Platelet-Activating Factor (PAF) Induces Strong Mechanical Activities Accompanied by Basal Tension Increases in Esophageal and Gastric Fundus Smooth Muscles from Rat

Abstract

In rats, platelet-activating factor (PAF) has been reported to increase mechanical activity in various gastrointestinal smooth muscles (SMs) except for esophagus SM. The aim of this study was to examine whether PAF increases mechanical activity in rat esophagus longitudinal SM (LSM) and to compare PAF actions in esophagus LSM with those in other gastrointestinal LSMs. PAF (10⁻⁹–10⁻⁶ M) increased esophagus LSM mechanical activities in a concentration-dependent manner; PAF mainly elicited basal tension increases that were almost eliminated by a PAF receptor antagonist CV-6209 (10⁻⁵ M; against 10⁻⁶ M PAF). In the LSM of the gastric fundus, which is similar to esophagus LSM in that it is derived from the foregut during development, PAF (10⁻⁶ M) increased basal tension to a comparable, albeit significantly different, magnitude as in esophagus LSM. In contrast, in LSMs of the duodenum–jejunum, ileum, and ascending colon, which are derived from the midgut, and the descending colon, which is derived from the hindgut, the ability of PAF (10⁻⁶ M) to increase basal tension was less than that in esophagus and gastric fundus LSMs. Interestingly, in ascending colon LSMs, PAF (10⁻⁶ M) induced oscillatory contractions with a small increase in basal tension. PAF-induced contractions were positively correlated with the mRNA expression levels of the PAF-degrading enzymes Pafah2 (R = 0.82) and Pafah1b3 (R = 0.51). These results suggest that PAF strongly stimulates mechanical activities that are mainly accompanied by basal tension increases in rat LSMs of the gastrointestinal tracts that are derived from the foregut during embryogenesis.

INTRODUCTION

Platelet-activating factor (PAF), an alkyl-ether phospholipid that was first described for its ability to cause platelet aggregation and dilation of blood vessels, is known to be a potent mediator of inflammation, allergic responses, and shock.^1–3) PAF also produces strong contractions in smooth muscles (SMs) from various organs including gastrointestinal tracts. For example, in rats, PAF-induced contraction was first reported in intestinal tract SMs,⁴⁾ followed by subsequent reports in other gastrointestinal SMs, specifically the gastric fundus,^5–9) duodenum,^4,9) jejunum,^4,6,9) ileum,^4,6,9) and ascending/transverse/descending colon.^4,9–12) With regard to PAF effects in gastrointestinal SMs, we have recently reported that PAF produces a strong contraction in esophagus longitudinal SM (LSM) isolated from guinea pigs.¹³⁾ In the present study, we expanded our analysis and examined whether PAF induced contractions in esophagus LSM from rat. We also compared PAF actions in esophagus LSM with those in other gastrointestinal LSMs and found that this phospholipid induced strong basal tension increases (BTIs) in esophagus and gastric fundus, both of which are derived from the foregut during embryogenesis.

MATERIALS AND METHODS

Animals

We used male Wistar rats (age: 8–12 weeks, weight: 190–285 g; Japan SLC Inc., Shizuoka, Japan), which were housed under a fixed 12/12 h light/dark cycle (08:00 to 20:00) and controlled conditions (20–22°C, relative air humidity: 50 ± 5%) with food and water available ad libitum. This study was performed in compliance with the guidelines of the Laboratory Animal Center of the Faculty of Pharmaceutical Sciences, Toho University, and was approved by the Toho University Animal Care and User Committee (Approval Number: 20-444).

Gastrointestinal SM Preparations

Rats were anesthetized by isoflurane inhalation and exsanguinated from the carotid artery. The esophagus was denuded of the adventitia and muscularis propria layer, including skeletal muscles, leaving the esophagus muscularis mucosa layer (LSM layer), in Locke–Ringer solution containing (in mM) NaCl, 154; KCl, 5.6; CaCl₂, 2.2; MgCl₂, 2.1; NaHCO₃, 5.9; and d-(+)-glucose, 2.8. The esophagus SM (approximately 30 mm in length) interior was irrigated with Locke-Ringer solution.

