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Delayed gastric emptying and abnormal antral motility patterns are common in preterm and newborn infants(1–4). Although the factors involved in the postnatal maturation of gastric motility remain to be fully elucidated, we and others have reported that antral smooth muscle from newborn animals differs from that of the adult with respect to maximal active force development(5–9), sensitivity to agonist stimulation(8), involvement/mobilization of extracellular and intracellular calcium stores(6–9), and the characteristics of cell membrane calcium channels(7). These findings have led to the hypothesis that the postnatal maturation of gastric motility involves, at least in part, an overall increase in the force-generating capacity(contractility) of antral smooth muscle.

An essential first step in smooth muscle contraction is the calcium-dependent phosphorylation of the 20-kD regulatory myosin light chain by the enzyme, myosin light chain kinase (for reviews, seeRefs. 10 and 11). MLCP is brought about by the interaction of calcium, the calcium-activated protein calmodulin, and a calcium-calmodulin-dependent protein responsible for MLCP called myosin light chain kinase. Phosphorylation of the 20-kD myosin light chain on each myosin head brings about a conformational change which results in actin activation of the myosin-Mg2+-ATPase, and subsequent crossbridge cycling. In the muscle cell, crossbridge cycling produces a mechanical output, which is expressed either as force development or shortening, depending upon the physical constraints on the muscle.

It is well documented that agonist-mediated increases in cytosolic calcium lead to rapid increases in MLCP and subsequent force development in vascular and tracheal smooth muscle(10, 11). Although it is also recognized that increases in cytoplasmic calcium levels initiate contraction in gastrointestinal smooth muscle, the relationship between MLCP and force development is less well studied. However, the data available suggest that MLCP is essential to the contractile event and that a correlation exists between levels of MLCP and force output by the muscle(12–17). The purpose of this study was to evaluate the relationship between active force development and MLCP in circular smooth muscle from the gastric antrum of adult and neonatal rabbits. Specifically, we tested the hypothesis that the reduced stress-generating capacity of antral smooth muscle from neonatal rabbits can be accounted for by reduced levels of MLCP.

METHODS

Tissue preparation. Antral muscle strips were obtained from neonatal (4-6 d old) and adult (female, 5-6 kg) New Zealand White rabbits as described previously(5). The stomachs were removed through a midline incision, opened along the greater curvature, and rinsed free of residual food contents using room temperature (23°C), oxygenated(100% O2) HEPES buffer solution of the following composition (in mM): 137.5 NaCl, 5.0 KCl, 1.0 MgCl2, 2.5 CaCl2, and 10.0 glucose, at pH 7.4. The antrum was cut free from the remainder of the stomach and pinned mucosal side up in a dissecting dish filled with fresh room temperature HEPES buffer solution. With the aid of a stereomicroscope, full thickness muscle strips (1 mm × 5-10 mm) were prepared by careful separation of the mucosa from the underlying muscle. The microscope permitted clear visualization of the muscle bundles and ensured that the long axis of the muscle strip was parallel to the circular muscle layer.

The muscle strips were mounted in individual 10-mL tissue baths filled with warmed (37°C), aerated HEPES buffer solution. One end was attached via an inelastic wire to an isometric force transducer (Grass, FT-03C), and the other end attached to a glass rod that could be raised or lowered by adjustment of fine micrometer. After a 1-h equilibration period, the muscle strips were stretched in 0.5-mm increments and stimulated with ACh (10-4 M) until the length at which maximal active stress develops was achieved(5). A permanent record of the force developed by each muscle strip was obtained using a multichannel recorder.

Response to ACh stimulation. Each muscle strip was examined for its contractile response to ACh (10-8 M to 10-3 M). ACh was added to the baths in microliter amounts to achieve the final concentrations reported. All dose-response curves were constructed with ACh being given in random doses; no cumulative dose-response curves were obtained. The dose-response data for tissues from neonates and adults were analyzed for differences in active stress (N/m2) and ED50 (defined as the dose of ACh which elicits a one-half maximal response). Also, in some instances, chart recorder paper speed was increased to permit assessment of the relationship between time after agonist administration and the development of the contractile response. Specifically, we determined the time between addition of the agonist and the beginning of force development (lag time), and the time to one-half (T50) and maximal(T100) force development.

