bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effects of bradykinin on signal transduction, cell proliferation, and cytokine, prostaglandin E2 and collagenase‐1 release from human corneal epithelial cells
Authors: Wiernas T K. , Davis T L. , Griffin B W. , Sharif N A. 
Published: January 2005

We recently demonstrated the presence of phospholipase C‐coupled bradykinin (BK) B2‐receptors in human primary and SV40 virus‐immortalized corneal epithelial (CEPI) cells.
The aims of the present studies were to demonstrate the specific binding of [3H]‐BK to CEPI cell membranes and to study its pharmacological characteristics. In addition, we wished to study the functional coupling of the BK receptors to various physiological and pathological mechanisms in the CEPI cells, including phosphoinositide (PI) turnover, intracellular Ca2+‐mobilization ([Ca2+]i), cell proliferation (via [3H]‐thymidine incorporation), and the release of various cytokines, collagenase‐1 (matrix metalloproteinase‐1) and prostaglandin E2 (PGE2).
Specific [3H]‐BK binding comprised 83±2% of the total binding, and was of high affinity (Kd=1.66±0.52 nM, n=5), saturable (Bmax=640±154 fmol g−1 wet weight) and reversible. Competition studies yielded the following affinity values for BK and a number of BK‐related peptides: Hoe‐140 (D‐Arg‐[Hyp3,Thi5,D‐Tic7,Oic8]BK; icatibant): Ki=0.17±0.07 nM; BK: Ki=1.0±0.11 nM; [Tyr8]‐BK: Ki=12.9±2.3 nM; [des‐Arg9]‐BK: Ki>9,200 nM (all n=3–5)).
BK potently stimulated PI turnover (EC50=2.3±0.3 nM; n=7) and [Ca2+]i mobilization (EC50=8–20 nM) in CEPI cells and both responses were inhibited in a concentration‐dependent manner by 100 nM–10 μM Hoe‐140, a selective B2‐receptor antagonist, and also inhibited by the selective phospholipase C (PLC) inhibitor, U73122 (1‐(6‐((17β‐3‐methoxyestra‐1,3,5(10)‐trien‐17‐yl)amino)hexyl)‐1H‐pyrrole‐2,5‐dione) (IC50=3.0±1.6 μM). BK‐induced [Ca2+]i mobilization was reduced by about 30% in the presence of 4 mM EGTA, but was not significantly affected by 100 nM nifedipine.
BK (0.1 nM–10 μM) significantly (P<0.05–0.001) stimulated [3H]‐thymidine incorporation into CEPI cellular DNA. However, while interleukin‐1α (IL‐1α; 10 ng ml−1) potently stimulated the release of IL‐6, IL‐8 and granulocyte macrophage colony‐stimulating factor from CEPI cells, BK (0.1 nM–10 μM) was without effect.
Whilst phorbol‐12‐myristate‐13‐acetate (PMA; 3 μg ml−1) and 10% foetal bovine serum (positive control agents) significantly stimulated the release of both MMP‐1 and PGE2 from CEPI cells, BK (0.1 nM–10 μM) was without any significant effect under these conditions.
In conclusion, these data indicate that the CEPI cells express high‐affinity [3H]‐BK binding sites representing B2‐subtype BK receptors coupled to PI turnover and [Ca2+]i mobilization which appear to stimulate [3H]‐thymidine incorporation into cellular DNA. In contrast, BK failed to elicit the release of PGE2, various cytokines and MMP‐1 from CEPI cells. These results suggest that BK may have a potential role in corneal epithelium wound healing by stimulating cell proliferation.

British Journal of Pharmacology (1998) 123, 1127–1137; doi:10.1038/sj.bjp.0701700
Myogenic nitric oxide synthase activity in canine lower oesophageal sphincter: morphological and functional evidence
Authors: Salapatek Anne Marie F. , Wang Yu‐Fang , Mao Yu‐Kang , Lam Annette , Daniel Edwin E. 
Published: January 2005

