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Regions of the stomach are responsible for specific functions, and the roles that these regions play in the digestive process may change during growth and development. The fundus functions as a reservoir that generates slow tonic contractions and has the ability to relax upon distention, whereas the antrum is able to produce phasic contractions responsible for trituration and to coordinate peristaltic sequences involving the pylorus and duodenum. During infancy mammals are initially nursed and then weaned to a mixed solid/liquid diet. The changes in dietary constituents during growth and development from predominantly a liquid to a mixed solid/liquid places an increasing burden on the antrum, which is required to triturate solids and play a role in regulating their emptying from the stomach.

We have previously reported that isolated smooth muscle cells from the fundus of the adult cat use intracellular calcium stores to contract in response to acetylcholine, whereas adult antral cells from the circular smooth muscle use both influx of extracellular calcium and release of calcium from intracellular stores for contraction(1). Isolated circular smooth muscle cells from the newborn kitten antrum are unable to use intracellular calcium stores to support contraction in response to acetylcholine or IP3 and are therefore unable to contract in response to these agonists in the absence of extracellular calcium. To assess the relative inability of newborn antral cells to use intracellular calcium stores we examined IP3 receptors, IP3 production, and the presence of intracellular calcium stores. The data indicate there is a decrease in density of IP3 receptors in the newborn period, whereas there is no change in binding affinity.

METHODS

Preparation of tissue. Adult cats of either sex and newborn kittens between the 4th and 7th d of life were used in this study. The animals were anesthetized with xylazine and ketamine and then killed with an intracardiac injection of pentobarbital. The chest and the lower abdomen were opened with a midline incision, exposing the esophagus and stomach. The esophagus, stomach, and proximal duodenum were removed together and pinned on a wax block in their in vivo orientation. The preparation was opened along the lesser curvature. Tissue was obtained from the antrum immediately orad to the pylorus. After carefully dissecting away the mucosa, the underlying external circular muscle layer was sliced into 0.5-mm thick slices with a Stadie-Riggs tissue slicer (Thomas Scientific Apparatus, Philadelphia, PA). The first slice containing the myenteric plexus and the last slices containing longitudinal muscle and serosa were discarded. The slices of circular muscle were placed flat on a wax surface, and tissue squares were made by cutting twice with a 2-mm blade block, the second cut at right angles to the first.

Dispersion of smooth muscle cells. Tissues from the antrum were digested in HEPES-buffered physiologic solution containing 190 U/mL collagenase type II (Worthington Biochemicals, Freehold, NJ) for 2 h. The HEPES solution contained NaCl, 115 mmol/L; KCl, 5.8 mmol/L; KH2PO4, 12 mmol/L; glucose, 2.5 mmol/L; HEPES, 25 mmol/L; CaCl2, 2 mmol/L; MgCl2, 0.6 μmol/L; BME amino acid supplement(M. A. Bioproducts, Walkersville, MD), 0.3 mg/mL, soybean trypsin inhibitor(Sigma Chemical Co.), 0.1 mg/mL, and the pH was 7.4. The solution was oxygenated (100% O2) at a low gas-flow rate to avoid agitating the tissue. At the end of the digestion period, the tissue was placed over a 500-μm Nitex filter (Tetko, Monterey Park, CA), rinsed in collagenase-free HEPES buffer to remove any trace of collagenase, and then incubated in collagenase-free HEPES buffer. The cells were allowed to dissociate freely in collagenase-free solution for 10-20 min. It was important not to agitate the fluid to avoid cell contraction in response to mechanical stress. All the glassware was prerinsed in a 0.05% silicon solution to prevent the cells from adhering to the glass.

Cell contraction studies. All experiments were done in calcium-free medium. After collagenase digestion, the isolated cells were rinsed and then incubated in calcium-free HEPES buffer containing 2 mM EGTA for 20 min before each experiment. Aliquots of 0.2 mL were added to tubes containing appropriate concentrations of thapsigargin. The cells were allowed to react for the indicated time interval and were then fixed by adding acrolein to the tubes at a 0.1% final concentration. From each tube, a few drops of the fixed cells were placed on a microscope slide and covered with a coverslip. The edges of the coverslip were sealed with nail enamel to prevent evaporation. Slides so prepared, if refrigerated, could be kept for several days.

