Effects of Platelet-Activating Factor (PAF) on the Mechanical Activities of Lower Urinary Tract and Genital Smooth Muscles

Abstract

Since its first discovery as a bioactive phospholipid inducing potent platelet aggregation, platelet-activating factor (PAF) has been shown to be involved in a wide variety of inflammatory and allergic disease states. Many pharmacological studies in the 1980s and 1990s also showed that PAF induces endothelium-dependent vascular relaxation and contraction of various smooth muscles (SMs), including those in the airway, gastrointestinal organs, and uterus. However, since the late 1990s, there have been few reports on the SM contractions induced by PAF. The lower urinary tract (LUT), particularly the urinary bladder (UB) has attracted recent attention in SM pharmacology research because patients with LUT dysfunctions including overactive bladder are increasing as the population ages. In addition, recent clinical studies have implicated the substantial role of PAF in the inflammatory state in LUT because its production increases with smoking and with cancer. However, the effects of PAF on mechanical activities of LUT SMs including UBSM have not been investigated to date. Recently, we found that PAF very strongly increased mechanical activities of UBSM in guinea pigs and mice, and partly elucidated the possible mechanisms underlying these actions of PAF. In this review, we describe the effects of PAF on LUT SMs by introducing our recent findings obtained in isolated UBSMs and discuss the physiological and pathophysiological significance. We also introduce our data showing the effects of PAF on the SM mechanical activities of genital tissues (prostate and vas deferens).

1. INTRODUCTION

Platelet-activating factor (PAF) was discovered in 1972 as a substance that aggregates platelets.¹⁾ In 1979, its structure was revealed as 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, a glyceryl ether-containing phosphoglyceride.²⁾ PAF belongs to the phosphatidylcholine group and contains (1) usually C16 or C18 fatty acids with ether bonds at the sn-1 position, (2) an acetyl group at the sn-2 position, and (3) phosphocholine at the sn-3 position³⁾ (Fig. 1). In particular, the acetyl group at the sn-2 position is important for the biological activity of PAF.³⁾

Fig. 1. Structure of Platelet-Activating Factor (PAF) and PAF Synthesis/Degradation Pathways

PLA₂: phospholipase A₂. LPCAT: lysophosphatidylcholine acyltransferase. PAF-AH: PAF acetylhydrolase.

PAF is known to exhibit a variety of biological activities in addition to platelet aggregation. For example, PAF acts as a potent inflammatory mediator that triggers pathological inflammatory conditions such as bronchial asthma, atherosclerosis, cardiovascular and kidney diseases, allergies, AIDS, and cancer.^4,5) PAF also plays a key role in the regulation of smooth muscle (SM) contractile activity. The specific effects of PAF on SM tissues are as follows: (1) endothelium-dependent relaxation of vascular SMs (aorta,^6–9) pulmonary artery,^6,10) and mesenteric artery^11,12)), (2) contraction of airway SMs (trachea^13–15) and bronchi^15–18)) and epithelium-dependent relaxation of trachea SM,^19–22) and (3) contraction of gastrointestinal SMs (esophagus,²³⁾ stomach,^24–28) duodenu,²⁹⁾ jejunum,^28,29) ileum,^25,28–31) colon,^25,29,32) gallbladder³³⁾) and uterine SM.^34,35) Among the contractions induced by PAF, it is noteworthy that PAF produces more potent contractions in uterine SM tissue isolated from pregnant females than in that isolated from non-pregnant females,³⁵⁾ suggesting that PAF substantially contributes to the progression of pregnancy. Consistent with this possible role of PAF in animal reproduction and birth, PAF is reported to play physiologically important functions in genital systems, including sperm motility, ovulation, fertilization, implantation, maintenance of pregnancy, and childbirth.^36–38) PAF is also suggested to play a role in pathological conditions, including menstrual pain, premature birth, and progression of prostate cancer.^38–42) The potential pathological role of PAF in the lower urinary tract (LUT) system has recently attracted attention. Specifically, the production of PAF in urinary bladder (UB) tissue was shown to be increased by smoking and cancer.^43–45) These PAF-associated findings obtained in LUT and genital tissues suggest that this phospholipid changes the physiological mechanical functions of LUT and genital SMs, including the UB SM (UBSM), into a state of disorder. However, except for the uterine SM, there has been little research on the effects of PAF on the contractile functions of these SM tissues.

