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Am J Physiol Gastrointest Liver Physiol 293: G979-G986, 2007. First published August 23, 2007; doi:10.1152/ajpgi.00310.2007
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HORMONES AND SIGNALING

Role of phospholipase A2 (group I secreted) in the genesis of basal tone in the internal anal sphincter smooth muscle

Márcio A. F. de Godoy and Satish Rattan

Department of Medicine, Division of Gastroenterology and Hepatology, Jefferson Medical College of Thomas Jefferson University, Philadelphia, Pennsylvania

Submitted 10 July 2007 ; accepted in final form 22 August 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The role of phospholipase A2 (PLA2) in the genesis of basal tone in the internal anal sphincter (IAS) is not known. We determined the effects of PLA2 and inhibitors on the basal tone and intraluminal pressures (IASP) in the rat IAS vs. rectal smooth muscles (RSM). In addition, we determined the correlations between the IAS tone, PLA2 levels, and the actual enzymatic activity. Inhibition of PLA2 by 4-bromophenacyl bromide (universal inhibitor of PLA2) and MJ33 [selective inhibitor of secreted isoform of PLA2 (sPLA2)] caused concentration-dependent decrease in the IAS tone and in the IASP. Maximal decreases in the IAS tone and IASP by 4-bromophenacyl bromide and MJ33 were 58.8 ± 6.9 and 51.5 ± 6.3%, and 66.7 ± 5.1 and 79.8 ± 8.2%, respectively. The sPLA2 inhibitors were ~100 times more potent in decreasing the IASP than the mean blood pressure. Conversely, the selective inhibitors of the cytosolic and calcium-independent PLA2 arachidonyl trifluoromethyl ketone and bromoenol lactone, respectively, produced no significant effect. The IAS had characteristically higher levels of sPLA2 activity (26.5 ± 4.9 µmol·min–1·ml–1) vs. the RSM (3.2 ± 0.4 µmol·min–1·ml–1), and higher levels of sPLA2 as shown by Western blot and RT-PCR. Interestingly, administration of sPLA2 transformed RSM into the tonic smooth muscle like that of the IAS: it developed basal tone and relaxed in response to the electrical field stimulation. From the present data, we conclude that sPLA2 plays a critical role in the genesis of tone in the IAS. PLA2 inhibitors may provide potential therapeutic target for treating anorectal motility disorders.

smooth muscle tone; rectoanal incontinence

THE INTERNAL ANAL SPHINCTER (IAS) smooth muscle remains in a state of sustained contraction and relaxes to allow the passage of fecal contents during the rectoanal inhibitory reflex. The rectal smooth muscle (RSM), on the contrary, contracts briefly during the reflex. The contractions of these smooth muscles are described as tonic (IAS) and phasic (RSM) and are myogenic in nature (5, 6, 24).

Recent reports comparing the IAS and RSM have shown that the tonic smooth muscle of the IAS expresses significantly higher levels of the renin-angiotensin system (RAS) components and angiotensin II (ANG II) than in the RSM (7–9). In addition, inhibitors of ANG II receptor subtype 1 and ANG II converting enzyme cause a significant fall in the IAS tone. Interestingly, similar data have been obtained in the human lower esophageal sphincter (LES) (3). These data suggest that the RAS pathway may be responsible for ~30% of the basal tone in the IAS. However, factors responsible for the majority of the basal tone in the IAS are not known. Based on the studies of Cao et al. (2, 12) in the LES, we considered it important to determine the role of phospholipase A2 (PLA2) pathway in the IAS tone.

PLA2 plays an important role for a broad range of diverse cellular responses, including digestion and metabolism of membrane phospholipids, signal transduction, and the generation of eicosanoid precursors (27). PLA2 belongs to a large group of enzymes that hydrolyze fatty acids from the sn-2 position of glycerol-based phospholipids. Mammalian cells generally contain more than one PLA2, each of which is regulated independently and exerts a distinct action (19).