Studies on canine lower oesophageal sphincter (LOS) evaluated the existence and function of a myogenic, nitric oxide synthase (NOS) by use of immunocytochemistry for NOS isozymes, NADPH‐d histochemistry, [3H]‐L‐arginine to [3H]‐L‐citrulline transformation. In addition, functional studies in the muscle bath were performed.
Smooth muscle bundles or freshly isolated smooth muscle cells of LOS were NADPH‐d reactive but did not recognize some antibodies against neural, endothelial or inducible NOS. NADPH‐d reactivity and immunoreactivity to a neural NOS antibody were colocalized in LOS enteric nerves. Muscle plasma membrane‐enriched fractions from fresh and cultured LOS cells converted [3H]‐L‐arginine to [3H]‐L‐citrulline; activity was mostly Ca2+/calmodulin‐dependent.
N‐Nitro‐L‐arginine (L‐NOARG) persistently increased tone (blocked by L‐arginine) in muscle strips despite blockade of nerve function. Nifedipine prevented or abolished L‐NOARG‐induced, but not carbachol‐induced, contraction showing that tone increase by L‐NOARG required functional L‐Ca channels.
Membrane‐bound, myogenic NOS in canine LOS may release NO continuously when Ca2+ entry through L‐Ca channels occurs under physiological conditions and thereby modulate tone in LOS.

British Journal of Pharmacology (1998) 123, 1055–1064; doi:10.1038/sj.bjp.0701701
Induction of nitric oxide synthase in vivo and cell injury in rat duodenal epithelium by a water soluble extract of Helicobacter pylori
Authors: Lamarque Dominique , Kiss József , Tankovic Jacques , François Flejou Jean , Delchier Jean‐Charles , Whittle Brendan J R. 
Published: January 2005

Helicobacter pylori (Hp) infection, which involves the gastric antrum and duodenal mucosa, may be involved in peptic ulceration by stimulating the local release of cytoxic or pro‐inflammatory factors.
Nitric oxide (NO) is known to be cytotoxic at high concentration. The aim of the present study was therefore to investigate the ability of a water soluble extract of Hp to induce NO synthase in duodenal mucosa and epithelial cells following its administration in vivo in rats and determine its association with cell damage.
Administration of Hp water extract (4 ml kg−1) led to the expression of the calcium‐independent inducible nitric oxide synthase (iNOS) after 4 h in the duodenum, determined as [14C]‐arginine conversion to citrulline.
This iNOS activity was not reduced by pretreatment with anti‐neutrophil serum (0.4 ml kg−1, i.p., 3 h before challenge). However, dexamethasone pretreatment (1 mg kg−1, i.v., 2 h before the extract), or administration of the NO synthase inhibitor NG‐nitro‐L‐arginine methyl ester (L‐NAME, 5 mg kg−1, i.v., 2.5 h after the extract) reduced this activity.
Furthermore, iNOS was expressed in duodenal isolated epithelial cells 4 h after the i.v. challenge with the extract, at a time when the cellular viability was also reduced, as assessed by trypan blue exclusion.
Dexamethasone pretreatment, administration of L‐NAME, or pretreatment with polymyxin B (1 mg kg−1, i.v.) which binds endotoxin, reduced both the iNOS activity and epithelial cell damage.
The induction of NO synthase by the Hp extract thus results in duodenal epithelial cell injury and such actions could play a role in pathogenesis of peptic ulcer disease.

British Journal of Pharmacology (1998) 123, 1073–1078; doi:10.1038/sj.bjp.0701706
Pharmacological detection of AMPA receptor heterogeneity by use of two allosteric potentiators in rat hippocampal cultures
Authors: Sekiguchi Masayuki , Takeo Jiro , Harada Takayuki , Morimoto Takako , Kudo Yoshihisa , Yamashita Shinya , Kohsaka Shinichi , Wada Keiji 
Published: January 2005