In this review, we focus on LUT and genital SMs and describe the changes in contractile activity induced by PAF (Table 1). In particular, we present our recent findings, which demonstrate the powerful stimulatory activity of PAF on UBSM mechanical activities (basal tone and spontaneous contractile activity (SCA)), together with the underlying mechanisms.^46–48) In addition, we discuss the possible physiological and pathophysiological roles of this phospholipid.

Table 1. Effects of Platelet-Activating Factor (PAF) on Lower Urinary Tract and Genital Smooth Muscles

Tissue	Species	Responses	Ref./Fig.
Urinary bladder	Guinea pig	Contraction	46,47)
	Rat	No/slight contraction	Fig. 5A
	Mouse	Contraction	46,48)
Urethra (female)	Guinea pig	No/slight contraction	Fig. 5B
Vas deferens	Rat	No contractile response to 10−6 M PAF	Fig. 6A
Prostate	Rat	No contractile response to 10−6 M PAF	Fig. 6B
Corpus cavernosum	Rabbit	No contractile response to 10−7 M PAF	89)
Non-pregnant uterus	Guinea pig	Contraction (dependent on cyclooxygenase/lipoxygenase pathways)	34)
	Rat	Contraction (very weak)	35)
Pregnant uterus	Rat	Contraction (strong)	35)

2. PAF SYNTHESIS AND DEGRADATION

PAF is synthesized via two biosynthetic pathways: de novo and remodeling^3,49) (Fig. 1). The remodeling pathway contributes to the majority of PAF biosynthesis, while the de novo pathway plays a smaller role. Therefore, the remodeling pathway is the major pathway of PAF biosynthesis that maintains physiological functions and causes pathological responses.⁴⁹⁾

In the de novo pathway, PAF is biosynthesized from 1-alkyl-2-lyso-sn-glycero-3-phosphate in three enzymatic steps^49,50) (Fig. 1). In contrast, in the remodeling pathway, the first step is the generation of lyso-PAF from 1-alkyl-phosphatidylcholine through the hydrolytic activity of phospholipase A₂ (PLA₂). Lyso-PAF is subsequently converted into PAF by lysophosphatidylcholine acyltransferase (LPCATs)³⁾ (Fig. 1). LPCATs, enzymes that catalyze the formation of PAF from lyso-PAF, are composed of two subtypes: LPCAT1 (constitutively active type) and LPCAT2 (inducible type).³⁾ The basic function of LPCATs is to introduce acyl-CoA fatty acids into lysophosphatidylcholines including lyso-PAF. Therefore, these enzymes catalyze the biosynthesis of phosphatidylcholine-grouped chemicals containing various fatty acids at the sn-2 position, which includes PAF.³⁾ LPCAT1 and LPCAT2 also catalyze the conversion of lyso-PAF to 1-alkyl-phosphatidylcholine³⁾ (Fig. 1).

PAF is degraded to lyso-PAF by PAF acetylhydrolases (PAF-AHs), which catalyze the hydrolysis of the acetyl group attached to the sn-2 position of PAF⁵¹⁾ (Fig. 1). The sn-2 acetyl group is indispensable for PAF to exhibit its biological activities; thus, PAF-AH-mediated deacetylation leads to the loss of the biological activities of the phospholipid.⁵²⁾ PAF-AHs in mammals are divided into two types based on their location: plasma PAF-AH and intracellular PAF-AH. The latter type is further subdivided into two subtypes: intracellular type I PAF-AH [PAF-AH (I)], and intracellular type II PAF-AH [PAF-AH (II)].^51,52) Therefore, there are three subtypes of PAF-AHs in mammals. Regarding PAF-AH in human plasma, 70% of it is bound to low-density lipoprotein (LDL), and the remainder is bound to high-density lipoprotein (HDL).⁵¹⁾ Therefore, plasma PAF-AH is also called lipoprotein-associated PLA₂ (LpPLA₂).⁵³⁾ Regarding intracellular PAF-AHs, PAF-AH (I) resides in the cytosol and forms a G protein-like complex consisting of two catalytic α subunits (α1 and α2) and a regulatory β subunit.⁵²⁾ These α1, α2, and β subunits are encoded by the PAFAH1B3, PAFAH1B2, and PAFAH1B1 genes, respectively.⁵¹⁾ PAF-AH (II) is composed of a single polypeptide chain present in both the cytosolic and membrane fractions, the latter after myristoylation.⁵²⁾

