Mechanism of internal anal sphincter smooth muscle relaxation by phorbol 12,13-dibutyrate

Sushanta Chakder, D. N. K. Sarma, and Satish Rattan

Department of Medicine, Division of Gastroenterology and Hepatology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We investigated the mechanism of the inhibitory action of phorbol 12,13-dibutyrate (PDBu), one of the typical protein kinase C (PKC) activators, in in vitro smooth muscle strips and in isolated smooth muscle cells of the opossum internal anal sphincter (IAS). The inhibitory action of PDBu on IAS smooth muscle (observed in the presence of guanethidine + atropine) was partly attenuated by tetrodotoxin, suggesting that a part of the inhibitory action of PDBu is via the nonadrenergic, noncholinergic neurons. A major part of the action of PDBu in IAS smooth muscle was, however, via its direct action at the smooth muscle cells, accompanied by a decrease in free intracellular Ca2+ concentration ([Ca2+]i) and inhibition of PKC translocation. PDBu-induced IAS smooth muscle relaxation was unaffected by agents that block Ca2+ mobilization and Na+-K+-ATPase. The PDBu-induced fall in basal IAS smooth muscle tone and [Ca2+]i resembled that induced by the Ca2+ channel blocker nifedipine and were reversed specifically by the Ca2+ channel activator BAY K 8644. We speculate that a major component of the relaxant action of PDBu in IAS smooth muscle is caused by the inhibition of Ca2+ influx and of PKC translocation to the membrane. The specific role of PKC downregulation and other factors in the phorbol ester-mediated fall in basal IAS smooth muscle tone remain to be determined.

smooth muscle tone; smooth muscle cells; phorbol esters; calcium influx; calcium channel activator


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE BASAL TONE in internal anal sphincter (IAS) smooth muscle plays a major role in anorectal continence. IAS smooth muscle tone has been suggested to be primarily myogenic in nature (7, 33). However, the cellular mechanism(s) governing the distinct myogenic properties of IAS smooth muscle cells (SMC) has not been identified. Knowledge of the intracellular mechanism responsible for basal tone in the IAS is critical to understanding the pathophysiology of anorectal continence and incontinence. In the lower esophageal sphincter (LES), a majority of the basal tone has also been suggested to be myogenic in nature (10), although other neurohumoral factors may contribute to basal tone in intact humans and animals (2, 11).

In both the LES (13, 35) and the IAS (4), it has been suggested that the major determinants of the basal tone in the sphincteric smooth muscles are protein kinase C (PKC) activation and higher levels of free intracellular Ca2+ concentration ([Ca2+]i). The higher levels of [Ca2+]i are considered to be under the control of the inositol trisphosphate (IP3) pathway. Investigations were carried out to examine the effects of the typical PKC activators phorbol esters and diacylglycerol (DAG) analogs in smooth muscle strips and in isolated SMC of the IAS. The effects of the classic PKC activators phorbol esters and DAG analogs on isolated smooth muscle preparations that are spontaneously tonic have not been examined previously.

Surprisingly, in our studies, the PKC stimulators, rather than producing the expected contraction, caused a concentration-dependent fall in basal tone of IAS smooth muscle. The purpose of the present investigation, therefore, was to determine the site and mechanism of action of the smooth muscle relaxation caused by phorbol esters.

A number of possibilities exist for the phorbol ester-induced fall in basal tone in IAS smooth muscle. We focused primarily on identifying the site (neural vs. smooth muscle) and the role of changes in [Ca2+]i in IAS smooth muscle relaxation by phorbol esters. The higher levels of [Ca2+]i in sphincteric smooth muscle may be caused by either the intracellular mobilization of Ca2+ by IP3 or the influx of Ca2+. To determine the role of intracellular mobilization versus the influx of Ca2+, we investigated the effects of the Ca2+-ATPase inhibitor thapsigargin (37), the L-type Ca2+ blocker nifedipine, and a Ca2+ channel activator BAY K 8644 (16). Direct measurements of [Ca2+]i levels were also made. In addition, we examined the role of Na+-K+-ATPase, which may play a critical role in the maintenance of ionic gradients across the membrane (31), by the use of the Na+-K+-ATPase inhibitor ouabain. Phorbol 12,13-dibutyrate (PDBu) was used as a prototype for the detailed analysis of the actions of phorbol esters in IAS smooth muscle.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation of smooth muscle strips. Adult opossums (Didelphis virginiana) of either sex weighing 2.6-4.5 kg were killed by exsanguination after pentobarbital sodium anesthesia (40-50 mg/kg ip). The anal canal was dissected out of the animals and transferred immediately into oxygenated (95% O2-5% CO2) Krebs solution (pH 7.4). The composition of the Krebs solution was as follows (mM): 118.07 NaCl, 4.69 KCl, 2.52 CaCl2, 1.16 MgSO4, 1.01 NaH2PO4, 25 NaHCO3, and 11.10 glucose. The anal canal was cleaned of extraneous connective tissue, blood vessels, and adjoining skeletal muscles and opened flat by an incision along the longitudinal axis while placed on a dissecting tray containing oxygenated Krebs buffer. The tissue was then pinned flat, the mucosa and submucosa were removed by a sharp dissection, and circular smooth muscle strips (~1 × 10 mm) of the IAS were prepared. Initially, three IAS smooth muscle strips were obtained along the entire circumference of the anal canal, which then were divided into six smooth muscle strips. The smooth muscle strips were tied at both ends with silk sutures (5-0; Ethicon, Somerville, NJ) for measurements of isometric tension.