The stomach was separated into the gastric fundus and gastric body in Locke-Ringer solution. After irrigating its interior with Locke-Ringer solution, the gastric fundus was cut longitudinally (approximately 25 mm in length).

After irrigating the interior of each intestinal tissue, an acrylic/glass rod was inserted into its lumen, and the LSM (approximately 25 mm in length) was peeled off using a tweezer and cotton swab. Since the boundaries between duodenum and jejunum were difficult to distinguish, preparations containing both were used.

Effects of PAF on Gastrointestinal LSMs

The gastrointestinal LSM preparations were suspended longitudinally under a 1.0-g resting tension in a 20-mL organ bath containing Locke-Ringer solution, oxygenated with 95% O₂ and 5% CO₂ and maintained at 32 ± 1°C. Tension changes were isometrically recorded with PowerLab™ and LabChart™ (Version 7) software (ADInstruments Pty., Ltd., Bella Vista, NSW, Australia). After a 60-min incubation, the preparations were contracted with acetylcholine (ACh, 3 × 10⁻⁶ M) for 20 min at least twice with a 20-min interval.

Subsequently, an inhibitor cocktail (atropine [10⁻⁶ M, a muscarinic receptor antagonist], phentolamine [10⁻⁶ M, an α-adrenoceptor antagonist], propranolol (10⁻⁶ M, a β-adrenoceptor antagonist), N^G-nitro-l-arginine methyl ester [l-NAME, 10⁻⁴ M, a nitric oxide synthase inhibitor], and tetrodotoxin (3 × 10⁻⁷ M, a Na⁺ channel inhibitor]) was added to the bath solution to prevent potential neurotransmitter action. Bovine serum albumin (BSA, 0.25%) and anti-foam agent were then added to the bath solution. When using CV-6209 (10⁻⁵ M, a PAF receptor antagonist), it was added before the inhibitor cocktail administration. After a 30-min incubation, the preparations were contracted with PAF (10⁻⁹–10⁻⁶ M) for 20 min. Afterward, the strips were relaxed with papaverine (10⁻⁴ M).

All tension recordings were performed in the presence of indomethacin (3 × 10⁻⁶ M) to prevent the potential effects of endogenous prostaglandins. The concentrations of the antagonists and inhibitors used in this study were sufficient to inhibit the targeted receptors and channels based on previous reports.^14–17)

RT-Quantitative PCR (RT-qPCR) Analysis

RT-qPCR was performed as previously described.¹³⁾ Briefly, total RNA was isolated from gastrointestinal tissue preparations. First-strand cDNA was synthesized using ReverTra Ace^® qPCR RT Master Mix with gDNA Remover (TOYOBO Co., Ltd., Osaka, Japan). RT-qPCR was performed on a CronoSTAR™ 96 Real-Time PCR System (TaKaRa Bio Inc., Shiga, Japan) using Taq Pro Universal SYBR^® qPCR Master Mix (NIPPON Genetics Co., Ltd., Tokyo, Japan). The primers are shown in Supplementary Table 1.

Drugs

The following drugs were used in this study: PAF C-16 and CV-6209 (Cayman Chemical Co., Ann Arbor, MI, U.S.A.), BSA (fatty acid free, pH 7.0, Nacalai Tesque Inc., Kyoto, Japan), ACh chloride (Daiichi Sankyo Co., Ltd., Tokyo, Japan), atropine sulfate salt monohydrate, dl-propranolol hydrochloride, and indomethacin (Sigma-Aldrich Co., LLC, St. Louis, MO, U.S.A.), l-NAME hydrochloride (Dojindo Laboratories, Kumamoto, Japan), phentolamine mesylate (Novartis Pharma K.K., Tokyo, Japan), and tetrodotoxin and anti-foam PE-H (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan).

PAF was dissolved in ethanol to prepare a 2 × 10⁻³ M stock solution and stored at −80°C to prepare 2 × 10⁻⁴–2 × 10⁻⁷ M solutions, the ethanol solvent was evaporated, and the PAF was redissolved in 0.25% BSA. Indomethacin was dissolved in ethanol to prepare a 10⁻² M solution. All other drugs were dissolved in and diluted with distilled water.