In order to examine whether a recently developed allosteric potentiator for AMPA receptors, 4‐[2‐(phenylsulphonylamino)ethylthio]‐2,6‐difluoro‐phenoxyacetamide (PEPA), can be utilized as an indicator of AMPA receptor heterogeneity, the action of PEPA upon the increase of intracellular free calcium ion concentration ([Ca2+]i) elicited by AMPA was investigated in rat hippocampal cultures, and the action was compared with that of cyclothiazide, a well characterized allosteric modulator of AMPA receptors.
PEPA dose‐dependently potentiated AMPA‐induced increase of [Ca2+]i. In 90% (72 out of 80) of the cells in which cyclothiazide acts, PEPA potentiated the increased [Ca2+]i induced by AMPA with pronounced cell‐to‐cell variation in rat hippocampal cultures.
The ratio of the potentiation by PEPA to the potentiation by cyclothiazide (P/C ratio) also varied with cells between 0 and 2.15. It was found that the cultured hippocampal cells consisted of multiple populations with different P/C ratios. Among them two populations exhibited characteristic P/C ratios; low (0 to 0.15; 27 out of 80 cells, 34%) and high (2.00; 1 out of 80 cells, 1%) P/C ratios. The P/C ratios of the other populations were between 0.25 and 1.20, and these cells constituted 65% (52 out of 80 cells) of the cells tested.
Reverse transcriptase‐polymerase chain reaction analysis suggested that GluR2‐flip, GluR1‐flip, GluR2‐flop, and GluR1‐flop were abundantly expressed (in this rank order) in the cultures used.
In Xenopus oocytes expressing GluR1, GluR3, or these subunits plus GluR2, the potentiation of AMPA response by PEPA and by cyclothiazide varied with subunit and splice‐variant combinations, and the P/C ratio was between 0.19 and 2.20. Oocytes with low P/C ratios (0.19 to 0.50) and low sensitivity to PEPA potentiation (1.9 fold to 6.41 fold) were those expressing flip variants predominantly, and oocytes with high P/C ratios (1.8 to 2.2) were those expressing flop variants predominantly. Oocytes with intermediate P/C ratios (0.51 to 1.20) were those expressing various combinations of flip and flop variants, and it was impossible to specify the relative abundance of flip and flop variants in these cells. Therefore, the P/C ratio can be used to infer subunit/splice variant expression only when the ratio is low or high.
These results suggest that the potentiation by PEPA alone reveals cell‐to‐cell heterogeneity of AMPA receptors, but a comparison of the actions of PEPA and cyclothiazide further facilitates the detection of the heterogeneity.

British Journal of Pharmacology (1998) 123, 1294–1303; doi:10.1038/sj.bjp.0701707
Influence of potassium channel modulators on cognitive processes in mice
Authors: Ghelardini Carla , Galeotti Nicoletta , Bartolini Alessandro 
Published: January 2005

The effect of i.c.v. administration of different potassium channel openers (minoxidil, pinacidil, cromakalim) and potassium channel blockers (tetraethylammonium, apamin, charybdotoxin, gliquidone, glibenclamide) on memory processes was evaluated in the mouse passive avoidance test.
The administration of minoxidil (10 μg per mouse i.c.v.), pinacidil (5–25 μg per mouse i.c.v.) and cromakalim (10–25 μg per mouse i.c.v.) immediately after the training session produced an amnesic effect.
Tetraethylammonium (TEA; 1–5 μg per mouse i.c.v.), apamin (10 ng per mouse i.c.v.), charybdotoxin (1 μg per mouse i.c.v.), gliquidone (3 μg per mouse i.c.v.) and glibenclamide (1 μg per mouse i.c.v.), administered 20 min before the training session, prevented the potassium channel opener‐induced amnesia.
At the highest effective doses, none of the drugs impaired motor coordination, as revealed by the rota rod test, or modified spontaneous motility and inspection activity, as revealed by the hole board test.
These results suggest that the modulation of potassium channels plays an important role in the regulation of memory processes. On this basis, the potassium channel blockers could be useful in the treatment of cognitive deficits.

British Journal of Pharmacology (1998) 123, 1079–1084; doi:10.1038/sj.bjp.0701709
Protein kinase C‐mediated inhibition of transmembrane signalling through CCKA and CCKB receptors
Authors: Smeets R L L. , Fouraux M A. , van Emst‐de Vries S E. , De Pont J J H H M. , Willems P H G M. 
Published: January 2005