PLA2 are generally classified into two groups based on their location in the cell: extracellular or secreted (sPLA2; low molecular mass, 14–29 kDa) (10) and intracellular or cytosolic (cPLA2; high molecular mass, 80–85 kDa) (27). The cPLA2 are further subdivided into two isoforms, the 85-kDa Ca2+ sensitive cPLA2 and the 80-kDa Ca2+-insensitive PLA2 (iPLA2).

In the present studies, we evaluated the role of PLA2 in the genesis of IAS tone via the use of the selective inhibitors of cPLA2 and sPLA2 isoforms. We also examined the ability of PLA2 to generate tone in the RSM. We tested the presence of cPLA2 and sPLA2 in IAS and RSM at the genetic and protein levels. Because the anorectal smooth muscle activities are entirely Ca2+ dependent (5), studies focused on the cPLA2 and sPLA2 isoforms in the IAS vs. RSM. Results showing higher PLA2 levels and activity in the IAS vs. RSM, selective decrease in the IAS tone by the sPLA2 inhibitors, and a number of other systematic studies suggest that sPLA2 plays an important role in the genesis of spontaneous tone in the IAS.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Tissue preparation. Male Sprague-Dawley rats (300–350 g) were killed by decapitation, and IAS and RSM strips were prepared as described before (8). Briefly, circular IAS and RSM strips (~0.5 mm x 7 mm) were prepared in the oxygenated Krebs physiological solution (KPS). The composition of KPS was as follows (in mM): 118.07 NaCl, 4.69 KCl, 2.52 CaCl2, 1.16 MgSO4, 1.01 NaH2PO4, 25 NaHCO3, and 11.10 glucose.

The experimental protocol of the study was approved by the Institutional Animal Care and Use Committee of Thomas Jefferson University in accordance with the recommendations of the American Association for the Accreditation of Laboratory Animal Care.

Measurement of isometric tension. The smooth muscle strips were transferred to 2-ml muscle baths containing oxygenated KPS at 37°C. One end of the smooth muscle strips was anchored at the bottom of the muscle bath, while the other end was connected to a force transducer (model FT03; Grass Instruments, Quincy, MA). Isometric tension was measured by the PowerLab/8SP data-acquisition system and recorded with Chart 4.1.2 (ADInstruments, Australia). Each smooth muscle strip was initially stretched to a tension of 0.7 g, followed by 60 min of equilibration period. During this period, the smooth muscle strips were replenished with fresh KPS every 20 min. The identity of the IAS smooth muscle strips was confirmed by the development of spontaneous tone and relaxation in response to electrical field stimulation (EFS; 0.5–20 Hz, 0.5-ms pulse, 10 V, 4-s train) (23).

Drug responses. We examined the effects of different PLA2 inhibitors: 4-bromophenacyl bromide (BPB); 1-hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol lithium (MJ33), a selective inhibitor of the sPLA2; arachidonyl trifluoromethyl ketone (AACOCF3), a selective inhibitor of the cPLA2; and of bromoenol lactone (BEL), a selective inhibitor of iPLA2 (all 0.01–100 µM). Decreases in the IAS tone were normalized by the 50 mM EDTA responses (percent maximal or 100% decrease), as described previously, at the end of each experiment (8).

To test the ability of PLA2 to generate tone, we examined the effect of exogenously administered sPLA2 (0.0001–1 U/ml) in IAS and RSM strips before and after the inhibitors. To compare results with purely phasic smooth muscle model, we also included anococcygeus smooth muscle (ASM) in these series of experiments. Responses were measured and normalized by the maximal contraction induced by 100 µM bethanechol (8).

