feeding. Telmisartan-induced weight loss in SD rats was partially antagonized after a high, but not a low dose of A779. CONCLUSIONS AND IMPLICATIONS: Angiotensin-(1-7) regulated food intake and body weight and contributed to the weight loss after AT1 receptor blockade. Angiotensin-(1-7)-like agonists may be drug candidates for treating obesity.

Soluble guanylate cyclase redox state under hypoxia or hypoxia/reoxygenation in isolated monkey coronary arteries.

Hypoxia or hypoxia/reoxygenation impairs nitric oxide (NO)-mediated relaxation through the increase in superoxide generation in monkey coronary arteries. Soluble guanylate cyclase (sGC), the target enzyme of NO, has been shown to change from the NO-sensitive reduced form to the NO-insensitive oxidized/heme-free form under substantial oxidative stress, so the present study investigated whether hypoxia or hypoxia/reoxygenation influences sGC redox equilibrium. In isolated monkey coronary arteries without endothelium, the relaxation caused by the sGC stimulator BAY 41-2272 (Emax: 93.3% ± 2.2%) was somewhat impaired under hypoxia (Emax: 86.3% ± 2.6%) or hypoxia/reoxygenation (Emax: 86.1% ± 3.2%), whereas that by the sGC activator BAY 60-2770 (Emax: 86.0% ± 3.2%) was significantly augmented under hypoxia (Emax: 94.4% ± 1.3%) or hypoxia/reoxygenation (Emax: 95.5% ± 1.1%). In addition, cGMP formation in response to BAY 41-2272 and BAY 60-2770 was inhibited and stimulated, respectively, under hypoxia or hypoxia/reoxygenation. The effects of hypoxia or hypoxia/reoxygenation on BAY 41-2272- and BAY 60-2770-induced vasorelaxation were completely canceled by the treatment with the superoxide dismutase mimetic tempol. These findings suggest that sGC redox equilibrium in the coronary artery is shifted towards the NO-insensitive form under hypoxia or hypoxia/reoxygenation and that superoxide seems to play an important role in this shift.

Role of haem oxygenase in the renoprotective effects of soluble epoxide hydrolase inhibition in diabetic spontaneously hypertensive rats.

We have shown previously that inhibition of sEH (soluble epoxide hydrolase) increased EETs (epoxyeicosatrienoic acids) levels and reduced renal injury in diabetic mice and these changes were associated with induction of HO (haem oxygenase)-1. The present study determines whether the inhibition of HO negates the renoprotective effect of sEH inhibition in diabetic SHR (spontaneously hypertensive rats). After 6 weeks of induction of diabetes with streptozotocin, SHR were divided into the following groups: untreated, treated with the sEH inhibitor t-AUCB {trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid}, treated with the HO inhibitor SnMP (stannous mesoporphyrin), and treated with both inhibitors for 4 more weeks; non-diabetic SHR served as a control group. Induction of diabetes significantly increased renal sEH expression and decreased the renal EETs/DHETEs (dihydroxyeicosatrienoic acid) ratio without affecting HO-1 activity or expression in SHR. Inhibition of sEH with t-AUCB increased the renal EETs/DHETEs ratio and HO-1 activity in diabetic SHR; however, it did not significantly alter systolic blood pressure. Treatment of diabetic SHR with t-AUCB significantly reduced the elevation in urinary albumin and nephrin excretion, whereas co-administration of the HO inhibitor SnMP with t-AUCB prevented these changes. Immunohistochemical analysis revealed elevations in renal fibrosis as indicated by increased renal TGF-ß (transforming growth factor ß) levels and fibronectin expression in diabetic SHR and these changes were reduced with sEH inhibition. Co-administration of SnMP with t-AUCB prevented its ability to reduce renal fibrosis in diabetic SHR. In addition, SnMP treatment also prevented t-AUCB-induced decreases in renal macrophage infiltration, IL-17 expression and MCP-1 levels in diabetic SHR. These findings suggest that HO-1 induction is involved in the protective effect of sEH inhibition against diabetic renal injury.

[The role of thiol antioxidants in restoring mitochondrial functions, modified by microbial metabolites].