Plasma PAF-AH (I) and PAF-AH (II) show similar substrate specificity; in addition to PAF, they are able to catalyze the hydrolysis of acyl groups (e.g., propionyl, butyroyl, succinyl, and glutaroyl groups) introduced into the sn-2 position of PAF derivatives.⁵¹⁾ Furthermore, these PAF-AHs (plasma PAF-AH and PAF-AH (II)) can hydrolyze short-chain diacylglycerols, triacylglycerols, and acetylated alkanols.^51,52) In contrast, PAF-AH (I) is unable to catalyze the hydrolysis of the abovementioned non-acetyl acyl groups introduced into PAF derivatives.⁵¹⁾ It has also been shown that the substrate specificity of PAF-AH (I) strongly reflects it subunit composition, as follows: (1) a PAF analog 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylethanolamine (AAGPE) can be hydrolyzed by α2/α2 homodimers but not by α1/α1 homodimers or α1/α2 heterodimers⁵¹⁾; and (2) another PAF analog 1-O-alkyl-2-acetyl-sn-glycero-3-phosphoric acid is hydrolyzed more efficiently than PAF by α1/α1 homodimers and α1/α2 heterodimers.^51,52) The role of the β subunit is to regulate the enzymatic activity of the α subunit dimer. Specifically, the β subunit strongly enhances the activity of α2/α2 homodimers and suppresses that of α1/α1 homodimers. The β subunit has little effect on the activity of α1/α2 heterodimers.^51,52)

3. PAF RECEPTOR

The actions of PAF are mediated by the PAF receptor composed of single polypeptides.⁵⁴⁾ PAF receptor subtypes have not been identified.⁵⁵⁾ The PAF receptor is a G protein-coupled receptor involving G_q and G_i proteins⁵⁴⁾; stimulation with PAF enhances inositol 1,4,5-trisphosphate (IP₃) synthesis and Ca²⁺ mobilization through the G_q protein and suppresses cAMP synthesis through the G_i protein.⁵⁵⁾ The stimulation of PAF receptor also leads to (1) activation of extracellular signal-regulated kinase (Erk) and p38 mitogen-activated protein kinase (p38 MAPK), and (2) regulation of the activities of phospholipase D, phospholipase C-γ, and small G proteins (Ras, Ral, and Rap).⁵⁵⁾

4. IMPORTANCE OF ALBUMIN ON THE EFFECTS OF PAF

Albumin is essential for the pharmacological actions of PAF in vitro. For example, the powerful endothelium-dependent relaxation of rat aorta in response to PAF requires the presence of bovine serum albumin.⁹⁾ Furthermore, the renal vasodilatory effect of PAF has not been observed in albumin-free solutions.⁵⁶⁾ As to the role of albumin in PAF activity, the PAF receptor is stimulated by PAF that forms a complex with albumin, but not by the albumin-free form.⁵⁷⁾ Therefore, the pharmacological activities of PAF in vitro should be evaluated using PAF prepared by dissolution in an albumin-containing solution.

5. EFFECTS OF PAF ON CONTRACTILE ACTIVITY OF LUT SMS

In this section, we describe an interesting new pharmacological activity of PAF on isolated UBSM, which was discovered by our research group, and the possible molecular mechanisms involved.^46–48)

We first found that PAF increased mechanical activities (basal tone and SCA) in isolated guinea pig (GP) UBSM in a concentration-dependent manner⁴⁶⁾ (Figs. 2A–E). We determined that this PAF action reflects direct stimulation of UBSM receptors by this phospholipid from the following evidence: (1) changes in UBSM mechanical activities were recorded in the presence of inhibitors and antagonists (inhibitors of cyclooxygenases and voltage-dependent Na⁺ channels, and antagonists of muscarinic receptors, purinoceptors, α-, and β-adrenoceptors) necessary to eliminate the effect of possible endogenous prostanoids and neurotransmitters; (2) PAF-induced responses were observed similarly in both UBSM preparations in which epithelium was preserved and mechanically removed; and (3) PAF-induced responses were abolished by apafant, a PAF receptor antagonist⁴⁶⁾ (Fig. 2F).