PLA2 activity. PLA2 activity in the IAS and RSM was determined using a dithiolester analog of phosphatidylcholine (PC) (diheptanoyl thio-PC substrate) as the substrate. Upon hydrolysis, the thio ester bond at the sn-2 position by PLA2 releases free thiols that can be measured by colorimetric assay using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), as described previously (13). Briefly, IAS and RSM tissues were homogenized in PLA2 assay buffer (25 mM Tris·HCl, pH 7.5, containing 10 mM CaCl2, 100 mM KCl, 0.3 mM Triton X-100, and 1 mg/ml BSA). This assay buffer was also used for reconstitution of substrate and dilution of samples before assaying. A 10 mM DTNB solution was prepared in 0.4 M Tris·HCl (pH 8.0). Following protein determination, the samples were diluted to a final loading of 0.1 µg/µl.

For assaying samples, 10-µl DTNB, 10-µl sample (or 10 µl of assay buffer for blank), and 5-µl assay buffer (when performing inhibitor experiments, 5 µl of inhibitor were added instead of assay buffer) were mixed. Reactions were initiated by adding 200 µl of substrate solution. Absorbance was recorded at 414 nm (A414) at different times. A standard curve for determining the optimum substrate concentrate was obtained using 0.5 µg/ml of PLA2. PLA2 activity was calculated based on the following equation:

Formula
where {Delta}A414 = [A414 (time 2) – A414 (time 1)] ÷ (time 2 – time 1). Here, 10.66 mM–1 represents the DTNB extinction coefficient. Data were expressed as the extinction of micromoles per minute per milliliter of diheptanoyl thio-PC or international units.

Experiments designed to determine the appropriate assay conditions show that the optimum substrate concentration ranges between 1.5 and 2.0 mM. In addition, results also show that time 1 and time 2 are best if read at 30 s and 1 min from the reaction starting point (time 0).

RNA isolation and RT-PCR analysis. Total RNA was isolated from the IAS and RSM, purified by the acid guanidine-phenol-chloroform method (4), and then quantified by measurement of absorbance at 260 nm in a spectrophotometer. Total RNA (2.0 µg) was subjected to first-strand cDNA synthesis using oligo(dT) primers (Promega, Madison, WI) and Omniscript RT Kit (Qiagen, Germantown, MD) in a final volume of 20 µl at 42°C for 60 min. PCR primers specific for cPLA2, sPLA2, and {alpha}-actin cDNA were designed as shown in Table 1.


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  		Table 1. Primers used in RT-PCRs for amplification of mRNA encoding cPLA2, sPLA2, and beta-actin

 
PCR was performed in a Promega 2x master mix (Promega, Madison, WI) in a final volume of 25 µl, using a Perkin-Elmer thermal cycler (PerkinElmer Life and Analytical Sciences). The PCR conditions consisted of 94°C for 2 min, followed by 35 cycles of 94°C for 30 s (denaturation), 59°C for 30 s (annealing), and 72°C for 1 min (extension). In the end, it was allowed a final extension at 72°C for 7 min.

The PCR products were separated on 1.5% (wt/vol) agarose gel containing ethidium bromide and were visualized with UV light. The relative densities of cPLA2 and sPLA2 were calculated by normalizing the integrated optical density (IOD) of each blot with that of {alpha}-actin.

Western blot analysis. Western blot studies were performed to determine the relative distribution of cPLA2 and sPLA2, as described before (9). Briefly, the IAS and RSM tissues were subjected to homogenization and protein extraction and determination by the method of Lowry et al. (16). Protein bands were separated by gel electrophoresis and transferred onto a nitrocellulose membrane (NCM) at 4°C.

Nonspecific binding on the NCM was blocked with nonfat milk (5%) in Tris-buffered saline-Tween [composed of 20 mM Tris, pH 7.6, 137 mM NaCl, and 0.1% Tween 20 (TBS-T)] overnight at 4°C. Then the NCM was incubated with the specific primary antibody (rabbit anti-sPLA2 Ib or rabbit anti cPLA2 at 1:1,000) for 2 h at room temperature. After washing with TBS-T, the NCM was incubated with horseradish peroxidase-labeled secondary antibody (1:10,000) for 1 h at room temperature. The corresponding bands were visualized with enhanced chemiluminescence substrate using the SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL) and Hyperfilm MP (Amersham Bioscience).