For some studies, smooth muscle strips from the carotid arteries of the opossums were also prepared as explained elsewhere (34). This was done to directly compare the effects of PKC activators and inhibitors on different types of smooth muscle that are basically nontonic and are well known to produce the established changes in muscle tension in response to PKC activators, i.e., contraction.

Measurement of isometric tension. The smooth muscle strips were transferred to 2-ml muscle baths containing oxygenated Krebs solution maintained at 37°C. The Krebs solution was oxygenated continually by bubbling with a 95% O2-5% CO2 mixture. One end of the muscle strip was fixed at the bottom of the muscle bath with the help of a tissue holder, and the other end was attached to an isometric force transducer (model FT03; Grass Instruments, Quincy, MA) for the measurement of isometric tension. The tensions of the smooth muscle strips at rest and in response to different stimuli were recorded on a Dynograph recorder (model R411; Beckman Instruments, Schiller Park, IL). At the start, the smooth muscle strips were stretched at 10-mN tension and allowed to equilibrate for at least 1 h with intermittent washings. During this period of equilibration, the muscle strips developed additional steady-state tension. Baseline and optimal length (Lo) of the smooth muscle strips were determined as described previously (19). In the case of IAS, only the smooth muscle strips that developed steady tension and relaxed in response to electrical field stimulation (EFS) were considered sphincteric and were included in the study.

Nonadrenergic, noncholinergic nerve stimulation with EFS. EFS was delivered from a Grass stimulator (model S88; Grass Instruments) connected in series to a Med-Lab Stimu-Splitter II (Med-Lab Instruments, Loveland, CO). The Stimu-Splitter was used to amplify and measure the stimulus intensity by using the optimal stimulus parameters for neural stimulation (12 V, 0.5-ms pulse duration, 200-400 mA, 4-s train) at varying frequencies of 0.5-20 Hz. The electrodes used for EFS were a pair of platinum wires fixed at both sides of the smooth muscle strip. Neurally mediated relaxation of the IAS smooth muscle strips was quantified in response to different frequencies of EFS. The parameters of EFS used cause relaxation of IAS smooth muscle via the activation of nonadrenergic, noncholinergic (NANC) myenteric neurons because the relaxation was maintained in the presence of adrenergic and cholinergic blockade by guanethidine plus atropine (25).

Drug responses. Responses to different agonists on the basal tension of the tissues were examined using cumulative concentrations or single injections. After a drug concentration was applied, the response was allowed to stabilize before the next higher concentration was applied. The exposure time of the tissues to an agent was determined by the stabilization of response to the previous concentration. Otherwise, the drugs were in contact with the tissues for a minimum of 5 min. In our earlier studies, because of the unexpected effects of the phorbol esters, we closely monitored the effects of PDBu in different concentrations for up to 30 min. At no time was an effect other than the relaxation of the IAS smooth muscle by PDBu observed. After the maximal effect of the agonist was achieved, the smooth muscle strips were washed for at least 1 h before the next agonist was added. To examine the effects of the antagonists, the antagonists were added 10-20 min before the concentration-response curve to the test agonist was examined. All experiments were performed in the presence of guanethidine (3 × 10-6 M) plus atropine (1 × 10-6 M).

At the end of each experiment, the smooth muscle strips were treated with 3 × 10-5 M phenylephrine or 1 × 10-4 M bethanechol followed by 10 mM EGTA to determine the maximum contraction and relaxation of the smooth muscles. These responses to phenylephrine or bethanechol and EGTA formed the bases for the calculation of contraction and relaxation, respectively, of the smooth muscles on a percentile basis. Responses to 10 mM EGTA also were used to establish the actual baseline and the maximal relaxation (3). Each smooth muscle strip served as its own control in all of these experiments.