Fig. 2. Representative Traces Showing the Effects of PAF on the Basal Tone and Spontaneous Contraction Activities in Isolated Guinea Pig Urinary Bladder Smooth Muscle

The effects of vehicle (0.25% bovine serum albumin (BSA)) (A), the PAF receptor antagonist apafant (10⁻⁵ M) (F), and PAF at 10⁻⁹ M (B), 10⁻⁸ M (C), 10⁻⁷ M (D), and 10⁻⁶ M (E). These experiments were carried out in the presence of indomethacin (3 × 10⁻⁶ M). ACh: acetylcholine (10⁻⁴ M). Inhibitors: atropine (10⁻⁶ M), suramin (10⁻⁴ M), phentolamine (10⁻⁶ M), propranolol (10⁻⁶ M), tetrodotoxin (3 × 10⁻⁷ M), BSA (0.25%), and anti-foam. w: wash out. These data were reproduced from ⁴⁶⁾.

Second, the UBSM mechanical activities (UBSM basal tone and SCA) increased by PAF were shown to be dependent on extracellular Ca²⁺ influx, since PAF actions were abolished in Ca²⁺-free solution⁴⁷⁾ (Figs. 3A, B). Subsequently, we pharmacologically investigated the functional Ca²⁺ entry routes (channels) that supply this cation from the extracellular space and that are responsible for the PAF-induced increase in UBSM mechanical activities. Two types of Ca²⁺ channels were revealed to be involved in the PAF-induced effects: voltage-dependent Ca²⁺ channels (VDCCs) and store-operated Ca²⁺ channels (SOCCs).

Fig. 3. Representative Traces Showing the Effects of Ca²⁺ and Various Ca²⁺ Channel Blockers on the Basal Tone and Spontaneous Contraction Activities Increased by PAF (10⁻⁶ M) in Isolated Guinea Pig Urinary Bladder Smooth Muscle

The effects of normal solution (A, control), Ca²⁺-free solution (containing 0.2 mM EGTA) (B), and various Ca²⁺ channel blockers [diltiazem (10⁻⁵ M) (C); verapamil (10⁻⁵ M) (D); verapamil plus LOE-908 (3 × 10⁻⁵ M) (E); verapamil plus SKF-96365 (3 × 10⁻⁵ M) (F)] were analyzed. These experiments were carried out in the presence of indomethacin (3 × 10⁻⁶ M). ACh: acetylcholine (10⁻⁴ M). Inhibitors: atropine (10⁻⁶ M), suramin (10⁻⁴ M), phentolamine (10⁻⁶ M), propranolol (10⁻⁶ M), tetrodotoxin (3 × 10⁻⁷ M), bovine serum albumin (0.25%), and anti-foam. EGTA: ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N'-tetraacetic acid. w: wash out. These data were reproduced from ⁴⁷⁾.

The crucial role of VDCCs as the principal Ca²⁺ entry route to supply this cation responsible for PAF-induced increases in UBSM mechanical activities was evidenced by the finding that inhibitors of this channel (diltiazem and verapamil) strongly suppressed PAF-induced increases in both basal tone and frequency of SCA⁴⁷⁾ (Figs. 3C, D). However, the PAF-induced increases in UBSM mechanical activities were not completely suppressed by VDCC inhibitors, and the amplitude of SCA remained elevated by PAF even in the presence of these channel inhibitors. The enhancing effects of PAF on the amplitude of SCA in the presence of verapamil were not affected by LOE 908 (an inhibitor of receptor-operated Ca²⁺ channels (ROCCs)) (Fig. 3E) but were strongly suppressed by SKF-96365 (an inhibitor of both ROCCs and SOCCs)⁴⁷⁾ (Fig. 3F). Therefore, SOCCs were shown to be responsible for the PAF-induced increase in the amplitude of SCA in guinea pig UBSM preparations. Similar results were observed in mouse UBSM.⁴⁸⁾