NCM was then stripped of antibodies using the Restore Western Blot Stripping Buffer (Pierce) for 10 min at room temperature and then reprobed for {alpha}-actin using the specific primary (mouse IgG 1:10,000 for {alpha}-actin) and secondary (1:10,000) antibodies. Bands corresponding to different proteins were scanned (SnapSacn.310; Agfa, Ridgefield Park, NJ), and the IODs were determined using Image-Pro Plus 4.0. The relative densities were calculated by normalizing the IOD of each blot with that of {alpha}-actin.

Intraluminal manometry of the IAS and mean blood pressure. The high-pressure zone of the IAS was identified using slow station pull-through, and IAS pressures (IASPs) were monitored by the modified approach of de Godoy and Rattan (9) and Terauchi et al. (28). Mean blood pressure (MBP) was recorded via a catheter (inner diameter 0.38 mm; Clay Adams, Parsippany, NJ), attached to a Statham transducer (Medex, Carlsbad, CA), as previously described (9).

Data were collected in the basal state and after administration (intravenous) of BPB and MJ33 in doses ranging from 0.1 to 100 nmol/kg. All recordings and analyses were carried out using Chart 5 PowerLab (ADInstruments). Results were calculated as the percent maximal decrease by 60 µmol/kg sodium nitroprusside.

Drugs and antibodies. AACOCF3 (IC50 = 1–8 µM), BPB, DTNB, sPLA2 Ib (porcine pancreas), and the antibody for {alpha}-actin were from Sigma-Aldrich (St. Louis, MO). Anti-sPLA2 Ib was from Millipore (Billerica, MA). All other antibodies were from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, CA). MJ33 (IC50 = 1–6 µM) was from Axxora (Axxora Life Sciences, San Diego, CA). Diheptanoyl thio-PC and BEL (EC50 = 1–5 µM) were from Cayman (Cayman Chemical, Ann Arbor, MI).

Data analysis. Results are expressed as means ± SE. Concentration-response curves were analyzed using a nonlinear interactive fitting program (GraphPad Prism 3.0, Graph Pad Software, San Diego, CA). Inhibitor potencies and maximum responses were expressed as pIC50 (negative logarithm of the molar concentration of inhibitor producing 50% of the maximal effect) and Imax (maximum inhibition). For in vivo studies, ID50 (the dose that causes 50% inhibition of the IAS tone) of PLA2 inhibitors was also determined.

PLA2 potency and maximum response were expressed as pEC50 (negative logarithm of the molar concentration producing 50% of the maximum) and Emax (maximum effect).

Statistical significance was tested by one-way ANOVA, followed by the Dunnett post hoc test when three or more different groups were compared. To compare only two different groups, the unpaired Student t-test was used. A P value <0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of PLA2 inhibitors on the basal IAS tone. To evaluate the role of the different PLA2 isoforms in the basal IAS tone, we first determined the effects of selective and nonselective inhibitors of PLA2. The nonselective PLA2 inhibitor BPB produced a significant and concentration-dependent decrease in IAS basal tone (Imax of 60.6 ± 4.0%; pIC50 of 5.4 ± 0.2; n = 5; Fig. 1A). The selective inhibitor of the sPLA2 isoform MJ33 produced a similar effect in the IAS (Imax = 53.1 ± 2.1%; pIC50 = 4.9 ± 0.1, n = 5). However, the selective inhibitors of cPLA2 and iPLA2, AACOCF3 and BEL, respectively, produced no significant effect (P > 0.05; n = 5). Actual tracings of the effect of BPB and MJ33 are shown in Fig. 1, B and C. These data suggest that, among different isoforms, sPLA2 plays a major role in the IAS tone.