The effects of phenolic acids of microbial origin on mitochondrial functions and the possibility of removing their effects by thiol antioxidants dithiotreitol and N-acethylcysteine were studied. The action of some phenolic acids on the redox state of NADH, the membrane potential and calcium capacity of mitochondria is due to their interaction with thiol groups. The partial restoration of mitochondrial functions occurred in the presence of dithiotreitol and N-acethylcysteine, the full recovery (short-term duration) was promoted by the combined action of dithiotreitol and menadione (vitamin K3). It was found that the protective effect of thiol antioxidants became prooxidant one, if the medium contained free iron and compounds with a quinone structure, capable of entering into a redox cycle with thiols. It is shown that the interaction of thiols with iron and menadione is accompanied by absorption of oxygen to form superoxide anion. Prooxidant effect of thiol antioxidants may explain the absence of the protective effect at the later stages of sepsis and systemic inflammatory syndrome.

Protective effect of a molecular chaperone inducer, paeoniflorin, on the HCl- and ethanol-triggered gastric mucosal injury.

AIMS: We previously found that paeoniflorin, a major constituent of Paeonia lactiflora Pall, could induce heat shock proteins (HSPs) in cultured mammalian cells without apparent toxicity (Yan et al. 2004). We here investigated the induction of HSPs by paeoniflorin in mouse stomach and the effect of paeoniflorin on the HCl- and ethanol-triggered gastric mucosal injury in mouse. MAIN METHODS: Paeoniflorin and quercetin were intraperitoneally administered in mouse and Hsp70 and other proteins in mouse tissues were detected by western blotting. KEY FINDINGS: The intraperitoneal administration of paeoniflorin clearly induced Hsp70 in mouse stomach, and paeoniflorin had a protective effect on the HCl- and ethanol-triggered gastric mucosal injury. When quercetin was injected before paeoniflorin administration, the induction of Hsp70 was suppressed and the protective effect of paeoniflorin was also diminished. Thus, the expression level of Hsp70 was well correlated with the extent of protection against irritant-induced gastric mucosal injury. Oral injection of HCl activated nuclear factor kappa B (NF-κB) and elicited the expression of cyclooxygenase-2 (COX-2) in gastric mucosa. Prior administration of paeoniflorin, however, suppressed these effects. No apparent systemic side effect of paeoniflorin has been observed so far. Hsp70 was also induced in the liver, heart, and brain by paeoniflorin. SIGNIFICANCE: From these results, it is suggested that paeoniflorin and paeoniflorin-containing herbal medicines might be used clinically as HSP inducers for the prevention and treatment of diseases associated with protein conformation and of various other pathological states, such as stress ulcers and irritant- or ischemia-induced injuries.

Transepithelial transport of telmisartan in caco-2 monolayers.

Telmisartan is the most recently marketed angiotensin II type 1 receptor antagonist. Drug-drug interactions involving transporters can directly affect the therapeutic safety and efficacy of many important drugs. In clinical practice, telmisartan is coadministered with many kinds of drugs. However, little is known about the contribution of transporters to the intestinal transport of telmisartan. The aim of this study was to determine the transport mechanism of telmisartan across intestinal epithelial cells. In the presence of an inwardly directed proton gradient, the apical-to-basal transport of telmisartan was greater than basal-to-apical transport. Thus, we focused on the uptake mechanism of telmisartan across brush-border membranes. The uptake of telmisartan by Caco-2 cells was shown to be energy- and proton-dependent. Although some monocarboxylates inhibited the uptake of telmisartan, L-lactic acid, which is a typical substrate of the monocarboxylate transporter (MCT) 1-MCT4, did not affect the uptake of telmisartan. Preloading of acetic acid enhanced the uptake of telmisartan, showing a trans-stimulation effect. These results suggest that the carrier-mediated transport system is involved in the uptake of telmisartan by Caco-2 cells and that the apical-localized transport system is similar to MCTs, but not MCT1-MCT4. It is possible that telmisartan reduce the absorption of coadministered drugs by sharing the MCTs. Since MCTs have an important role in the intestinal absorption of pharmacologically active compounds, it is important to be aware of the potential of telmisartan-drug interactions involving MCTs and to act in order to prevent undesirable and harmful consequences.