SOCCs are plasma membrane Ca²⁺ entry channels whose opening is triggered by the depletion of Ca²⁺ in intracellular Ca²⁺ storage sites. Orai channels are molecular candidates of SOCCs.⁵⁸⁾ To date, three types of Orai channels (Orai1–3) have been identified, and these channels are generally activated through stromal interaction molecules (Stims), which are Ca²⁺ sensor proteins of the sarco/endoplasmic reticulum (SER).⁵⁸⁾ Two Stim proteins (Stim1 and Stim2) have been identified, and their activation was deemed to be triggered by the depletion of stored Ca²⁺ in the SER.⁵⁸⁾ In guinea pig UB tissues, our investigation showed that Orai1 and Orai3 are abundantly expressed at the mRNA level among the three Orai channels, and Stim2 is the major Stim mRNA expressed⁵⁹⁾ (Fig. 4A). Therefore, the intracellular Ca²⁺ responsible for the PAF-induced increase in the amplitude of SCA in guinea pig UBSM is speculated to be supplied through Orai1 and/or Orai3, the activation of which is triggered through Stim2 after the stimulation of the UBSM PAF receptor. The Orai-STIM pathway is also involved in the generation of contractions in human UBSM. Therefore, molecules associated with this pathway are potential new targets for drug therapy for overactive bladder (OAB).⁶⁰⁾

Fig. 4. mRNA Expression Levels of Orai Channels (Orai1, Orai2, and Orai3)/Stromal Interaction Molecule (Stim) Homologs (Stim1 and Stim2) (A) and PAF Receptor (Ptafr)/PAF-Synthesizing Enzymes (Lpcat1 and Lpcat2)/PAF-Degrading Enzymes (Pafah1b3 and Pafah2) (B) in Guinea Pig Urinary Bladder Tissues as Assessed by RT-qPCR

The expression level of each mRNA is shown relative to that of Gapdh, which is arbitrarily set as 1. Data are expressed as the means ± standard error of the mean [n = 4–5 (A) and n = 8 (B)]. These data were reproduced from ⁵⁹⁾ (A) and ⁴⁶⁾ (B).

Consistent with our pharmacological findings that PAF strongly increased UBSM mechanical activities, the PAF receptor was found to be expressed at the mRNA level (Ptafr) in guinea pig UB tissue⁴⁶⁾ (Fig. 4B). Furthermore, in guinea pig UB tissue, enzymes responsible for PAF biosynthesis and degradation were shown to be expressed at the mRNA level, namely the LPCATs (Lpcat1 and Lpcat2) and intracellular types of PAF-AHs (Pafah1b3 and Pafah2), respectively⁴⁶⁾ (Fig. 4B). In support of our biochemical findings on PAF-associated enzymes, PAF has been reported to be produced in microvascular endothelial and urothelial cells in UB tissues.^43,44) Similarly, we found mRNA expression of PAF-associated proteins (receptors and enzymes involved in biosynthesis and degradation) in mouse UB tissue.⁴⁶⁾

Regarding the production of PAF in UB tissues, it may be increased by smoking; smoking has been reported to enhance PAF production in UB microvascular endothelial and urothelial cells.^43–45) Specifically: (1) exposure to cigarette smoke extract (CSE) leads to increased PAF accumulation and PAF receptor expression with decreasing PAF-AH activity in epithelial-line cells (human UB microvascular endothelial cells, human UB urothelial cells, human UB urothelial cancer cells (HTB-9), human UB cancer cells (HT-1376), and mouse UB endothelial cells)^43–45); and (2) long-term exposure to cigarette smoke increases PAF production and PAF receptor expression in mouse UB.⁴⁴⁾ These effects of cigarette smoke exposure on increasing PAF levels and PAF-associated functional proteins are suppressed by inhibitors of Ca²⁺-independent phospholipase A₂β (iPLA₂β) or by its gene knockout,^43–45) suggesting an obligatory role of iPLA₂β in smoking-induced increases in PAF production.