Figure 1
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  		Fig. 1. A: cumulative concentration-response curves (CRC) for selective and nonselective inhibitors of phospholipase A2 (PLA2) in the basal tone of the rat internal anal sphincter (IAS). Among different inhibitors examined, only 1-hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol lithium (MJ33) and 4-bromophenacyl bromide (BPB) decrease the IAS tone. AACOCF3, arachidonyl trifluoromethyl ketone; BEL, bromoenol lactone. Data represent the means ± SE of 5 independent determinations. This suggests sPLA2 is the primary PLA2 isoform responsible for tone maintenance in the rat IAS. B and C: representative tracings on the effects of the BPB and MJ33, respectively, on the IAS tone.

 
In contrast to the IAS, none of the PLA2 inhibitors examined in concentration range from 10–7 M to 10–4 M produced any significant effect in the RSM (data not shown).

Effect of exogenous sPLA2 on the RSM: influence of PLA2 inhibitors. Exogenous administration of active sPLA2 Ib from porcine pancreas produced a significant (P < 0.05) and concentration-dependent increase in the force of IAS (Emax = 0.015 ± 0.002 g/mg; pIC50 of 2.3 ± 0.1; n = 5) and RSM (Emax = 0.013 ± 0.004 g/mg; pIC50 of 2.0 ± 0.1; n = 4) in the basal state, as shown in Fig. 2, A and B. This effect of sPLA2 Ib was significantly (P < 0.05) inhibited by BPB and MJ33. Exogenous sPLA2 Ib, however, produced no significant effect in the ASM.


Figure 2
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  		Fig. 2. Typical tracings of the effects of sPLA2 (Ib) in the phasic RSM (A) and tonic IAS smooth muscles (B). Exogenous sPLA2 causes increase in the tonic activity of the RSM. Not shown, exogenous sPLA2 produces no significant effect in the anococcygeus smooth muscle (ASM). A and B: quantitative data on the right sides show that BPB and MJ33 (not AACOCF3 and BEL) inhibit contractions by sPLA2 in the RSM and IAS, respectively. These results denote the specificity of the effects of both sPLA2 and MJ33. Data represent the means ± SE of 4 independent determinations.

 
Data suggest that exogenous sPLA2 Ib selectively converts RSM into a tonic smooth muscle comparable to the IAS. To evaluate it further, we compared the effect of EFS on sPLA2 Ib (1 U/ml) increased force in the IAS vs. RSM and ASM. The experiments revealed that, in the presence of sPLA2 Ib, EFS caused relaxation of the RSM (somewhat similar to the IAS). For control to prove the selectivity of sPLA2 Ib, we examined the effects of sPLA2 Ib on the ASM. As shown, the sPLA2 had no significant effect on the ASM (Fig. 3).


Figure 3
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  		Fig. 3. Top: control force in the IAS [basal tone and relaxation with electrical field stimulation (EFS); A], RSM (no tone and contraction with EFS; B), and ASM (no activity and frank contraction with EFS; C), and their responses to EFS. Bottom: responses of these respective tissues to EFS in the presence of sPLA2 (1 U/ml). A: in the IAS, sPLA2 increases tone that relaxes normally in response to EFS. B: in the RSM, sPLA2 causes the development of tone that undergoes relaxation following EFS. C: in contrast, in the ASM, sPLA2 produces no tone, and there is frank contraction in response to EFS similar to the control. Based on this data, we suggest that sPLA2 has the ability to generate tone in selected smooth muscle.

 
These data suggest that the basal tone in the IAS may depend on the higher levels and activity of sPLA2. Data also suggest the tissue selectivity of sPLA2, because the phenomenon cannot be duplicated in the adjoining phasic smooth muscle of the ASM, which lacks the basic machinery for the generation of tone (20). This discriminative phenomenon has important implications in the genesis of the basal tone, as discussed below.