Isolation of SMC. SMC from the circular smooth muscle layer of the opossum IAS were isolated as described previously (5). Briefly, after the IAS was isolated, the mucosal and longitudinal muscle layers were removed from the circular layer by sharp dissection. The circular muscle layer was cut into small pieces and incubated at 31°C in Krebs buffer (pH 7.4) containing 0.1% collagenase (Worthington Biochemicals), soybean trypsin inhibitor (0.01%), and mixtures of amino acids and vitamins. After the first incubation of 45 min, the solution was replaced with fresh enzyme solution and the incubation continued for another 45 min. The incubation medium was filtered through a Nitex mesh (500 µm), and the tissues were washed in enzyme-free Krebs buffer. The partly digested tissues then were incubated in oxygenated Krebs buffer at 31°C for 30 min. The SMC dispersed spontaneously in the buffer and were isolated by filtration.

Measurement of length of IAS SMC isolated from circular smooth muscle layer. The length of the individual SMC was measured by using phase-contrast microscopy and a personal computer with an image analysis system (Image Pro Plus 4.0; Media Cybernetics, Silver Spring, MD). SMC were treated with different concentrations of PDBu for 60 s and then fixed with acrolein (1% final concentration). For the measurement of relaxation, SMC first were treated with different concentrations of PDBu for 60 s and then were treated with bethanechol (1 × 10-6 M) for 30 s, at which time the cells were fixed with acrolein. Relaxation was expressed as the percent inhibition of bethanechol-induced maximal shortening of the SMC. In each of these experimental protocols, data from 25-30 SMC were averaged before and after the corresponding treatments in each animal. Final data were calculated (means ± SE) from four animals.

Measurement of [Ca2+]i levels of IAS smooth muscle strips. [Ca2+]i levels of the IAS smooth muscle strips were measured with a JASCO calcium analyzer (model CAF-100; JASCO, Easton, MD) using fura 2-AM as the fluorescent indicator. The smooth muscle strips were treated with fura 2 according to the method of Tognarini and Moulds (38) with some modifications for our application. Briefly, Krebs solution containing 2.5% BSA, 0.5% cremophor EL, and fura 2-AM was mixed vigorously. Cremophor EL is a high-molecular-weight surfactant that facilitates the tissue uptake of fura 2-AM. Fura 2-AM is a cell-permeant acetoxymethyl ester form of fura 2. The tissues were loaded with fura 2-AM in this cocktail in the dark at room temperature (24-26°C) for 2-3 h. Fura 2-AM was dissolved previously in 40 µl of 15 µM DMSO. After being loaded with fura 2, the tissues were washed frequently with Krebs solution for 30 min to remove any extracellular fura 2-AM until the fluorescence intensity readings at 340 and 380 nm (F340 and F380) were stable. The [Ca2+]i levels of the tissues were determined from the F340 and F380 readings as described by Grynkiewicz et al. (12). Minimum and maximum fluorescence ratios were determined by treating the tissues with EGTA (3 mM) and Triton X-100 (0.4%), respectively, at the end of each experiment, and the dissociation constant of fura 2 in the physiological solution was considered to be 224.

Smooth muscle strips prepared as described above were transferred in the horizontal position to thermostatically controlled 2-ml muscle baths (37°C) containing Krebs solution bubbled with 95% O2-5% CO2. The muscle bath was a part of the equipment specially designed (CB-01; JASCO) for the purpose of simultaneous recordings of changes in [Ca2+]i and isometric tension in the basal state and in the presence of different stimuli. One end of the muscle strip was fixed to the bottom of the muscle bath with a tissue holder, and the other end was attached to a isometric force transducer (model FT03; Grass Instruments) via a specially designed pulley system for the simultaneous measurement of [Ca2+]i and isometric tension. The muscle strips were adjusted to lie exactly in the path of the optical axis, and the Lo and baseline of the smooth muscle strips were determined as described previously (19).

PKC activity assay. PKC activity in the circular smooth muscle of the IAS in the basal state and after PDBu was determined by using standard PKC enzyme assay system RPN 77 (Amersham, Arlington Heights, IL). The PKC activity assay method described by the supplier, essentially derived from Parker et al. (22), was followed for the extraction and PKC assay procedures. Untreated smooth muscle strips (basal state) and strips treated with a single maximal effective concentration of PDBu (1 × 10-5 M) were quickly frozen at the point of maximal relaxation, using liquid N2-precooled forceps for the purpose. PKC activity in the cytosolic as well as the particulate fractions of the IAS and the rectal smooth muscles was expressed in picomoles per minute per milligram of protein. The protein contents of the extracts were determined by the method of Lowry et al. (17), using BSA as the standard.