The rat CCKA and CCKB receptors were stably expressed in Chinese hamster ovary (CHO‐09) cells in order to compare modes of signal transduction and effects of protein kinase C (PKC) thereupon.
Spectrofluorophotometry of Fura‐2‐loaded cells revealed that both receptors retained their pharmacological characteristics following expression in CHO cells. Sulphated cholecystokinin‐(26‐33)‐peptide amide (CCK‐8‐S) increased the cytosolic Ca2+ concentration ([Ca2+]i) in CCKA cells, measured as an increase in Fura‐2 fluorescence emission ratio, 1000 fold more potently than its non‐sulphated form (CCK‐8‐NS) (EC50 values of 0.19 nM and 0.18 μM, respectively). By contrast, CCK‐8‐S and CCK‐8‐NS were equally potent in CCKB cells (EC50 values of 0.86 nM and 1.18 nM, respectively). The CCKA receptor agonist JMV‐180 increased [Ca2+]i only in CCKA cells. Likewise, pentagastrin increased [Ca2+]i only in CCKB cells. Finally, CCK‐8‐S‐induced Ca2+ signalling through the CCKA receptor was most potently inhibited by the CCKA receptor antagonist L364,718, whereas the CCKB receptor antagonist L365,260 was more potent in CCKB cells.
Receptor‐mediated activation of adenylyl cyclase was measured in the presence of the inhibitor of cyclic nucleotide phosphodiesterase activity, 3‐isobutyl‐1‐methylxanthine. CCK‐8‐S and, to a lesser extent, CCK‐8‐NS, but not JMV‐180 or pentagastrin, stimulated the accumulation of cyclicAMP in CCKA cells. By contrast, none of these agonists increased cyclicAMP in CCKB cells.
Short‐term (3 min) pretreatment with the PKC activator 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) evoked a rightward shift of the dose‐response curve for the Ca2+ mobilizing effect of CCK‐8‐S in both cell lines. In addition, short‐term TPA pretreatment markedly reduced CCK‐8‐S‐induced cyclicAMP accumulation in CCKA cells. In both cases, the inhibitory effect of TPA was abolished by the PKC inhibitors, GF‐109203X and staurosporine, whereas no inhibition was observed with the inactive phorbol ester, 4‐α‐phorbol 12‐myristate 13‐acetate.
During prolonged TPA treatment, the cells gradually recovered from phorbol ester inhibition and in the case of CCK‐8‐S‐induced Ca2+ mobilization complete recovery was achieved after 24 h of TPA treatment. Western blot analysis revealed that this recovery was paralleled by down‐regulation of PKC‐α, suggesting the involvement of this PKC isotype in the inhibitory action of TPA.
This study demonstrates that following expression in CHO cells (i) both CCKA and CCKB receptors are coupled to Ca2+ mobilization, (ii) only CCKA receptors are coupled to cyclicAMP formation and (iii) with both receptors signalling is inhibited by PKC.

British Journal of Pharmacology (1998) 123, 1189–1197; doi:10.1038/sj.bjp.0701713
Effect of 17β‐oestradiol on cytokine‐induced nitric oxide production in rat isolated aorta
Authors: Kauser Katalin , Sonnenberg Dagmar , Diel Patrick , Rubanyi Gabor M. 
Published: January 2005

Studies were performed on isolated aortic rings without endothelium to investigate the effect of 17β‐oestradiol on cytokine‐induced nitric oxide production by the inducible nitric oxide synthase (iNOS).
Treatment of the isolated aortic rings with interleukin‐1β (IL‐1β, 20 μ ml−1) led to the expression of iNOS mRNA and protein, as well as significant nitrite accumulation in the incubation media and suppression of phenylephrine (1 nM–10 μM)‐evoked contraction.
Cycloheximide (1 μM), a protein synthesis inhibitor, prevented iNOS protein expression, nitrite accumulation and the suppression of contractility by IL‐1β on the isolated aortic rings. 17β‐oestradiol (1 nM–10 μM) and the partial oestrogen receptor agonist 4‐OH‐tamoxifen (1 nM–10 μM) produced concentration‐dependent inhibition of IL‐1β‐induced nitrite accumulation and restored vasoconstrictor responsiveness to phenylephrine, similar to the iNOS inhibitor aminoguanidine (100 μM).
Semiquantitative PCR demonstrated decreased iNOS mRNA in the IL‐1β‐induced and 17β‐oestradiol‐treated rings. Western blot analysis of rat aorta homogenates revealed that 17β‐oestradiol treatment resulted in a reduction in IL‐1ß‐induced iNOS protein level.
Incubation with tumour necrosis factor α (TNFα, 1 ng ml−1) resulted in significant nitrite accumulation in the incubation media and suppression of the smooth muscle contractile response to phenylephrine, similar to IL‐1β. The effects of TNFα were also inhibited by co‐incubation of the rings with 17β‐oestradiol and 4‐OH‐tamoxifen (1 μM).
The anti‐transforming growth factor‐β1 (TGF‐β1) antibody, which inhibited TGF‐β1‐induced suppression of nitrite production from IL‐1β‐treated vascular rings, did not affect the inhibitory action of 17β‐oestradiol, suggesting that the effect of oestrogen on iNOS inhibition was not mediated by TGF‐β1.
These results show that the ovarian sex steroid, 17β‐oestradiol is a modulator of cytokine‐induced iNOS activity in rat vascular smooth muscle and its mechanism of action involves decrease of iNOS mRNA and protein.