Data Analysis

The area under the contraction curve (AUC) induced by PAF and ACh (3 × 10⁻⁶ M) for 20 min was analyzed using LabChart™. The AUC of PAF-induced contractions, shown as a relative value (%) to that of ACh-induced contractions (=100%), was regarded as PAF-induced mechanical activity.

Data are expressed as means ± standard error of mean, where n refers to the number of experiments. Statistical analyses were performed with t-tests or one-way ANOVA, followed by Tukey’s tests using GraphPad Prism™ (Version 6) (GraphPad Software Inc., San Diego, CA, U.S.A.). Statistical significance was set at p < 0.05.

RESULTS

Effects of PAF on Esophagus LSM

Figure 1 depicts representative traces (Figs. 1A–1D) and summarized data (Fig. 1E) of the effects of PAF on esophagus LSM. PAF (10⁻⁹–10⁻⁶ M) contracted esophagus LSM in a concentration-dependent manner.

Fig. 1. Representative Traces (A–D) and Summarized Data (E) Showing the Effects of PAF (10⁻⁹ M, A; 10⁻⁸ M, B; 10⁻⁷ M, C; 10⁻⁶ M, D) on Esophagus Smooth Muscle from Rat

Data are expressed as means ± standard error of the mean with each data point plotted (n = 6 [10⁻⁷ and 10⁻⁶ M], n = 5 [10⁻⁹ and 10⁻⁸ M]). ACh (3 × 10⁻⁶ M). Inhibitors: atropine (10⁻⁶ M), phentolamine (10⁻⁶ M), propranolol (10⁻⁶ M), l-NAME (10⁻⁴ M), tetrodotoxin (3 × 10⁻⁷ M), bovine serum albumin (0.25%), and anti-foam. ACh: acetylcholine; l-NAME: N^G-nitro-l-arginine methyl ester; PAF: platelet-activating factor; PPV: papaverine (10⁻⁴ M); w: wash out; AUC: area under the contraction curve.

Effects of CV-6209 on PAF-Induced Contractions in Esophagus LSM

Figure 2 depicts representative traces (Figs. 2A, 2B) and summarized data (Fig. 2C) of the effects of CV-6209 (a PAF receptor antagonist) on PAF (10⁻⁶ M)-induced contractions in esophagus LSM. PAF-induced contractions were almost completely suppressed by CV-6209 (10⁻⁵ M).

Fig. 2. Representative Traces (A, B) and Summarized Data (C) Showing the Effects of CV-6209 (10⁻⁵ M, B) on PAF (10⁻⁶ M)-Induced Contractions in Esophagus Smooth Muscles from Rat

Data are expressed as means ± standard error of the mean with each data point plotted (each n = 5). **p < 0.0001 vs. control (Welch’s t-test). ACh (3 × 10⁻⁶ M). Inhibitors: atropine (10⁻⁶ M), phentolamine (10⁻⁶ M), propranolol (10⁻⁶ M), l-NAME (10⁻⁴ M), tetrodotoxin (3 × 10⁻⁷ M), bovine serum albumin (0.25%), and anti-foam. ACh: acetylcholine; l-NAME: N^G-nitro-l-arginine methyl ester; PAF: platelet-activating factor; PPV: papaverine (10⁻⁴ M); w: wash out; AUC: area under the contraction curve.

In contrast, CV-6209 (10⁻⁵ M) did not affect ACh-induced contractions (3 × 10⁻⁶ M) (Supplementary Fig. 1).

Effects of PAF on Various Gastrointestinal LSMs

Figure 3 depicts representative traces (Figs. 3A–3F) and summarized data (Fig. 3G) of the effects of PAF (10⁻⁶ M) on esophagus (A), gastric fundus (B), duodenum-jejunum (C), ileum (D), ascending colon (E), and descending colon (F) LSMs. Among the tested gastrointestinal LSMs, PAF (10⁻⁶ M) strongly increased mechanical activities in those from esophagus (A), gastric fundus (B), and ascending colon (E); in esophagus and gastric fundus, the PAF effects were characterized as potent BTIs while oscillatory contractions (OCs) occurred in the ascending colon.