Drugs and chemicals. Fura 2-AM and fura 2 were obtained from Molecular Probes (Eugene, OR). Cremophor EL, bethanechol, BSA, 12-deoxyphorbol 13-tetradecanoate (DPT), DMSO, 1,2 dioctanoyl-sn-glycerol, EGTA, ionomycin, Triton X-1000, NG-nitro-L-arginine (L-NNA), 1-oleolyl-2-acetyl-sn-glycerol (OAG), tetrodotoxin (TTX), and omega -conotoxin GVIA were from Sigma (St. Louis, MO). BAY K 8644, nifedipine, ouabain, phorbol 12-myristate 13-acetate (PMA), PDBu, thapsigargin, ouabain, and calphostin C were from RBI (Natick, MA). All chemicals were of the highest purity available. Whenever possible, solutions of all of the chemicals were prepared fresh in Krebs solution on the day of the experiment; otherwise, they were dissolved in minimum amounts of DMSO (BAY K 8644, DAG analogs, nifedipine, ouabain, phorbols, thapsigargin, and calphostin C) and diluted in Krebs solution. The initial stock solution of L-NNA (1 × 10-2 M) was prepared by adding 10 µl of 1 N HCl to a 1-ml suspension of L-NNA in physiological saline, and the subsequent dilutions were made with physiological saline. The effect of the solvent in the concentrations used to attain the final concentrations of the respective agent in the muscle bath was used routinely to determine the nonspecific action of the solvent. Such concentrations of the solvent produced no effect on the basal tone of the IAS smooth muscle in any experiment.

Data analysis. [Ca2+]i values were calculated as described in Measurement of [Ca2+]i levels of IAS smooth muscle strips and were expressed as nanomolar. The relative changes in response to different agonists were found to be reproducible. The responses of basal IAS tension to EFS and the other relaxants were expressed as the percentage of maximal relaxation by 10 mM EGTA, and the contractile responses were expressed as the percentage of maximal contraction by ionomycin (2 × 10-5 M), phenylephine (3 × 10-5 M), or bethanechol (3 × 10-4 M), as the case may be.

The results are expressed as means ± SE. The statistical significance between different groups was determined by ANOVA and by paired or unpaired t-test. A P value <0.05 was considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of different phorbol esters and DAG analogs on basal tension of IAS smooth muscle. Of the three phorbol esters tested, PDBu was found to be the most potent, followed by PMA and DPT in causing IAS smooth muscle relaxation (Fig. 1). PDBu in concentrations ranging from 1 × 10-8 to 3 × 10-5 M caused falls ranging from 8.7 ± 3.07% to 76.4 ± 4.7% in the basal tension of the IAS. PMA at the same concentrations caused falls in the basal IAS tension ranging from 5.0 ± 3.2% to 36.4 ± 4.2%. The third phorbol ester, DPT, was less potent, causing only a 16.0 ± 4.9% fall in the basal IAS tension at a concentration of 1 × 10-5 M. OAG had no significant effect, whereas 1,2-dioctanoyl glycerol caused a modest fall in basal IAS smooth muscle tension (Fig. 1).


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Fig. 1.   Concentration-response curves showing percent fall (means ± SE; n = 5-8) in basal internal anal sphincter (IAS) smooth muscle tension by phorbol esters phorbol 12,13-dibutyrate (PDBu), phorbol 12-myristate 13-acetate (PMA), and 12-deoxyphorbol 13-tetradecanoate (DPT) and diacylglycerol (DAG) analogs 1,2 dioctanoyl-sn-glycerol (DOG) and 1 oleolyl-2- acetyl-sn-glycerol (OAG). Note that PDBu, PMA, DPT, and OAG cause a concentration-dependent fall in basal IAS tone, and DOG has a limited effect on IAS smooth muscle. None of these agents causes a contraction of IAS smooth muscle.

Effect of neurotoxin TTX, neuronal Ca2+ channel blocker omega -conotoxin, or nitric oxide synthase inhibitor L-NNA on PDBu-induced relaxation of IAS smooth muscle. TTX, omega -conotoxin, and L-NNA were used at concentrations of 1 × 10-6, 1 × 10-6, and 3 × 10-5 M, respectively, which were shown previously to be effective in inhibiting NANC relaxations in the IAS smooth muscle and other systems. TTX caused a significant but only partial inhibition of the fall of the IAS tension caused by PDBu (P > 0.05; n = 5-8; Fig. 2).


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Fig. 2.   Influence of the neurotoxin tetrodotoxin (TTX), the neuronal Ca2+ channel blocker omega -conotoxin, and the nitric oxide synthase inhibitor NG-nitro-L-arginine (L-NNA) on the fall in basal IAS tension caused by PDBu. Note that TTX causes only a partial but significant attenuation of the PDBu-induced fall in basal tension of the IAS (*P < 0.05; n = 5). The NOS inhibitor L-NNA and omega -conotoxin, on the other hand, have no significant effect on the PDBu-induced fall in basal tension of IAS smooth muscle (P > 0.05; n = 5). * Statistically significant differences between the entire curves obtained during control vs. a specific treatment in this figure and in Figs. 3 and 5-7.