A possible causal role for PAF in the development of UB-related diseases has also been suggested as follows: (1) Involvement in the exacerbation of interstitial cystitis/bladder pain syndrome (IC/BPS). The amount of PAF is increased in the urine of patients with IC/BPS, and this increase is more pronounced in patients who smoke.⁴⁴⁾ Furthermore, when exposed to CSE, immortalized urothelial cells generated from patients with IC/BPS produce much higher amounts of PAF than those generated from healthy individuals.⁴⁴⁾ (2) PAF production increases with increasing malignancy in cancerous UB tissues obtained from patients with a history of smoking.⁴⁵⁾ (3) The relative risk of OAB increases in smokers.^61,62) These findings suggest that smoking-induced increases in PAF production/PAF receptor expression in the UB lead to OAB in addition to causing UB inflammation. Therefore, PAF receptor antagonists and PAF synthase (LPCAT1/2) inhibitors may prevent or improve these UB functional abnormalities and are expected to be useful as new therapeutic agents for UB-related diseases. Commercially available PAF receptor antagonists and LPCAT1/2 inhibitor are shown in Table 2.^63–87) However, in rat UBSM tissues, the effects of PAF on mechanical activity were marginal, and contraction effects were not detected in many cases (Fig. 5A). Although we do not have a reasonable explanation for the results in rat UBSM, PAF effects may differ depending on the species. Therefore, whether human UBSM can be induced to contract in response to PAF requires further investigation.

Table 2. Commercially Available PAF Receptor Antagonists and Lysophosphatidylcholine Acyltransferase (LPCAT) 1/2 Inhibitor

Reagents	IC50/Ki values	Inhibitory effects	Species	Ref.
1. Synthesized PAF receptor antagonists
ABT-299	Ki = 0.3 nM	Specific binding of [3H]PAF	Human	63)
ABT-491	Ki = 0.57 nM	Specific binding of [3H]PAF	Human	64)
Apafant (WEB 2086)	Ki = 30 nM	Specific binding of [3H]apafant	Human	65)
Bepafant (WEB 2170)	IC50 = 300 nM	PAF-induced platelet aggregation	Human	66)
BN 50739	IC50 = 127 nM	Specific binding of [3H]PAF	Human	67)
CL 184,005	IC50 = 600 nM	PAF-induced platelet aggregation	Human	68)
CV-3988	Ki = 120 nM	Specific binding of [3H]PAF	Rabbit	69)
CV-6209	IC50 = 170 nM	PAF-induced platelet aggregation	Human	70)
Foropafant (SR27417)	Ki = 50 pM	Specific binding of [3H]PAF	Human	71)
Israpafant (Y-24180)	IC50 = 0.84 nM	PAF-induced platelet aggregation	Human	72)
L659989	Ki = 14.3 nM	Specific binding of [3H]PAF	Human	73)
Lexipafant (BB-882)	IC50 = 0.14 nM	PAF-induced platelet aggregation	Rabbit	74)
Modipafant (UK-80067)		((+)-enantiomer of UK-74505)		75)
PCA 4248	IC50 = 1.05 µM	PAF-induced platelet aggregation	Rabbit	76)
Rocepafant (BN 50730)	IC50 = 461 nM	PAF-induced [3H]-serotonin release	Human	77)
RP-52770	Ki = 7.0 nM	Specific binding of [3H]PAF	Human	78)
Rupatadine (UR-12592)	IC50 = 0.68 µM	PAF-induced platelet aggregation	Human	79)
SCH 37370	IC50 = 0.61 µM	PAF-induced platelet aggregation	Human	80)
SCH 40338	IC50 = 0.59 µM	PAF-induced platelet aggregation	Human	80)
SDZ 64-412	IC50 = 0.06 µM	Specific binding of [3H]PAF	Human	81)
TCV-309	IC50 = 58 nM	PAF-induced platelet aggregation	Human	82)
UK-74505	IC50 = 4.3 nM	PAF-induced platelet aggregation	Rabbit	83)
UR-12670	IC50 = 0.30 nM	PAF-induced platelet aggregation	Rabbit	74)
2. Natural products and their derivatives with PAF receptor antagonistic activities
Epiyangambin	IC50 = 610 nM	PAF-induced platelet aggregation	Rabbit	84)
Ginkgolide A	Ki = 1.46 µM	Specific binding of [3H]apafant	Mouse	85)
Ginkgolide B (GB)	Ki = 0.56 µM	Specific binding of [3H]apafant	Mouse	85)
7α-Cl-GB	Ki = 0.11 µM	Specific binding of [3H]apafant	Mouse	86)
7α-F-GB	Ki = 0.99 µM	Specific binding of [3H]apafant	Mouse	86)
7α-N3-GB	Ki = 0.55 µM	Specific binding of [3H]apafant	Mouse	86)
7α-NH2-GB	Ki = 8.64 µM	Specific binding of [3H]apafant	Mouse	86)
7α-NHMe-GB	Ki = 0.61 µM	Specific binding of [3H]apafant	Mouse	86)
7α-NHEt-GB	Ki = 1.62 µM	Specific binding of [3H]apafant	Mouse	86)
7α-OAc-GB	Ki = 7.84 µM	Specific binding of [3H]apafant	Mouse	86)
7α-OCOCH2Ph-GB	Ki = 2.40 µM	Specific binding of [3H]apafant	Mouse	86)
10-OBn-GB	Ki = 0.12 µM	Specific binding of [3H]apafant	Mouse	86)
10-OBn-7α-F-GB	Ki = 0.10 µM	Specific binding of [3H]apafant	Mouse	86)
Ginkgolide C (GC)	Ki = 12.6 µM	Specific binding of [3H]apafant	Mouse	85)
7-epi-GC	Ki = 4.26 µM	Specific binding of [3H]apafant	Mouse	86)
10-OBn-GC	Ki = 1.67 µM	Specific binding of [3H]apafant	Mouse	86)
10-OBn-epi-GC	Ki = 0.61 µM	Specific binding of [3H]apafant	Mouse	86)
Ginkgolide J	Ki = 9.90 µM	Specific binding of [3H]apafant	Mouse	85)
3. LPCAT1/2 inhibitor
TSI-01	IC50 = 0.42 µM	PAF production by LPCAT1	Human	87)
	IC50 = 3.02 µM	PAF production by LPCAT2	Human	87)