Myosin light chain phosphorylation determination. MLCP levels in antral smooth muscle from neonatal and adult rabbits were measured under basal (unstimulated) conditions and after ACh (10-4 M) stimulation using techniques described previously(12–14). Stimulated tissues were studied at 5, 10, 15, 30 and 60 s of stimulation. At the appropriate time, each muscle strip was rapidly frozen in a dry ice-acetone slurry and then allowed to thaw in this solution for 60 min at 22°C. This procedure denatures endogenous phosphatases and kinases thereby preserving the levels of MLCP that existed at the time of freezing. The muscle strips were homogenized at 4°C in 1% SDS, 10% (wt/vol) glycerol, and 20 mM DTT. The phosphorylated and unphosphorylated myosin light chains were resolved using a two-dimensional gel electrophoretic system involving isoelectric focusing in the first dimension and SDS-PAGE electrophoresis in the second dimension(18). MLCP was quantitated by densitometric scanning of Coomassie Blue-stained gels using a Helena Labs Quick-scan Jr. densitometer (Beaumont, TX). The amount of phosphorylated myosin light chain was calculated by dividing the area of the phosphorylated peak by the total area of the phosphorylated and unphosphorylated peaks and expressing the results as a percent myosin light chain phosphorylation. The acidic satellite forms of the phosphorylated light chains were rarely observed and therefore excluded from the analysis.

Data analysis. Upon completion of each experiment, the length and weight of each muscle strip was determined and used to calculate tissue cross-sectional area as described previously(5). All contractions were normalized for tissue cross-sectional area, and the data were reported as stress in N/m2. Values of ED50 were obtained from a probit analysis of the dose-response curves(19). Statistical analyses utilized analysis of variance and the Student'st test for unpaired observations to compare data from neonatal and adult animals. Differences were considered statistically significant whenp was <0.05. All data are presented as mean ± SEM.

RESULTS

Response to ACh stimulation. ACh stimulation of antral smooth muscle from neonates and adults produced a rapid increase in tension (phasic response) followed by reduced but sustained contraction (tonic response). No statistically significant differences existed between tissues from neonatal and adult animals with respect to lag time (3.4 ± 0.4 s, neonate; 3.2± 0.3 s, adult), the time to one-half peak response (3.9 ± 0.5 s, neonate; 4.5 ± 0.6 s, adult) or the time to peak response (11.2± 0.7 s, neonate; 12.4 ± 1.0 s, adult).

Antral smooth muscle from neonates and adult rabbits responded to increasing concentrations of ACh with increased force (Fig. 1). At each concentration above 10-6 M ACh, the force developed by muscle strips from adult rabbits was significantly greater than the force developed by tissues from neonates. For each age group, maximal active stress developed at an ACh concentration of 10-4 M and was 1.86 ± 0.24 N/m2 for the adult and 0.95 ± 0.05 N/m2 for the neonate(p < 0.05).

Figure 1
figure 1

ACh dose-response curves of gastric antral smooth muscle from neonatal and adult rabbits. Each data point represents the mean± SE of at least 12 experiments.

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Normalization of the contractile data from neonates and adult animals to 100% of their maximal responses produced virtually identical dose-response relationships (Fig. 2). No statistical difference was found between the two age groups with respect to the ED50 of the dose-response curves; 3.9 ± 1.2 × 10-6 M for the adult and 5.7 ± 1.4 × 10-6 M for the neonate (p > 0.05).

Figure 2
figure 2

Normalization of data shown in Figure 1 to 100% of respective maximal responses. Each data point represents the mean ± SE of at least 12 experiments.

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MLCP. Figure 3 summarizes the MLCP data obtained from antral smooth muscle of neonatal and adult rabbits. Basal levels of MLCP were similar in neonates and adult animals, 7.4 ± 1.4% and 8.2± 0.9%, respectively (p > 0.05). When stimulated with 10-4 M ACh, tissues from both the neonate and the adult evidenced identical time-dependent changes in MLCP. In antral smooth muscle from the neonate and the adult, MLCP reached peak levels at 10 s of stimulation(neonate, 48.7 ± 1.9%; adult, 44.6 ± 2.3%; p 0.05) and returned to near basal levels by 60 s of stimulation (neonate 13.3 ± 2.4%; adult, 12.0 ± 2.3%).

Figure 3
figure 3

Levels of MLCP in circular smooth muscle from the gastric antrum of neonatal and adult rabbits under basal conditions and after ACh (10-4 M) stimulation. Each data point represents the mean ± SE of at least six experiments.