British Journal of Pharmacology (1998) 123, 1089–1096; doi:10.1038/sj.bjp.0701715
Differential effects of cyclosporine A after acute antigen challenge in sensitized catsin vivoandex vivo
Authors: Mitchell Richard W. , Cozzi Phillip , Maurice Ndukwu I , Spaethe Stephen , Leff Alan R. , Padrid Philip A. 
Published: January 2005

We determined the effect of cyclosporine A (CsA) treatment on mast cell degranulation and lung resistance (RL)in vivo, and tracheal smooth muscle (TSM) contractionex vivoafter antigen challenge in sensitized cats. We also determined the direct effects of addition of CsA to the tissue bath on antigen‐induced responses of TSMin vitro.Cats (n=10) were sensitized by i.m. injection ofAscaris suumantigen (AA); 5 cats (CsA+) received CsA twice daily for 2 weeks before acute antigen challenge in doses sufficient to suppress interleukin‐2 secretion from feline peripheral blood mononuclear cellsex vivo.Lung resistance increased comparably within 10 min of exposure to AA (P<0.03). Histamine content in bronchoalveolar lavage fluid from both groups increased comparably within 30 min of antigen challenge, from undetectable levels to 542±74 pg ml−1post AA for CsA+ and from 74±19 pg ml−1at baseline, to 970±180 pg ml−1post AA CsA− (P<0.05;P=NS vs CsA+).In excised TSM, active tension elicited by exposure to AAin vitrowas 107±38% KCl in the CsA+ group vs 144±56% KCl in the CsA− group (P=NS). However, contraction of TSM (n=4) harvested from both groups was abolished or greatly diminished after AA challenge when tissues were pre‐incubated with 1 μMCsAin vitro(8±8% KCl,P<0.05 vs CsA+ and CsA−). This was associated with inhibited release of 5‐hydroxytryptamine into the organ bath fluid of tissues treated with CsAin vitroonly.We demonstrated that CsA treatmentin vivodoes not inhibit the early phase asthmatic response or mast cell degranulation following antigen challenge in sensitized cats. Additionally, the effects of CsA on mast cell functionex vivodo not reflect lack of effects of CsA on mast cell functionin vivoin this animal model of atopic asthma.British Journal of Pharmacology(1998)123, 1198–1204; doi:10.1038/sj.bjp.0701716
Prostaglandin E2 suppression of acetylcholine release from parasympathetic nerves innervating guinea‐pig trachea by interacting with prostanoid receptors of the EP3‐subtype
Authors: Spicuzza Lucia , Giembycz Mark A. , Barnes Peter J. , Belvisi Maria G. 
Published: January 2005

We have demonstrated recently that exogenous prostaglandin E2 (PGE2) inhibits electrical field stimulation (EFS)‐induced acetylcholine (ACh) release from parasympathetic nerve terminals innervating guinea‐pig trachea. In the present study, we have attempted to characterize the pre‐junctional prostanoid receptor(s) responsible for the inhibitory action of PGE2 and to assess whether other prostanoids modulate, at a prejunctional level, cholinergic neurotransmission in guinea‐pig trachea. To this end, we have investigated the effect of a range of both natural and synthetic prostanoid agonists and antagonists on EFS‐evoked [3H]‐ACh release.
In epithelium‐denuded tracheal strips pretreated with indomethacin (10 μM), PGE2 (0.1 nM–1 μM) inhibited EFS‐evoked [3H]‐ACh release in a concentration‐dependent manner with an EC50 and maximal effect of 7.62 nM and 74% inhibition, respectively. Cicaprost, an IP‐receptor agonist, PGF2α and the stable thromboxane mimetic, U46619 (each at 1 μM), also inhibited [3H]‐ACh release by 48%, 41% and 35%, respectively. PGD2 (1 μM) had no significant effect on [3H]‐ACh release.
The selective TP‐receptor antagonist, ICI 192,605 (0.1 μM), completely reversed the inhibition of cholinergic neurotransmission induced by U‐46619, but had no significant effect on similar responses effected by PGE2 and PGF2α.
A number of EP‐receptor agonists mimicked the ability of PGE2 to inhibit [3H]‐ACh release with a rank order of potency: GR63799X (EP3‐selective)>PGE2>M&B 28,767 (EP3 selective)>17‐phenyl‐ω‐trinor PGE2 (EP1‐selective). The EP2‐selective agonist, AH 13205 (1 μM), did not affect EFS‐induced [3H]‐ACh release.
AH6809 (10 μM), at a concentration 10 to 100 times greater than its pA2 at DP‐, EP1‐ and EP2‐receptors, failed to reverse the inhibitory effect of PGE2 or 17‐phenyl‐ω‐trinor PGE2 on [3H]‐ACh release.
These results suggest that PGE2 inhibits [3H]‐ACh release from parasympathetic nerves supplying guinea‐pig trachea via an interaction with prejunctional prostanoid receptors of the EP3‐receptor subtype. Evidence for inhibitory prejunctional TP‐ and, possibly, IP‐receptors was also obtained although these receptors may play only a minor role in suppressing [3H]‐ACh release when compared to receptors of the EP3‐subtype. However, the relative importance of the different receptors will depend not only on the sensitivity of guinea‐pig trachea to prostanoids but on the nature of the endogenous ligands released locally that have activity on parasympathetic nerves.