IC₅₀, 50% inhibitory concentration; K_i, inhibitory constant.

Fig. 5. Representative Traces Showing the Effects of PAF (10⁻⁶ M) on Isolated Rat Urinary Bladder (A) and Guinea Pig (Female) Urethra (Ba, Bb) Smooth Muscles

These experiments were carried out in the presence of indomethacin (3 × 10⁻⁶ M). ACh: acetylcholine. NA: noradrenaline. BSA: bovine serum albumin. w: wash out. PPV: papaverine.

We also examined whether PAF could contract urethral SM to increase its resistance, and the results obtained in female GP urethral SM are shown in Fig. 5B. The results show that PAF stimulation (10⁻⁶ M) induced only a small magnitude contraction (Fig. 5Ba) or had a practically negligible effect (Fig. 5Bb). Therefore, we conclude that PAF exhibits insignificant effects on the mechanical activity of urethral SM.

6. EFFECTS OF PAF ON CONTRACTILE ACTIVITY OF GENITAL SMS

PAF is also produced in male genital organs. The PAF-generating male organs reported to date include the testes, epididymis, and vas deferens in both the GP and rat, as well as rat prostate.⁸⁸⁾ PAF production in these organs decreases due to castration and increases with the administration of androgens, suggesting that PAF production is largely controlled by male hormones, and this phospholipid substantially regulates the function of the male genital system.⁸⁸⁾ To clarify whether PAF affects the SM mechanical activities of male genital organs, we tested the effects of PAF on rat vas deferens and prostate SMs (Figs. 6A, B). However, we did not observe any prominent effects of PAF on either tissue. Similarly, the rabbit corpus cavernosum does not generate contractions in response to PAF.⁸⁹⁾ Therefore, in male genital organs containing SM, the contribution of PAF may be in the regulation of their functions other than the control of muscle tension, although the physiological significance of PAF is still unknown in this regard. PAF may also be associated with disease progression. Possible roles for PAF in the progression of prostate cancer have been reported.^39–41) However, PAF is suggested to be more important in the control of sperm motility and fertilization than in the SM mechanical activity of male genital organs. This is evidenced by the finding that the PAF content in human sperm is positively correlated with semen parameters and pregnancy outcomes.³⁶⁾