Azaphilones, furanoisophthalides, and amino acids from the extracts of Monascus pilosus-fermented rice (red-mold rice) and their chemopreventive effects.

Six azaphilones, monascin (1), ankaflavin (2), rubropunctatin (3), monascorburin (4), rubropunctamine (5), and monascorburamine (6), two furanoisophthalides, xanthomonasin A (7) and xanthomonasin B (8), and two amino acids, (+)-monascumic acid (9) and (-)-monascumic acid (10), isolated from the extracts of Monascus pilosus-fermented rice (red-mold rice) were evaluated for their inhibitory effects on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation in mice, on the induction of Epstein-Barr virus early antigen (EBV-EA) by TPA in Raji cells, and on the activation of (+/-)-(E)-methyl-2[(E)-hydroxy-imino]-5-nitro-6-methoxy-3-hexemide (NOR 1), a nitric oxide (NO) donor. Among the compounds tested, seven compounds (1-6 and 10) on TPA-induced inflammation, and six compounds (1, 3-5, 9, and 10) on EBV-EA activation, exhibited potent inhibitory effects. All of the compounds tested showed moderate inhibitory effects on NOR 1 activation.

Restorative effect of repetitive administration of Shaoyao-Gancao-tang on bioavailability of paeoniflorin reduced by antibacterial synthetic drugs treatment in rats.

Paeoniflorin (PF) is an active glucoside in Shaoyao (peony root), and is transformed into an antispasmodic metabolite, paeonimetabolin-I (PM-I), by intestinal bacteria in the gut after oral administration of Shaoyao or Shaoyao-Gancao-tang (SGT, Shakuyaku-Kanzo-To in Japanese). SGT is a pain-relieving traditional Chinese formulation (Kampo-medicine in Japanese) and is often used together with antibacterial synthetic drugs, such as amoxicillin and metronidazole (AMPC-MET), in peptic ulcer therapy. Since the bioavailability of PF in SGT has been reported to be significantly reduced by co-administered antibacterial drugs, we investigated how to minimize this reducing effect of antibacterial treatment in the present study. We found that repetitive administration of SGT starting 24 h after AMPC-MET treatment rapidly restored the plasma PM-I concentration from SGT reduced by AMPC-MET, due to its restorative effect on the decreased PF-metabolizing activity of intestinal bacteria in rat feces. The present findings suggest that it may be clinically useful to administer SGT repetitively, starting 1 or 2 d after treatment with a mixture of AMPC-MET during their combination therapy, to accelerate the recovery of the reduced bioavailability of PF in SGT. Similar administration regimens may also be useful in other combination therapies involving traditional Chinese formulations and antibacterial synthetic drugs to ensure the efficacy of the bioactive glycosides in the formulations.

Ethyl 4-[2-(6-Methyl-3-pyridyloxy)butyloxy]benzoate, a novel anti-juvenile hormone agent.

Ethyl 4-[2-(6-methyl-3-pyridyloxy)butyloxy]benzoate (2) was prepared as a novel anti-juvenile hormone (anti-JH) agent. Compound 2 induced precocious metamorphosis in larvae of the silkworm and black pigmentation of the larval cuticle, which are clearly recognized as JH-deficiency symptoms. The 4-ethoxycarbonyl group on the benzene ring was indispensable for activity. The activity of compound 2 could be fully counteracted by methoprene, a JH agonist, but not by the dietary administration of 20-hydroxyecdysone.

Phenylcyclohexene and phenylcyclohexadiene substituted compounds having retinoid antagonist activity.

Retinoids are natural and synthetic analogues of the hormone retinoic acid. Systemic retinoid agonist therapy is usually associated with toxic side effects, such as mucocutaneous toxicity, which may be alleviated by the use of topical retinoid antagonists. We report the synthesis and biological activity of a new series of potent, RAR-specific antagonists substituted with phenylcyclohexene and phenylcyclohexadiene groups.

Benzoate degradation via the ortho pathway in Alcaligenes eutrophus is perturbed by succinate.