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DISCUSSION

Smooth muscle contains an endogenous enzyme, myosin light chain kinase, capable of phosphorylating the 20-kD light chains of myosin. It is now well documented that contraction of smooth muscle is initiated by phosphorylation of the 20-kD light chain of myosin(10, 11). Phosphorylation of myosin is the direct result of an increase in the cytoplasmic concentration of free calcium which occurs as a result of agonist-mediated calcium influx and/or calcium mobilization from intracellular stores. Calcium-dependent MLCP results in actin activation of myosin ATPase activity, the development of force, and muscle shortening. Evidence exists in support of a close correlation between the levels of activator calcium, maximal active stress, and peak levels of MLCP for a number of smooth muscles(10, 20, 21).

The primary purpose of this study was to test the hypothesis that the decreased force-generating capacity of antral smooth muscle from neonatal rabbits is due in part to reduced levels of MLCP. This hypothesis is based on two assumptions. First, a close correlation exists between the amount of calcium available for activation of the contractile process and peak levels of MLCP. Second, antral smooth muscle from neonatal animals develops less active stress than tissues from adult animals in part as the result of reduced amounts of calcium available for activation of the contractile machinery. The results of the present study indicate that MLCP also is a regulatory step in antral smooth muscle contraction, in as much as unstimulated muscle had low levels of MLCP, and ACh stimulation was followed by rapid increases in MLCP. The observed basal and peak levels of MLCP are similar to values reported for gallbladder(12–14), uterine(22–24), tracheal(10, 25), and vascular(11, 21, 26) smooth muscle. However, the findings of the present study do not support our hypothesis. Although we did observe a significant difference in the maximal active stress developed by antral smooth muscle from neonates and adults, no significant differences were observed with respect to the time dependency or the levels of MLCP attained in response to ACh stimulation. The data suggest that at equivalent levels of MLCP tissues from neonatal animals develop less active stress than tissues from adult animals. Thus, as compared to the adult, gastric antral smooth muscle from the neonate is characterized by a decreased stress/MLCP ratio.

To date, little information is available concerning how alterations in the physiologic status of a smooth muscle affects the stress/MLCP relationship of the muscle. Xie et al.(15) compared this relationship in colonic smooth muscle from healthy rabbits and rabbits with documented experimental colitis and observed that the decreased stress generating capacity of colonic smooth muscle from colitic rabbits was associated with decreased levels of MLCP, with no change in the stress to MLCP ratio. They explained their results by assuming that the inflammatory process reduced the amount of calcium available for activation of the contractile process. Recently, Word et al.(24) demonstrated that the increased stress-generating capacity of myometrial smooth muscle from pregnant women occurred despite reduced levels of MLCP. They observed that the stress to MLCP ratio of uterine smooth muscle was increased more than two times in tissues from pregnant women. The findings of Xie et al.(15) and Word et al.(24), as well as the results of this study suggest that factors in addition to MLCP levels must be accounted for when considering the force-generating capacity of a smooth muscle.

It is possible that differences in tissue thickness between the neonate and the adult may have affected our data and compromised our ability to determine differences in MLCP levels between the age groups. Kamm and Murphy(27) have reported that peak MLCP levels depend upon the rate of tissue activation and, as such, are affected by the thickness of the muscle strip preparation. In their study, significant increases in the thickness of an in vitro hog carotid artery preparation decreased peak MLCP values, and prolonged the lag period and the times to one-half and maximal contraction. In our study, we found that the lag time and the times to one-half maximal and maximal contraction were not different between the age groups, suggesting similar rates of tissue activation. Thus, we do not believe that our data were influenced by differences in muscle strip thickness between the neonate and the adult.

In summary, despite comparable levels of MLCP upon Ach stimulation, circular smooth muscle from the gastric antrum of neonatal rabbits developed less active stress than tissues from adult animals. Although we did not examine the factors responsible for the decreased stress to MLCP ratio in tissues from neonatal rabbits, we recently reported that the postnatal increase in antral smooth muscle contraction in the rabbit is paralleled by changes in myosin heavy chain isoform distribution(28). Ayas et al.(29) reported similar data for the fundus and antrum of the cat. They also reported that the postnatal period is associated with an increase in the cellular concentration of actin and myosin. The latter could account for increased stress development despite similar levels of MLCP. The data presently available suggest that the postnatal maturation of force in smooth muscle is a complex dynamic involving changes in contractile protein content and isoform distribution, as well as possibly changes in other cytostructural and regulatory proteins.