Fig. 3. Representative Traces (A–F) and Summarized Data (G) Showing the Effects of PAF (10⁻⁶ M) on Smooth Muscles of Eso (A), Fun (B), Duo (C), Ile (D), Asc (E), and Des (F) from Rat

Data are expressed as means ± standard error of the mean with each data point plotted (n = 6 [Eso and Ile], n = 7 [Fun], and n = 5 [Duo, Asc, and Des]). **p < 0.01 vs. Eso; ^##p < 0.01 vs. Fun; ^$p < 0.05, ^$$p < 0.01 vs. Asc (p > 0.9999 [Fun vs. Asc and Duo vs. Ile/Des], p = 0.2268/0.1653 [Eso vs. Fun/Asc], p = 0.0006 [Duo vs. Asc], p = 0.0002/0.9994/0.0017 [Fun/Ile/Asc vs. Des], p < 0.0001 [Eso vs. Duo/Ile/Des, Fun vs. Duo/Ile, and Ile vs. Asc]; one-way ANOVA followed by Tukey’s test). ACh: 3 × 10⁻⁶ M. Inhibitors: atropine (10⁻⁶ M), phentolamine (10⁻⁶ M), propranolol (10⁻⁶ M), l-NAME (10⁻⁴ M), tetrodotoxin (3 × 10⁻⁷ M), bovine serum albumin (0.25%), and anti-foam. Eso: esophagus; Fun: gastric fundus; Duo: duodenum-jejunum; Ile: ileum; Asc: ascending colon; Des: descending colon; PAF: platelet-activating factor; ACh: acetylcholine; l-NAME: N^G-nitro-l-arginine methyl ester; PAF: platelet-activating factor; PPV: papaverine (10⁻⁴ M); w: wash out.

Supplementary Figure 2 summarizes the BTIs (A) and OC amplitudes (B) over 20 min after PAF (10⁻⁶ M) administration in the various gastrointestinal LSMs shown in Fig. 3. The BTI magnitudes were significantly larger in the esophagus and gastric fundus than in other gastrointestinal LSMs including the ascending colon (Supplementary Fig. 2A). The OC amplitude was significantly larger in the ascending colon than in other gastrointestinal LSMs including the esophagus and gastric fundus (Supplementary Fig. 2B).

Although ACh (3 × 10⁻⁶ M)-induced contractions were slightly but significantly smaller in duodenum-jejunum and ileum LSMs than in esophagus LSM, we concluded that ACh (3 × 10⁻⁶ M)-induced contractions were roughly similar in the tested gastrointestinal LSMs (Supplementary Fig. 3).

Correlation between PAF-Induced Contractions and mRNA Expression of PAF-Related Molecules in Gastrointestinal Tissues

Figure 4 depicts the correlations in various gastrointestinal tissues between the PAF-induced contractions shown in Fig. 3 and the mRNA expression levels of PAF receptor (Ptafr, A), PAF-synthesizing enzymes (lysophosphatidylcholine acyltransferase; LPCAT) (Lpcat1, B; Lpcat2, C), and PAF-degrading enzymes (PAF acetylhydrolase; PAFAH) (Pafah1b3, D; Pafah2, E) shown in Supplementary Fig. 4. PAF-induced contractions were strongly positively correlated with Pafah2 levels (R = 0.82) (Fig. 4E) and positively correlated with Pafah1b3 levels (R = 0.51) (Fig. 4D).

Fig. 4. Correlation between PAF-Induced Contractions Shown in Fig. 3 and mRNA Expression Levels of PAF Receptor (Ptafr, A), PAF-Synthesizing Enzymes (Lpcat1, B; Lpcat2, C), and PAF-Degrading Enzymes (Pafah1b3, D; Pafah2, E) Shown in Supplementary Fig. 4 in Various Gastrointestinal SMs

ACh: 3 × 10⁻⁶ M. Eso: esophagus SM; Fun: gastric fundus SM; Duo: duodenum-jejunum SM; Ile: ileum SM; Asc: ascending colon SM; Des: descending colon SM; PAF: platelet-activating factor; AUC: area under the contraction curve; Gapdh: glyceraldehyde-3-phosphate dehydrogenase; ACh: acetylcholine; SMS: smooth muscles.