Cell measurements. Fifty consecutive intact cells from each slide were observed through a phase-contrast microscope (Carl Zeiss), a television camera (model WV-1550; Panasonic, Secaucus, NJ) and a television screen (model WV-5410, Panasonic). The camera was connected to a video microscaler (model IV-550, For-A Co., West Newton, MA). The microscaler superimposed two vertical and one horizontal line onto the television screen. Cells were oriented along the horizontal line on the television screen by rotating the slide on a rotating microscope stage. Cell length was obtained by placing the vertical lines on the right and left ends of the cell. The microscaler measured the distance between the vertical lines, providing a measurement of cell length.

IP3 binding studies. IP3 determinations were performed by following a previously described specific radioreceptor assay(2) and modified for calculation of IP3 receptors in smooth muscle(3). Smooth muscle homogenates were prepared by placing isolated antral tissue in iced buffer containing 50 mM Tris-HCl, 1 mM DTT, and 1 mM EDTA at pH 8.3 at 30 × volume. Minced tissue was then homogenized twice for 20 s each at Polytron setting 8. The homogenate was next centrifuged for 12 min at 35 000 ×g and resuspended in fresh buffer. Assay tubes were prepared in triplicate and contained 500 μg of protein of smooth muscle homogenate, 2 nM D-myo-[3H]IP3, and 100 μL of varying concentrations of cold IP3 (0-200 μM) in a total of 500 μL in the above buffer. Tubes were then incubated for 30 min at 4 °C. Membrane-bound and -free labeled IP3 were then separated by centrifugation in a microcentrifuge. After the supernatant was aspirated, radioactivity associated with the pellet was counted in a liquid scintillation counter. Binding affinity (Kd) and receptor density(Bmax) were determined by Scatchard analysis, with the Bmax values standardized to tissue protein content. Protein content was measured by the Bio-Rad assay.

Measurement of IP3. Antral muscle cell suspensions were exposed to agents and agonists, and incubation was terminated at the times indicated by addition of one-half the volume of 20% trichloroacetic acid. The resultant precipitate was centrifuged at 3000× g for 10 min. The supernatant was then extracted with trioctylamine-Freon(4) to remove the acid. The mass of IP3 was measured by a radioreceptor binding assay using rat cerebellar protein as previously described(5, 6). Briefly, 100-μL aliquots of prepared samples of authentic IP3 (0.3125-200 pmol) were incubated with a rat cerebellar IP3-binding protein preparation in the presence of 5 nCi of D-myo-[3H]IP3 in 50 mM Tris-Cl, 5 mM 2-mercaptoethanol, 1 mM EDTA buffer, pH 8.4, and incubated for 30 min at 4 °C. Bound and free labeled IP3 were then separated by centrifugation in a microcentrifuge. After the supernatant was aspirated, radioactivity associated with the pellet was counted in a liquid scintillation counter. The IP3 content of the extract, and thus of the muscle cell preparation, was determined by comparing the extent of the inhibition of D-myo-[3H]IP3 binding with that observed with known amounts of authentic IP3.

Drugs and chemicals. Collagenase was obtained from Worthington Biochemical; D-myo-[3H]inositol 1,4,5-trisphosphate was obtained from Amersham Corp. (Arlington Heights, IL). All other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO).

RESULTS

IP3 receptor. Receptor binding was determined in antral smooth muscle membranes isolated from the circular smooth muscles of newborn and adult antrum. Scatchard analysis revealed a linear relationship in all studies, typical examples of which are shown for the kitten and the adult (Fig. 1).Kd or measurement of binding affinity was similar in both age groups with the value of 28.6 ± 4.2 nM in the kitten versus 30.7 ± 3.8 nM in the adult (Fig. 2). Receptor density was significantly different at 647 ± 181 fmol/mg of protein in the kitten compared with 1755 ± 275 fmol/mg of protein in the adult (Fig. 3).