PLA2 activity in the IAS vs. RSM in the basal state and following PLA2 inhibitors. For the specific determinations of sPLA2 activity, we used a novel reaction approach that measured the effect of sPLA2 (Ib) at the sn-2 position of glycerophospholipids. This reaction provides precursors for the generation of eicosanoids, with important biological activity in gastrointestinal smooth muscles (2, 13, 21). Data show that sPLA2 activity in the IAS is significantly higher than in the RSM. One microgram of IAS sPLA2 metabolizes 26.5 ± 4.9 µmol·min–1·ml–1 of diheptanoyl thio-PC vs. 3.2 ± 0.4 µmol·min–1·ml–1 in the case of RSM (*P < 0.05; n = 4; Fig. 4, A and B). Incubation with BPB and MJ33 specifically and significantly decreased the activity of sPLA2 in the IAS (*P < 0.05; n = 4; Fig. 4A), but not in the RSM (P > 0.05; Fig. 4B).


Figure 4
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  		Fig. 4. Comparison of PLA2 activity in the IAS (A) vs. the RSM (B) reveals higher levels of the activity in the IAS in the basal state (*P < 0.05; ANOVA). The assay was optimized as described in MATERIALS AND METHODS. Only MJ33 (a selective sPLA2 inhibitor) and BPB (nonselective PLA2 inhibitor) produce a significant (*P < 0.05; ANOVA) decrease in PLA2 activity in the IAS. Based on this data, we suggest the PLA2 activity in the IAS is primarily because of the sPLA2.

 
Results show that the IAS sPLA2 is more effective than the RSM sPLA2 in converting membrane phospholipids. The specificity of the activity assay was further confirmed by the lack of significant effect of other c- and iPLA2 inhibitors on the basal PLA2 activity of the IAS.

RT-PCR analyses of sPLA2 and cPLA2 in the IAS vs. RSM. Total RNA from the IAS and RSM extracted by the acid guanidine-phenol-chloroform method (13) was reversely transcribed into cDNA and then amplified by PCR using specific primers for the sPLA2 and cPLA2 (described in Table 1). RT-PCR products were separated by electrophoresis in 1.5% agarose gel, and the relative density was calculated.

The IAS expressed significantly (P < 0.05; n = 3) higher levels of sPLA2 as well as cPLA2 in IAS vs. the RSM at the transcriptional level (shown by the RT-PCR analysis; Fig. 5, A and C). These data suggest the role of sPLA2 in the IAS vs. the RSM in converting membrane phospholipids and that the differences in sPLA2 expression are regulated at the transcriptional level. The significance of higher levels of cPLA2 at the present time is not known.


Figure 5
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  		Fig. 5. RT-PCR (A and C) and Western blot analyses (B and D) demonstrate higher levels of sPLA2 (Ib) in the IAS vs. the RSM (*P < 0.05; n = 3). cPLA2, cytosolic PLA2.

 
Western blot analyses of sPLA2 and cPLA2 in the IAS vs. RSM. We evaluated the presence of sPLA2 and cPLA2 in protein extracts from IAS and RSM. After separation by gel electrophoresis, protein bands specific for the sPLA2 and the cPLA2 were identified by specific antibodies, revealed by chemiluminescence, and recorded in X-ray films.

The levels of the sPLA2 and cPLA2 in the IAS were significantly (P < 0.05) higher than in the RSM (*P < 0.05; n = 3; Fig. 5, B and D). These data suggest that expression of both sPLA2 and cPLA2 is also regulated at the translational level. The significance of higher levels of cPLA2 is not known at the present time.

Effect of BPB on IASP and MBP in vivo studies. BPB and MJ33 decreased the IASP (Imax = 66.7 ± 5.1 and 79.8 ± 8.2%, respectively) in a dose-dependent manner. In this regard, MJ33 (ID50 = 0.55 nmol/kg, n = 4) was significantly (P < 0.05) more potent than BPB (ID50 = 2 nmol/kg, n = 5). In decreasing the IASP, these agents were ~100 times more potent than in decreasing the MBP (Fig. 6; n = 5). BPB at 0.5 nmol/kg caused a 25 ± 4.2% decrease in IASP (n = 5) without having any significant decrease in MBP (P > 0.05; 0.7 ± 0.7%; n = 5). Similarly, MJ33 at 0.3 nmol/kg caused a 22.4 ± 5.1% decrease in IASP (n = 4) without affecting the MBP (P > 0.05; 0.7 ± 0.7%; Fig. 6; n = 4). Even if compared at the 10 nmol/kg maximal dose, BPB and MJ33 had limited effects on the MBP (Imax 28.6 ± 8.5 and 36.2 ± 5.2%, respectively; n = 5).