DISCUSSION

In this study, we found that PAF induced strong mechanical activity accompanied by BTIs in esophagus LSM isolated from rats. We previously reported that PAF showed BTIs in esophagus LSM from guinea pig.¹³⁾ Therefore, this characteristic seems to be conserved across species.

PAF also induced strong mechanical activity accompanied by BTIs in gastric fundus, and the BTI magnitudes in both esophagus and gastric fundus LSMs were nearly equivalent, although they were significantly different when compared. In contrast, the PAF-induced BTIs were significantly weaker in LSMs from other intestinal regions (Supplementary Fig. 2A). These regional differences in the PAF effects on BTIs may be associated with tissue differentiation. During embryogenesis, the gastrointestinal tract is derived from the archenteron and forms by 3 divisions into the foregut, midgut, and hindgut. The esophagus and gastric fundus are derived from the foregut; the duodenum-jejunum, ileum, and ascending colon from the midgut; and the descending colon from the hindgut.¹⁸⁾ Interestingly, the foregut is also the differentiation origin for respiratory tissues,¹⁹⁾ and SMs in these tissues such as rat trachea²⁰⁾ and guinea pig lung parenchyma (pleural SMs)²¹⁾ contract strongly in response to PAF. Therefore, PAF may be a strong constrictor in foregut-originating SM tissues although further studies are required to support this assumption. PAF-induced contractions in foregut-originated SM tissues could be a protective mechanism against foreign substances since these tissues play roles as “gateways” against foreign substances; when foreign substances are detected by foregut-originating SM tissues/immune cells, PAF is produced, which in turn prevents the entry of foreign substances by inducing SM tissue contractions. Indeed, PAF production increases in the esophagus due to acid exposure, which supports this assumption.^22–24) PAF-induced contraction of LSM may alleviate acid-induced injury of the esophagus by reducing its surface area. However, PAF is a major mediator of inflammatory responses. Therefore, long-term acid exposure and the resultant increase in PAF production may cause chronic tissue inflammation. In fact, the mRNA expression of PAF synthase (LPCAT) increases in patients with erosive esophagitis.²⁵⁾

The PAF-induced BTI was significantly smaller in midgut- and hindgut-originating intestinal tissues (duodenum-jejunum, ileum, ascending colon, and descending colon) than in foregut-originating tissues (esophagus and gastric fundus). However, in ascending colon, PAF (10⁻⁶ M) induced substantial mechanical activity, which was characterized by the generation of pronounced OCs (Supplementary Figs. 2A, 2B). The physiological/pathophysiological significance of PAF-induced OCs generated in the ascending colon is unclear. However, PAF is suggested to be associated with inflammatory bowel disease of the ascending colon.¹¹⁾ Thus, PAF-induced OCs generated in the ascending colon may be involved in this chronic disease.

The serum PAF concentration in healthy individuals is reported to be 127 ± 104 pg/mL (0.243 ± 0.199 nM), which increases up to 805 ± 595 pg/mL (1.54 ± 1.14 nM) in patients with anaphylaxis.²⁶⁾ This serum PAF concentration (approximately 1 nM) in patients with anaphylaxis is sufficient to produce substantial contraction of esophagus LSM, as shown in this study, and stomach and colon LSMs, as reported previously.^4,6,10) Therefore, in patients with anaphylaxis, mechanical activities of gastrointestinal SMs may be enhanced in response to PAF. When comparing PAF effects in esophagus, gastric fundus, and colon LSMs, we used 1 µM at which maximum basal tension changes were attained. This concentration is much higher than the serum PAF concentrations reported in patients with anaphylaxis. However, the local PAF concentration in gastrointestinal tissues may exceed its serum concentration (>nM levels) since PAF synthase is expressed in these tissues (Supplementary Figs. 4B, 4C). Therefore, PAF may induce enhanced mechanical activities in various gastrointestinal SM tissues as shown in Fig. 3. These abnormal activities enhanced by PAF could be blunted by anti-allergic drugs possessing PAF receptor antagonistic effects such as rupatadine.