The overall comparison of the concentration-response curves obtained with PDBu before and after omega -conotoxin and L-NNA revealed no significant differences (P > 0.05; n = 5-8; Fig. 2). TTX, omega -conotoxin, or L-NNA caused no significant effect on basal tone of IAS smooth muscle. The inhibitory effects of PDBu on IAS smooth muscle were not affected by the adrenergic and cholinergic blocking agents.

Actions of PDBu in opossum IAS vs. carotid artery smooth muscle and influence of PKC inhibitor calphostin C. In a separate series of experiments, the effects of PDBu in IAS and carotid artery smooth muscle were compared before and after calphostin C. In the opossum carotid artery smooth muscle, but not in IAS smooth muscle, PDBu caused a concentration-dependent contraction that was inhibited by calphostin C (1 × 10-7 M) (P < 0.05; n = 5; Fig. 3B). Calphostin C, on the other hand, had no significant effect on PDBu-induced relaxation of IAS smooth muscle (P > 0.05; n = 5; Fig. 3A). The main objective of the carotid artery smooth muscle experiments was to rule out the possibility of differences in the handling of tissues in our experiments that might explain the unusual inhibitory effects of phorbol esters on IAS smooth muscle.


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Fig. 3.   Effect of PDBu on opossum IAS (A) vs. carotid artery smooth muscle (B) before and after administration of the PKC inhibitor calphostin C (1 × 10-7 M). Note that unlike in the IAS smooth muscle, the phorbol ester causes a concentration-dependent increase in carotid artery smooth muscle tension. Furthermore, PDBu-induced carotid artery smooth contraction is significantly antagonized by calphostin C (1 × 10-7 M) (*P < 0.05; n = 5). The same concentration of calphostin C, on the other hand, has no significant effect on PDBu-induced relaxation of the IAS (P > 0.05; n = 5).

Effect of PDBu on SMC isolated from opossum IAS. PDBu caused concentration-dependent inhibition of bethanechol (1 × 10-6 M)-induced contraction of SMC (Fig. 4). Bethanechol caused maximal shortening of the SMC by 26.6 ± 2.3%, which was inhibited to 23.9 ± 2.8%, 18.0 ± 1.8%, 10.4 ± 1.5%, and 5.7 ± 0.3%, respectively, by 1 × 10-8, 3 × 10-8, 1 × 10-7, and 3 × 10-7 M PDBu. Some studies were carried out to examine the effect of PDBu alone on percent change in the cell length. SMC measured at different times after the administration of PDBu (1 × 10-6 M) showed no significant change in cell length at 20 and 40 s; however, at a 60-s interval, PDBu caused an increase in the cell length of 11.5 ± 1.6%.


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Fig. 4.   Percent decrease in bethanechol-induced maximal shortening of isolated smooth muscle cells (SMC) of the IAS by different concentrations of PDBu. Note that PDBu causes a significant and concentration-dependent decrease in the maximal shortening of the SMC contraction caused by bethanechol (*; n = 4 animals). Bethanechol (1 × 10-6 M) alone causes a 26.6 ± 2.3% shortening of the SMC (shown by open bar on left).

Effect of Ca2+-ATPase inhibitor thapsigargin and Na+-K+-ATPase inhibitor ouabain on fall of basal tension of IAS smooth muscle caused by PDBu. Thapsigargin caused no significant inhibition of the fall in basal IAS smooth muscle tension caused by PDBu (P > 0.05; n = 5; Fig. 5A). In control experiments, PDBu at concentrations ranging from 1 × 10-8 to 3 × 10-5 M caused falls ranging from 11.6 ± 4.6% to 82.8 ± 4.3% in basal IAS tension. After thapsigargin (1 × 10-6 M), the same concentrations of PDBu caused falls ranging from 5.3 ± 2.6% to 81.9 ± 8.6% in basal IAS tension. Interestingly, thapsigargin by itself caused an increase rather than a decrease in the basal tone of IAS smooth muscle from 14.6 ± 1.2 mN to 14.8 ± 1.6, 16.3 ± 1.8, 17.2 ± 2.0, 18.5 ± 1.8, and 18.6 ± 2.2 mN at 2, 4, 6, 8, and 10 min, respectively.


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Fig. 5.   Percent fall in IAS tension in response to PDBu before and after Ca2+-ATPase inhibitor thapsigargin (1 × 10-6 M; A) and the Na+-K+-ATPase inhibitor ouabain (1 × 10-5 M; B). Neither thapsigargin nor ouabain had any significant effect on the overall concentration-response curves of PDBu obtained during control experiments (P > 0.05; n = 5).