Figure 6
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  		Fig. 6. A: representative tracings of the effects of different doses of MJ33 in the IAS pressure (IASP). B: comparison of the dose-response curves of MJ33 and BPB in causing fall in the IASP and mean blood pressure (MBP) in in vivo studies. Data show that, in the lower doses (1 nmol/kg), inhibitors of the sPLA2 cause a significant decrease in the IASP (P < 0.05; n = 5) without significant effect on the MBP.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The IAS plays a major role in the anorectal continence and in the pathophysiology of incontinence, constipation, Hirschsprung's disease, anal fissures, and hemorrhoids (14, 15, 17, 18, 22, 26). Knowledge of the molecular mechanisms regulating the IAS tone may hold the key in the therapeutic management of such motility disorders. Using a multipronged approach of in vivo, in vitro, and molecular biology, present studies for the first time show that membranous sPLA2 plays a major upstream role for the basal tone in the IAS smooth muscle.

sPLA2 is secreted to the external side of the plasma membrane of mammalian cells (10, 19). In the plasma membrane, PLA2 converts glycerol-based membrane phospholipids into metabolites (e.g., thromboxanes and PGF2{alpha}) with potent contractile activity in smooth muscles via G protein-coupled receptor activation (2, 12, 27). In turn, downstream molecular mechanism events, such as RhoA/Rho kinase and PKC, may determine the basal tone in the IAS.

Data show that spontaneous tone in the IAS is directly related to the higher levels and activity of sPLA2. Experiments designed to determine the specific molecular nature of PLA2 (10) revealed the presence of both low molecular mass 14-kDa (sPLA2) and high molecular mass 85-kDa (cPLA2) isoforms in the IAS and the RSM. The IAS expresses significantly higher levels of cPLA2 and sPLA2 than the RSM, both at the translational and message levels, as determined by Western blot and RT-PCR analyses (Fig. 5). However, based on the functional data (discussed below), we speculate that sPLA2 (specifically group I secreted form) is important for the maintenance of IAS tone.

The IAS has characteristically approximately eightfold higher sPLA2 activity compared with the RSM (Fig. 4). Accordingly, in contrast to the potent inhibitory effect of sPLA2-selective inhibitor MJ33, the cPLA2 and iPLA2 inhibitors AACOCF3 and BEL produce no significant effect on the PLA2 activity and in the basal tone in the IAS. sPLA2 activity in the IAS and RSM was determined using diheptanoyl thio-PC substrate (13). The method offers particular advantage because it measures the PLA2 acting at the sn-2 position of glycerophospholipids. Catalysis at the sn-2 position provides precursors for the generation of eicosanoids, with important biological activity in gastrointestinal smooth muscles (2, 21).

In the IAS, the selective and nonselective inhibitors of PLA2, MJ33 and BPB, respectively, produce a significant and concentration-dependent decrease in the basal tone, with similar maximal effects of ~60% (Fig. 1). Data with the use of selective inhibitors suggest that intracellular high molecular mass PLA2 isoforms (iPLA2 and cPLA2) may not be important in the genesis of IAS tone, as their selective inhibitors produce no significant effect on the IAS tone (Fig. 1A). These data are in agreement with previous studies by Cao et al. on the LES of cats (2) and humans (1) and suggest that development of tone in IAS partly depends on the activity of the low molecular mass sPLA2. Simultaneous measurements of basal tone and sPLA2 activity in the present study suggest that the inhibitory effects of BPB on the basal tone in the IAS are because of sPLA2 inhibition.