Among PAF-related genes, a positive correlation with PAF-induced mechanical activities (shown as AUCs) was found for PAFAHs (Pafah2 and Pafah1b3), and the expression of PAFAH mRNAs was high in foregut-originating tissues (esophagus and gastric fundus). PAFAHs are enzymes that degrade PAF; PAFAH2 is localized in both the cytoplasm and cell membrane, whereas PAFAH1 is found in the cytoplasm.²⁷⁾ The high expression of PAFAH mRNAs in foregut-originating tissues might be obligatory for these tissues to prevent long exposure to PAF during inflammation. Substantial roles of functional PAFAHs in rat esophagus tissue may be supported by the finding that PAF-induced contraction was more potent in the presence of BSA than in its absence (Supplementary Fig. 5). This is because albumin, previously shown to increase the in vitro pharmacological action of PAF,²⁸⁾ could prevent PAF degradation by PAFAH.²⁹⁾

Unexpectedly, no correlation was found between PAF receptor mRNA expression and the actions of PAF (Fig. 4A), and the PAF-induced mechanical activities (contractions) of intestinal LSMs were much smaller than would be expected from Ptafr expression alone. Plausible explanations for this discrepancy are as follows: (1) The downstream signaling cascades following PAF receptor activation and stimulation of extracellular Ca²⁺ influx through plasma membrane Ca²⁺ channels could differ for each gastrointestinal SM tissue. PAF-induced mechanical activities in gastrointestinal SMs highly depend on extracellular Ca²⁺ influxes, which are mediated through voltage-dependent Ca²⁺ channels (VDCCs) and non-VDCCs. For example, in rat gastric fundus LSM, PAF-induced contractions depend on extracellular Ca²⁺ influx, and this influx may be mediated by VDCCs and receptor-operated Ca²⁺ channels (ROCCs).⁶⁾ In guinea pig esophagus LSM, PAF-induced contractions are mediated by extracellular Ca²⁺ influx through ROCCs and store-operated Ca²⁺ channels, but not VDCCs.¹³⁾ In guinea pig ileal circular SM, PAF-induced contractions are mediated by extracellular Ca²⁺ influx through VDCCs.³⁰⁾ In many cases, the molecular mechanisms responsible for the mechanical coupling between PAF receptors and Ca²⁺ channels still remain unknown. However, it is plausible that the Ca²⁺ channel opening in response to PAF receptor activation does not supply sufficient Ca²⁺ to induce mechanical activities. One of the explanations for such functional uncoupling would be the regional differences in the G protein molecules coupled with the PAF receptor. Although the PAF receptor is classified as a Gq protein-coupled receptor, it also associates with inhibitory Gi protein.³¹⁾ This PAF receptor–Gi protein coupling is reported to play a role in the regulation of neurotransmission and cell invasion.^32–34) Therefore, in gastrointestinal SM tissues such as the duodenum-jejunum and ileum in which PAF induces only marginal mechanical activities, the main PAF receptors might be coupled with SM contraction-unrelated Gi proteins. (2) PAF receptor mRNA expression does not correlate with its functional protein expression. In fact, both high and very low correlations have been reported between the mRNA expression of some genes and their protein levels.³⁵⁾ To clarify this issue, regional differences in PAF receptor protein expression should be examined. In this regard, it should be noted that PAF receptors are reportedly desensitized by protein kinase C in rat gastric fundus LSM.⁷⁾ Therefore, even if both PAF receptor mRNA and protein are highly expressed, if the receptor is phosphorylated before stimulation with PAF, SM mechanical activities cannot be enhanced. It is also plausible that the PAF receptor is localized in the cytoplasm rather than the cell membrane. To obtain further information about this possibility, the cell membrane/intracellular localization and expression level/phosphorylation state of the PAF receptor should be studied in more detail.