During batch growth of Alcaligenes eutrophus on benzoate-plus-succinate mixtures, substrates were simultaneously metabolized, leading to a higher specific growth rate (mu = 0.56 h-1) than when a single substrate was used (mu = 0.51 h-1 for benzoate alone and 0.44 h-1 for succinate alone), without adversely affecting the growth yield (0.57 Cmol/Cmol). Flux distribution analysis revealed that succinate dehydrogenase most probably controls the rate of total succinate consumption (the maximum flux being 9.7 mmol.g-1.h-1). It is postulated that the relative consumption rate of each substrate is in part related to modified levels of gene expression but to a large extent is dependent upon the presence of succinate, end product of the beta-ketoadipate pathway. Indeed, the in vitro beta-ketoadipate-succinyl coenzyme A transferase activity was seen to be inhibited by succinate, a coproduct of the reaction.

Retinobenzoic acids. 6. Retinoid antagonists with a heterocyclic ring.

Several candidate retinoid antagonists were designed on the basis of the ligand superfamily concept and synthesized. Retinoidal activities of these benzimidazole and benzodiazepine derivatives were examined by assay of differentiation-inducing activity on human promyelocytic leukemia cell line HL-60. The parent benzimidazole derivative, 4-(5,6,7,8-tetrahydro-5,5,8,8- tetramethylnaphth-[2,3-d]imidazol-2-yl)benzoic acid (7a), and related compounds with a small alkyl group instead of the hydrogen on the nitrogen (1N) atom of the imidazole ring exhibited retinoidal activity, and the potency strongly depended on the bulkiness of the substituent. The compounds having a phenyl or benzyl group on the nitrogen lacked differentiation-inducing activity on HL-60 cells and acted as antagonists to the potent retinoid 4-[(5,6,7,8-tetrahydro-5,5,8,8- tetramethyl-2-naphthalenyl)carbamoyl]benzoic acid (Am80). Among the compounds possessing a seven-membered heterocyclic ring as a linking group, 4-(5H-7,8,9,10-tetrahydro-5,7,7,10,10- pentamethylbenzo[e]- naphtho[2,3-b][1,4]diazepin-13-yl)benzoic acid (16) also exhibited the antagonistic activity. The binding abilities of these compounds to retinoic acid receptors alpha and beta were consistent with their potency for the inhibition of HL-60 cell differentiation induced by the retinoid Am80.

Effect of substituted benzoates on p-aminohippurate transport in dog renal membrane vesicles.

The effect of substituted benzoates on the transport of p-aminohippurate (PAH) was studied in basolateral (BLMV) and brush border membrane vesicles (BBMV) isolated from dog kidney cortex. For both membranes, kinetic analysis of [3H]PAH transport in the presence of a fixed concentration of two different benzoates, respectively, revealed an increase in the apparent Km for PAH, while the transport capacity (Vmax) was unaffected. This is compatible with competitive inhibition of a common transport pathway. A range of 19 monosubstituted benzoates were then tested as potential inhibitors by measuring the probenecid-sensitive fraction of 100 mumol/l PAH uptake into BLMV and BBMV in the presence of 5 mmol/l benzoate, and apparent inhibition constants (Ki) were calculated. For all benzoates the inhibitory potency in BBMV was lower than in BLMV, but the pattern of inhibition was similar; the most pronounced inhibition was found for 3-Cl- and 4-Cl-benzoate, while the least pronounced inhibition was found for the 3-NH2 and 4-NH2 substitutes. The inhibitory potency, expressed as logKi, correlated significantly with the relative hydrophobicity of the benzoates determined by reversed phase HPLC, whereas a poor correlation was found between pKa and logKi. This indicates that hydrophobic and electronic parameters are the main determinants of affinity for the PAH transport system. It is suggested that the PAH transport system present in the proximal tubules is responsible for the active secretion of benzoates by the mammalian kidney.

PUVA treatment inhibits nonimmunologic immediate contact reactions to benzoic acid and methyl nicotinate.