The Na+-K+-ATPase inhibitor ouabain (31) also had no significant effect on the overall concentration-response curve, showing a fall in basal IAS tension caused by PDBu (P > 0.05; n = 5; Fig. 5B). In control experiments, PDBu in concentrations ranging from 1 × 10-8 to 3 × 10-5 M caused falls in basal tension of IAS smooth muscle ranging from 4.4 ± 1.8% to 76.8 ± 7.2%, respectively. After pretreatment with 1 × 10-6 M ouabain, the same concentrations of PDBu caused falls in basal tension of IAS smooth muscle ranging from 7.2 ± 1.8% to 82.3 ± 9.7%.

Effect of Ca2+ channel blocker nifedipine on basal IAS tension and effect of BAY K 8644 on nifedipine-induced fall in basal IAS tension. Nifedipine caused a concentration-dependent fall in basal tension of IAS smooth muscle that was shifted to the right by the Ca2+ channel activator (16) BAY K 8644 (1 × 10-5 M; P < 0.05; n = 5; Fig. 6A). In control experiments, 1 × 10-7, 3 × 10-7, 1 × 10-6, and 3 × 10-6 M nifedipine caused 28.2 ± 10.6%, 61.7 ± 7.2%, 77.9 ± 5.6%, and 87.4 ± 3.2% falls in IAS tension, respectively, and after BAY K 8644, the same concentrations of nifedipine caused 22.0 ± 2.0%, 30.6 ± 2.8%, 45.5 ± 3.2% and 69.8 ± 3.0% falls, respectively, in basal IAS smooth muscle tension. The time course of the effect of nifedipine on basal IAS tension and its reversal to near control levels by BAY K 8644 suggest that nifedipine and BAY K 8644 compete for the same site at the Ca2+ channels of the IAS smooth muscle cell membrane.


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Fig. 6.   A: percent fall in IAS tension in response to nifedipine before and after administration of the Ca2+ channel activator BAY K 8644 (1 × 10-5 M). BAY K 8644 causes a significant rightward shift in the concentration-response curve of nifedipine (n = 5). B: time course of the fall in basal IAS tension by nifedipine (1 × 10-6 M) followed by its reversal by BAY K 8644 (1 × 10-5 M) given at the time of the plateau effect of nifedipine.

Effect of Ca2+ channel agonist BAY K 8644 on PDBu-induced fall of basal IAS tension. In these experiments, we examined the effect of BAY K 8644 (1 × 10-5 M) on the entire concentration-response curve that showed a fall in the basal IAS tension with PDBu. Figure 7A shows that BAY K 8644 caused a significant rightward shift in the concentration-response curve of PDBu as it does with nifedipine (P < 0.05; n = 5).


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Fig. 7.   A: percent fall in IAS tension by PDBu before and after Ca2+ channel activator BAY K 8644 (1 × 10-5 M). BAY K 8644 causes a significant rightward shift in the concentration-response curve of PDBu (n = 5). B: time course of the fall in basal IAS tension by different concentrations of PDBu followed by reversal by BAY K 8644 (1 × 10-5 M). BAY K 8644 was given after the peak fall in basal IAS tension with PDBu had stabilized.

Figure 7B shows the time course of the effect of different concentrations of PDBu on basal IAS tension and its reversal by BAY K 8644. The Ca2+ activator was given when the fall in the IAS tension with PDBu had plateaued. BAY K 8644 was found to be rather selective in causing a reversal of the fallen basal IAS tone by nifedipine and PDBu because it failed to cause a similar reversal of vasoactive intestinal polypeptide (VIP)- and isoproterenol-induced fall in the basal smooth muscle tone.

Effect of PDBu and nifedipine on [Ca2+]i in IAS smooth muscle and reversal by BAY K 8644. To obtain direct evidence for the effect of PDBu on the changes in [Ca2+]i levels, we examined [Ca2+]i levels before and after PDBu and nifedipine and their possible reversal by the Ca2+ channel agonist BAY K 8644. PDBu at concentrations of 1 × 10-6 and 1 × 10-5 M caused a significant concentration-dependent decrease in basal [Ca2+]i levels (P < 0.05; n = 5; Fig. 8A), which were restored to levels not significantly different from controls (P > 0.05; n = 5).


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Fig. 8.   Influence of PDBu (A) and nifedipine (B) on free intracellular Ca2+ ([Ca2+]i) levels in IAS smooth muscle and reversal by BAY K 8644. Both PDBu and nifedipine cause significant (*P < 0.05; n = 5) decreases in [Ca2+]i levels that are restored by BAY K 8644.

Similar to PDBu, nifedipine caused a concentration-dependent decrease in basal levels of [Ca2+]i that was reversed by BAY K 8644 to levels that were not significantly different from controls (P < 0.05; Fig. 8B). Thapsigargin (1 × 10-6 M) had no significant effect on the PDBu-induced decrease in [Ca2+]i levels in IAS smooth muscle (P > 0.05; n = 5).