British Journal of Pharmacology (1998) 123, 1246–1252; doi:10.1038/sj.bjp.0701720
Role of serine esterases in mast cell activation
Authors: Emadi Khiav B , Pearce F L. 
Published: January 2005

A variety of chymotryptic substrates and inhibitors prevented the release of histamine and prostaglandin D2 from rat peritoneal mast cells stimulated with anti‐IgE but not the calcium ionophore A23187 or a variety of polyamines.
The activity of the compounds was strikingly increased in cells reversibly permeabilized with ATP, indicating the importance of their effective incorporation into the cytosol.
The compounds produced a comparable inhibition of immunological, but not pharmacological, histamine release from human mast cells and basophils.
Treatment of rat mast cells with anti‐IgE led to a marked increase in the total chymotryptic activity expressed by the cells.
Immunological, but not pharmacological, stimulation of permeabilized rat mast cells loaded with a fluorescent chymotryptic substrate led to a pronounced and rapid increase in fluorescence, indicating activation of the enzyme and hydrolysis of the substrate. These changes were attenuated by chymotryptic inhibitors.
In total, these data provide compelling evidence for the direct involvement of a serine protease in IgE‐mediated histamine release from mast cells.

British Journal of Pharmacology (1998) 123, 1267–1273; doi:10.1038/sj.bjp.0701724
Acetylcholine sensitivity of biphasic Ca2+ mobilization induced by nicotinic receptor activation at the mouse skeletal muscle endplate
Authors: Dezaki Katsuya , Kimura Ikuko 
Published: January 2005

Acetylcholine (ACh) was locally applied onto the endplate region in a mouse phrenic nerve‐diaphragm muscle preparation to measure intracellular free calcium ([Ca2+]i) entry through nicotinic ACh receptors (AChRs) by use of Ca2+‐aequorin luminescence.
ACh (0.1–3 mm, 20 μl) elicited biphasic elevation of [Ca2+]i (fast and slow Ca2+ mobilization) in muscle cells. The peak amplitude of the slow Ca2+ mobilization (not accompanied by twitch tension) was concentration‐dependently increased by ACh, whereas that of the fast component (accompanied by twitch tension) reached a maximum response at a lower concentration (0.1 mm) of applied ACh.
A pulse of nicotinic agonists, (−)‐nicotine (10 mm) and 1,1‐dimethyl‐4‐phenyl‐piperazinium (10 mm), but not a muscarinic agonist pilocarpine (10 mm), also elicited a biphasic Ca2+ signal.
Even though ACh release from motor nerve endings was blocked by botulinum toxin (5 μg, bolus i.p. before isolation of the tissue), the generation of both a fast and slow Ca2+ component caused by ACh application was observed.
These results strongly suggest that ACh locally applied onto the endplate region of skeletal muscle induces a slow Ca2+ signal reflecting Ca2+ entry through a postsynaptic nicotinic AChR, which has a low sensitivity to transmitter ACh.

British Journal of Pharmacology (1998) 123, 1418–1424; doi:10.1038/sj.bjp.0701725