The effect of trioxsalen bath PUVA therapy on nonimmunologic immediate contact reactions (NIICRs) induced by benzoic acid (BA) and methyl nicotinate (MN) was studied in 12 dermatologic patients. One half of the back skin was covered with a cloth before each of ten irradiations on subsequent days. Four concentrations of each test substance were applied to the PUVA-exposed and nonexposed areas before any irradiation and after the 1st, 5th, and 10th PUVA treatment. Erythema and edema reactions were observed visually 40 minutes after BA and MN application, and changes in the skin blood flow were monitored using laser Doppler flowmetry (LDF). PUVA therapy suppressed the NIICRs in all concentrations of BA and MN on the PUVA-exposed area and occasionally on the nonexposed test sites. The suppressive effect was intesified during therapy.

Hypercalcemia induced with an arotinoid in thyroparathyroidectomized rats. New model to study bone resorption in vivo.

A model of stimulated bone resorption was developed using a synthetic retinoid in thyroparathyroidectomized rats. The retinoid induced an increase in bone resorption and in the number of vertebral subperiosteal osteoclasts. The resulting increase in plasma Ca could be used as an easily measured index of bone resorption. Three bisphosphonates produced a dose-related prevention and reversal of retinoid-induced hypercalcemia. Their potencies were similar to those previously obtained by histomorphometry. Irradiation (600 rad) of the rats prevented hypercalcemia but failed to reverse it, showing that proliferation of osteoclast precursor cells was important in inducing, but not in maintaining, bone resorption. Calcitonin produced similar effects on calcemia and prevented the increase in osteoclast number but failed to reverse the increase, suggesting that it inhibited precursor proliferation. This model represents a new tool to study mechanisms of bone resorption and the action of inhibitors in vivo.

Acetylsalicylic acid inhibits non-immunologic contact urticaria.

To investigate the mechanisms of non-immunologic contact urticaria (NICU), the effects of 1g + 1g of acetylsalicylic acid (ASA) on contact reactions to methyl nicotinate, diethyl fumarate, benzoic acid, cinnamic acid, cinnamic aldehyde and dimethyl sulfoxide were studied in 21 test subjects. Erythema and edema reactions were observed visually, and the changes in the skin blood flow were monitored using laser-Doppler flowmetry. ASA had a significant inhibitory effect on erythema from all 6 agents and also on edema from all substances except dimethyl sulfoxide. The mechanism of the effect may be a result of the inhibitory influence of ASA on prostaglandin bioformation. Thus, to avoid false negative test results, non-steroidal anti-inflammatory drugs should not be used during NICU tests.

[Interaction of kynurenine and diazepam]. / Vzaimodeistvie kinurenina i diazepama.

Intracerebroventricular injection of 1-kynurenine sulfate (10 micrograms) eliminated anticaffeine effect of diazepam (5, 10 and 20 mg/kg) in mice. Antipentylenetetrazole (0.5-2.5 mg/kg) and anticonflict (1-2.5 mg/kg) effects of diazepam were not altered by kynurenine (0.1-10 micrograms). The sedative effect of diazepam (1 and 4 mg/kg) was potentiated by kynurenine (1 microgram). Stimulation of locomotion induced by kynurenine (0.5 and 2.5 micrograms) was prevented or perversed by pretreatment with diazepam (1 mg/kg). In a conflict situation test, diazepam and kynurenine had an opposite action, respectively increasing and decreasing the number of transitions between light and dark compartments. The central effects of kynurenine can be related and not related to benzodiazepine receptors.

Effects of cholinomimetic agents given into the brain of fowls.

1 Effects of cholinomimetic agents, given into the IIIrd ventricle of adult fowls (Gallus domesticus) or infused into the hypothalamus of young chicks, were tested on behaviour, respiratory rate and body temperature.2 Carbachol evoked behavioural and electrocortical arousal but lacked postural and respiratory effects. Contrariwise, pilocarpine increased respiratory rate and induced postural changes, i.e. abduction of the wings, but lacked other behavioural effects and did not alter electrocortical activity. Benzoylcholine induced tachypnoea, postural changes and brief electrocortical arousal. Acetylcholine was ineffective unless given with physostigmine, when electrocortical arousal, postural changes and tachypnoea developed. Methacholine induced tachypnoea and postural changes.3 Effects of carbachol and pilocarpine were prevented by hyoscine and those of benzoylcholine by pempidine; hyoscine and pempidine were required together to prevent the effects of methacholine and to attenuate those of acetylcholine with physostigmine.