Effect of PDBu on PKC activity in IAS smooth muscle. In the basal state, the specific activity of PKC in the particulate fraction of IAS circular smooth muscle homogenate (70.0 ± 6.5 pmol · min-1 · mg protein-1) was found to be higher than in rectal smooth muscle (20.8 ± 4.3 pmol · min-1 · mg protein-1). PDBu (1 × 10-5 M) caused a significant decrease in basal PKC activity of IAS smooth muscle in the particulate fraction to 45.7 ± 3.8 pmol · min-1 · mg protein-1 and an increase in rectal smooth muscle to 48.4 ± 4.6 pmol · min-1 · mg protein-1 (P < 0.05; n = 5). The reverse was the case with the cytosolic fraction of IAS smooth muscle, which showed a corresponding significant increase in PKC activity (P < 0.05; n = 5). Conversely, the alpha -adrenoceptor agonist phenylephrine (1 × 10-6 M) caused a significant increase in translocation of PKC from the cytosolic to the particulate fraction. The data show that PDBu caused inhibition of PKC translocation to the membrane rather than an increase.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This is the first report to demonstrate an inhibition of spontaneously tonic smooth muscle of the IAS by PDBu, a typical PKC stimulant. The studies show that phorbol esters cause relaxation of IAS smooth muscle in small part by activation of NANC inhibitory neurons but in large part by direct action on IAS SMC. Closer examination revealed that the inhibitory action of PDBu on IAS smooth muscle was caused by inhibition of Ca2+ channels and decrease in PKC translocation to the membrane.

The inhibitory action of phorbol esters in IAS smooth muscle is considered to be specific to the tonic smooth muscle of the IAS. The smooth muscle relaxation caused by PDBu is reproducible and concentration dependent. Furthermore, the relaxant response is unique to the IAS because in carotid artery smooth muscle (phasic in nature and routinely used to study the effect of PKC stimulants and inhibitors), PDBu produces a concentration-dependent contraction. The relaxant action of PDBu is further verified in isolated SMC of the IAS. The fall in basal IAS smooth muscle tone may be similar to the specific inhibition of tonic but not phasic contraction of the smooth muscle of the guinea pig ileum caused by carbachol (30). The contractile and relaxant effects of PDBu may be present in the phasic and tonic smooth muscles, but being the dominant effect, only relaxation is expressed in the tonic smooth muscle of the IAS.

In the IAS, the potencies of different phorbol esters and DAG analogs in causing smooth muscle relaxation were different. PDBu was significantly more potent than PMA and other DAG analogs. Among different DAG analogs, OAG was found to produce no significant fall in basal tension of IAS smooth muscle. On the other hand, 1,2 dioctanoyl-sn-glycerol and DPT produced IAS smooth muscle relaxation that was almost comparable to that of PMA. Interestingly, none of the agents produced any contraction of IAS smooth muscle. The exact reason for the profound differences in the actions of these agents in IAS smooth muscle is not presently known.

Phorbol ester-induced inhibition of the contraction caused by muscarinic agonist, high K+, histamine, 5-hydroxytryptamine, and oxytocin in intestinal (18, 24, 30) and nonintestinal (1, 9, 39, 40) smooth muscle has been reported previously. However, inhibition of spontaneously tonic smooth muscle has not been reported previously.

A portion of the inhibitory action of PDBu on IAS smooth muscle is found to be caused by activation of NANC neurons because the responses are resistant to adrenergic and cholinergic blocking agents and are partly attenuated by the neurotoxin TTX. The neural component of phorbol ester in different systems is well known (6, 14). The neural component of the PDBu response in the IAS smooth muscle, however, does not involve the nitric oxide synthase (NOS) pathway because it is not modified by the NOS inhibitor L-NNA. The NOS pathway for the inhibitory actions of PDBu in IAS smooth muscle was considered because it is the predominant pathway for NANC relaxation in the IAS (25, 28). The possible involvement of other inhibitory neurotransmitters such as VIP (20), pituitary adenylate cyclase-activating polypeptide, ATP (27, 29), and CO (26) in the PDBu-induced fall in IAS tension remains undetermined.

A major portion of the inhibitory action of PDBu appears to be directed at the SMC level because a majority of the response is maintained in the presence of TTX and is not affected by the neuronal Ca2+ channel blocker omega -conotoxin. In addition, the response is directly demonstrable in the isolated SMC of the IAS. Another example in which PKC may promote relaxation of the smooth muscle cells has been shown in guinea pig stomach (21).

The exact relationship between PKC activation and PDBu-induced relaxation of IAS smooth muscle is not known. This issue was examined by the use of the selective PKC inhibitor calphostin C in a concentration that was successful in blocking PDBu-induced contraction in carotid artery smooth muscle. The data suggest that the inhibitory response of PDBu in IAS smooth muscle may be largely independent of PKC activation. These observations are somewhat similar to those made in rat uterus, in which oxytocin-induced tonic contractions of the smooth muscle were not modified by a PKC inhibitor (9, 23).