Non-correlation was also found between the mRNA expression of LPCATs and the actions of PAF (Figs. 4B, 4C). Plausible explanations for this lack of correlation are as follows: (1) As mentioned above, the protein expression of LPCATs could be lower than the corresponding mRNA expression. (2) This study used healthy animals and not animal models of inflammatory disease. In healthy animals, the mRNA expression of LPCATs may not be affected by regional differences in gastrointestinal tissues. In contrast, during inflammation, various cells (e.g., immune cells and mast cells) produce PAF, which subsequently stimulates PAF-releasing cells and other cells, leading to further PAF production.³⁶⁾ Therefore, during inflammation, the mRNA expression of LPCATs may be induced by PAF receptor stimulation, which could result in regional differences in gastrointestinal SM tissues. (3) PAF synthesis may be regulated by non-LPCAT enzymes. The PAF accumulation in urinary bladder tissues caused by exposure to cigarette smoke is reported to be suppressed in knockout mice lacking calcium-independent phospholipase A2β.³⁷⁾ Therefore, this enzyme could be the rate-limiting enzyme for PAF synthesis in the gastrointestinal tract. Further studies are needed to explain the lack of correlation between the mRNA expression of PAF receptors/LPCATs and the actions of PAF.

In addition to PAF, other physiologically active substances including neurotransmitters and autacoids show regional differences in their effects on the mechanical activities of gastrointestinal SM tissues. For example, ACh (3 × 10⁻⁶ M)-induced contractions were similar in all tested gastrointestinal tissues (Supplementary Fig. 3). However, esophageal LSM was found to be the most sensitive to ACh among the tested tissues. Specifically, the pD₂ values of ACh were in the following order: 6.30 ± 0.05 (esophagus) > 6.21 ± 0.09 (descending colon > 6.15 ± 0.11 (ascending colon) > 6.01 ± 0.06 (gastric fundus) ≈ 6.00 ± 0.14 (duodenum-jejunum) > ileum (5.78 ± 0.09) (Supplementary Fig. 3). Regarding histamine and serotonin, they show regional differences in rat gastrointestinal SMs. Histamine produces significant contractions of esophagus SM and duodenum LSM, whereas it induces only slight contractions of ileum LSM and does not contract gastric fundus and colon LSMs.^38–41) Serotonin induces contractions of gastric fundus, duodenum, ileum, and colon LSMs, whereas it relaxes esophagus LSM.^40,42–44) In addition, tachykinins show regional differences in musk shrews. Specifically, substance P contracts stomach LSM more strongly than neurokinin A (NKA) does, whereas its contractile effect is weaker than that of NKA in duodenum, ileum, and colon LSMs. NKA also does not affect OCs in stomach LSM, but increases their amplitude in duodenum, ileum, and colon LSMs.⁴⁵⁾ Therefore, various physiologically active substances, including PAF, show regional differences in their effects on gastrointestinal SM mechanical activities. These findings strongly suggest that the regulation of mechanical activities of gastrointestinal SMs occurs spatially and precisely by physiologically active substances, although the mechanisms of this fine tuning should be elucidated in future studies.

The present study has some limitations: (1) The changes in mechanical activities were measured at 32°C, which was determined according to previous pharmacological studies on gastrointestinal SM tissues.^46–49) This temperature is lower than the physiological temperature (37°C), and the characteristics of changes in PAF-induced mechanical activities might differ between these temperatures. (2) We used male rats in this study. In female animals, including humans, the effect of PAF on the uterus has been reported to increase during pregnancy¹⁾ and may be influenced by sex hormones. Therefore, it is necessary to examine whether similar results can be obtained in female rats, including during pregnancy.

In summary, this study revealed that PAF induces strong mechanical activities accompanied by BTIs in rat gastrointestinal tract LSMs that are derived from the foregut.

Acknowledgments

This work was supported in part by the JSPS KAKENHI Grants-in-Aid for Scientific Research (C) (24K14659 to K.O., Y.T., and K.Y.; 24K14637 to K.Y., Y.T., and K.O.; and 23K10856 to Y.T., K.O., and K.Y.).

Conflict of Interest

The authors declare no conflict of interest.

Data Availability

The data that support the findings of this study are available from the corresponding author, K.O., upon reasonable request.

Supplementary Materials

This article contains supplementary materials.

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