Fig. 6. Representative Traces Showing the Effects of PAF (10⁻⁶ M) on Isolated Rat Vas Deferens (A) and Prostate (B) Smooth Muscles

These experiments were carried out in the presence of indomethacin (3 × 10⁻⁶ M). NA: noradrenaline. Inhibitors: atropine (10⁻⁶ M), phentolamine (10⁻⁶ M), propranolol (10⁻⁶ M), tetrodotoxin (3 × 10⁻⁷ M), bovine serum albumin (0.25%), and anti-foam. w: wash out. PPV: papaverine

In the female genital tract, PAF has been postulated to play a significant role in ovulation, implantation, and childbirth.³⁷⁾ This is evidenced by the following findings. (1) In rats, the uterus during pregnancy exhibits strong contraction in response to PAF with the degree of contraction increasing with gestational age, whereas the uterus in females that are not pregnant produces only a very weak contraction.³⁵⁾ PAF-induced contractions in the uteruses of pregnant rats are highly dependent on extracellular Ca²⁺ influx through VDCCs.³⁵⁾ (2) The concentration of PAF in the uterus of a pregnant rat increases four-fold between days 15 and 21 of gestation, while uterine PAF-AH activity decreases significantly.⁹⁰⁾ (3) Plasma PAF-AH activity decreases during late pregnancy in humans, rabbits, and rats.⁹¹⁾ Estrogen is a key factor that induces these changes (increases/decreases in PAF concentration/PAF-AH activity) in the uterus during pregnancy.^91,92) These findings indicate the role of endogenous PAF as a labor inducer that promotes contraction of the uterus during pregnancy. However, excessive production of PAF may lead to uterine overcontraction, potentially interrupting the maintenance of pregnancy and leading to premature birth. Clinically, plasma PAF levels in women who are pregnant with threatening premature labor are lowered by the combination treatment of atosiban (an oxytocin receptor antagonist) and ritodrine (a β₂-adrenoceptor agonist), which are preterm-birth preventive drugs.³⁸⁾

PAF is also associated with menstrual pain. This is supported by the finding that plasma PAF concentrations are increased in women with primary dysmenorrhea and that this increase is correlated with the severity of the condition.⁴²⁾ PAF-associated menstrual pain may be partly attributed to uterine contractions induced by PAF-produced prostanoids, as well as contractions induced by the direct action of PAF. The possible mediation by prostanoids in PAF uterine activity is supported by the finding obtained in the uterus of non-pregnant GP that prostanoids and leukotrienes mediate PAF-induced rhythmic contractions of the myometrium with increased amplitude.³⁴⁾ Similarly, in the uterus of pregnant GP, PAF increases the production of prostaglandin (PG) F_2α during the second trimester (days 6–10), and those of PGF_2α, PGE₂, and 6-keto-PGF_1α during the third trimester.⁹³⁾ The PAF-induced increase in prostanoid production depends on an increase in intracellular Ca²⁺ but is independent of extracellular Ca²⁺ influx.⁹³⁾ In both women who are pregnant and those who are not, an increase in PAF leads to uterine contractions mediated by increased prostanoid production. However, in patients with dysmenorrhea who do not respond to prostaglandin synthase inhibitors, the degree of disease severity and increase in PAF are correlated with an increase in leukotrienes and not prostanoids.⁴²⁾ Therefore, PAF-associated dysmenorrhea is attributed to uterine contractions caused by leukotrienes, in addition to prostanoids.

7. CONCLUSION

PAF is a key player in the regulation of mechanical activities of various SM tissues. In LUT and genital systems, the mechanical activities of the UBSM and uterine SM increase in response to PAF, and these PAF actions may lead to pathological conditions in the LUT and genital tissues, including OAB, premature birth, and menstrual pain. Inhibitors of PAF synthase and/or antagonists of the PAF receptor may serve as new therapeutic agents to protect against and improve diseases of the LUT and genital tissues.

Acknowledgments

The authors would like to thank Ms. Azusa Murata and Ms. Mio Yamashita for their expert technical assistance.

Conflict of Interest

The authors declare no conflict of interest.

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