These studies also argue against the primary possibilities of intracellular mobilization of Ca2+ and Na+-K+-ATPase in the inhibitory action of the phorbol esters in the IAS. This was accomplished by using agents that block these events, specifically thapsigargin (37) and ouabain (31).

Closer scrutiny of the mechanism of the inhibitory action of PDBu in IAS smooth muscle reveals that the phorbol causes part of the smooth muscle relaxation by inhibiting Ca2+ influx. The inhibitory action of PDBu in IAS smooth muscle is reversed promptly and specifically by the classic Ca2+ channel activator BAY K 8644 (16). The fall in basal tone of IAS smooth muscle by PDBu and its reversal by BAY K 8644 are strikingly similar to those of nifedipine. In addition, the fall in basal IAS tension caused by PDBu and nifedipine is accompanied by a fall in basal levels of [Ca2+]i. Decreases in [Ca2+]i levels also are specifically restored by BAY K 8644. These findings suggest that phorbol esters may inhibit smooth muscle tone by inhibiting Ca2+ influx. Such a suggestion has been made before in limited studies of different systems (8, 15, 36), in which inhibition of the agonist-induced response was shown. The precise mechanism of Ca2+ influx blockade and the type of Ca2+ channel involved, however, are not known at the present time. Conversely, data exist to show that smooth muscle contraction to the phorbol esters is mediated by an increase in Ca2+ influx (32). In addition to decrease in Ca2+ influx, PDBu-induced IAS smooth muscle relaxation was also associated with a decrease in translocation of PKC to the membrane and an increase in translocation to the cytosol. The relationship between inhibition of Ca2+ influx and redistribution of PKC after administration of PDBu in the IAS is not known. Whether phorbol-induced IAS smooth muscle relaxation is related to PKC downregulation or its downregulation or to a mechanism that is independent of PKC also remains to be determined.

Previous studies in cat LES (13) and IAS (4) showed that, in the normal state, a major portion of the basal tone in the smooth muscle is regulated by PKC. The present studies showing a fall in basal IAS tone in response to phorbol ester do not minimize the role of PKC activity in the maintenance of IAS tone. In addition, we have also observed that the PKC inhibitor causes a concentration-dependent fall (especially at >1 × 10- 7 M) in basal tone of the IAS. Furthermore, IAS smooth muscle in the basal state was found to have higher levels of PKC activity in the particulate fraction. The fall in basal IAS tone elicited by phorbol esters may be caused by downregulation of PKC activity already at a higher level in the basal state or by a nonspecific action. It is conceivable that high levels of PKC activity in the basal state do not allow further activation and that attempts in that regard only lead to PKC downregulation by feedback inhibition. It is well known that PKC downregulation by phorbol ester is partly responsible for inhibiting the tonic component of the smooth muscle contraction by K+ depolarization (30). To accomplish this, however, requires several hours of exposure to the phorbol ester. The exact exposure time of phorbol esters for the downregulation of the spontaneously tonic smooth muscle has not been determined. The possibility of the nonspecific action of phorbol esters in the IAS cannot be ruled out because the final response of phorbol esters may be the sum total of the effect on multiple isozymes of PKC (35) and other kinases in the smooth muscle.

In summary, the classic PKC stimulators, phorbol esters, may cause relaxation of the smooth muscle of the IAS via inhibition of Ca2+ influx and decrease in PKC translocation to the membrane and increase in translocation to the cytosol. The exact role of other potential mechanisms, such as PKC downregulation and inhibition of phosphatidyl signaling pathways, in causing the smooth muscle relaxation remains to be determined. Further studies are needed to discover tissue-specific (tonic vs. phasic smooth muscle) PKC stimulants that cause an increase in the basal sphincteric smooth muscle tone. Such agents may have important therapeutic implications in gastrointestinal motility disorders such as anorectal incontinence.


    ACKNOWLEDGEMENTS

We thank Drs. Ya-Ping Fan and Rajinder Puri for valuable suggestions and technical assistance and Dr. John J. Gartland for editing the manuscript.


    FOOTNOTES

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

Address for reprint requests and other correspondence: S. Rattan, Div. of Gastroenterology, Thomas Jefferson Univ., 1025 Walnut St., Rm. 901 College, Philadelphia, PA 19107 (E-mail: Satish.Rattan{at}mail.tju.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.

Received 1 November 2000; accepted in final form 20 March 2001.


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Am J Physiol Gastrointest Liver Physiol 280(6):G1341-G1350
0193-1857/01 $5.00 Copyright © 2001 the American Physiological Society




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