Because of the minimal basal tone in the RSM (with the presence of low levels of intracellular molecular signal transduction machinery vs. the IAS), we explored the possibility of RSM (24) as a model for the tone generation by the exogenous sPLA2. Interestingly, active sPLA2 produces concentration-dependent tone in the RSM and an increase in the basal tone in the IAS that is selectively inhibited by BPB and MJ33 (Figs. 2 and 3). On the other hand, exogenous sPLA2 produces no significant effect on the purely phasic ASM. A possible explanation for the diverse effects by sPLA2 in the partially tonic vs. purely phasic smooth muscles is explained as follows.

While ASM is basically devoid of certain intracellular signal transduction proteins known to be critical for the genesis of myogenic tone, the IAS has high levels, and RSM only has modest levels (20). Data suggest that sPLA2 provides an important trigger to functionally upregulate otherwise lower levels of intracellular molecular signal transduction machinery in the RSM to the levels that are capable of producing sustained contraction. Because of the virtual absence of such machinery in the ASM, this phenomenon cannot be demonstrated in the ASM.

It is well known that the IAS smooth muscle relaxes in response to EFS because of nonadrenergic noncholinergic relaxation, and such relaxation in the RSM is depicted primarily in the form of decrease in the ongoing phasic activity (11, 25). An active relaxation in the RSM may be difficult to demonstrate because of the virtual absence of an active tone. Effects of sPLAs in the RSM offer a unique opportunity to test whether an increase in the tone in the RSM will allow the expression of nonadrenergic noncholinergic relaxation and development of true tone in this smooth muscle. Data reveal that, under control conditions, RSM contracts in response to EFS. Conversely, following treatment with sPLA2, because of the generated tone, the RSM relaxes in response to EFS. This suggests that sPLA2 causes the development of true tone in the RSM (Fig. 3B). This effect appears to be selective, because exogenous sPLA2 does not modify either the relaxation in the IAS (Fig. 3A) or contraction in the ASM caused by EFS (Fig. 3C).

Earlier experiments in the IAS have shown that the local synthesis of ANG II (by the RAS) and subsequent activation of ANG II receptor subtype 1 in the smooth muscle cells are important for ~30% of the basal tone (7–9). The present studies show that sPLA2 (most likely via the production of prostanoids) accounts for ~60% of the IAS basal tone. Relative contribution by sPLA2 and RAS and the sPLA2 pathways and the nature of interaction between them, if any (29, 30), remain to be determined.

To examine the clinical relevance of sPLA2, we monitored the IASP and the MBP before and after the administration of the PLA2 inhibitors BPB and MJ33. Data show that both inhibitors are significantly more effective in decreasing the IASP than the MBP (Fig. 6). The selective cPLA2 and iPLA2 inhibitors AACOCF3 and BEL did not produce any significant effect (data not shown). BPB and MJ33 are ~100-fold more potent in producing a 25% reduction (ID25) in the IASP than the corresponding decrease in MBP (Fig. 6B). The ID25 was used for these calculations because this was the maximal observed decrease in MBP with different inhibitors used in the study. In addition, selective inhibitor of sPLA2 MJ33 is more potent than the nonselective sPLA2 inhibitor BPB in decreasing the IASP.

In conclusion, the basal tone in the IAS and possibly in the other tonic smooth muscles is highly dependent on the activity of sPLA2. The efficacy and potency of sPLA2 inhibitors in causing decrease in IASP with minimal cardiovascular effects suggest therapeutic potential of such agents in treating certain motility disorders associated with the hypotensive IAS. Selective sPLA2 agonists, on the other hand, may have a therapeutic potential in the hypotensive IAS.


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The work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-35385 and an institutional grant from Thomas Jefferson University.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Rattan, Dept. of Medicine, Division of Gastroenterology and Hepatology, Thomas Jefferson Univ., 1025 Walnut St., Rm. #901 College, Philadelphia, PA 19107 (e-mail: Satish.Rattan{at}Jefferson.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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