Figure 1
figure 1

Satchard analysis of IP3 receptor binding in kitten and adult. Left: Scatchard analysis of a single representative experiment to identify IP3 receptors in the kitten antrum. Right: Scatchard analysis of IP3 receptors in the adult antrum. The linear plot suggests there is a single receptor for the newborn and the adult.

Figure 2
figure 2

Kd for IP3 receptor binding in kitten and adult. Compares the Kd for IP3 receptor binding in kitten and adult antral circular smooth muscle cells. There is no significant differences between the two. Data reported are n = 6 for adult and n = 4 for kitten and the SEM is shown.

Figure 3
figure 3

Bmax for IP3 receptor binding in kitten and adult. Comparion of the Bmax, which is calculated from the IP3 receptor binding studies on circular smooth muscle cells from both the kitten and adult cat. The Bmax of receptor density was significantly less in the kitten (*p ≤ 0.05) than in the adult cat. Data reported is n = 6 for adult and n = 4 for kitten. The SEM is shown.

IP3 production. We then measured production of IP3 and determined if the inability of the newborn's antral smooth muscle cells to respond to agonists in the absence of extracellular calcium might be related to the inability to increase IP3 production. As shown in Figure 4, in both newborn and adult antral smooth muscle a maximally effective dose of carbachol(10-6 M) results in equivalent production of IP3 at 15 s.

Figure 4
figure 4

IP3 production in kitten and adult. IP3 production as measured in circular smooth muscle antral tissue from newborn and adult at 30 s in response to maximally effective dose of carbachol. The values are not significantly different, and the data reported are n= 4 for adult and n = 3 for kitten. The SEM is shown.

Effects of thapsigargin on muscle contraction. Because newborn antral smooth muscle cells are unable to contract to agonists in the absence of extracellular calcium, we used thapsigargin in an attempt to determine whether intracellular calcium stores are present during the newborn period. Thapsigargin as a potent selective inhibitor of the calcium ATPase, which is responsible for replenishing depleted intracellular calcium stores and will result in an increase of intracellular calcium levels if intracellular calcium stores are dynamically active. As shown in Figure 5, thapsigargin caused dose-dependent contractions in both newborn and adult antral smooth muscle cells, which are equivalent in magnitude. In Figure 6 the kinetic response to thapsigargin is shown to be similar in both newborn and adult antral smooth muscle cells. This stoichiometrically and kinetically similar contraction in both age groups suggests that functionally dynamic intracalcium stores that are constantly being replenished are present in both age groups.

Figure 5
figure 5

Cell contraction in response to increasing concentrations of thapsigargin. Demonstrated is the dose-dependent contraction to thapsigargin in adult and kitten antral circular smooth muscle cells at 60 s. The maximally effective contractile response is shown. Shown are the average (n ≥ 3) for both adult and kitten. The SEM is shown.

Figure 6
figure 6

Kinetics of cell contraction in response to thapsigargin. Demonstration of the time course of the contractile process in response to a maximally effective dose of thapsigargin (10-6 M) in the circular smooth muscle cells from the antrum of both the kitten and the adult. Shown are the average (n ≥ 3) for both adult and kitten. The SEM is shown. The values between the kitten and adult are significantly different at the indicated points (*p ≤ 0.05 and **p ≤ 0.07).

DISCUSSION

The newborn's diet consists initially of a nutritionally complex liquid(i.e. breast milk or substitute) and then progresses to a mixture of solids and liquids. The antrum is the region of the stomach primarily in which mechanical transformation of solids into a form suitable for emptying from the stomach takes place. These different functional requirements of the antrum with advancing age could be associated with developmental differences in the manner in which the circular smooth muscle of the antrum contracts. It has been suggested that the ability of the newborn antral circular smooth muscle to generate force is not as well developed as in the adult(7), and we have shown that there are developmental differences in the structural contractile proteins that comprise circular smooth muscle(8).

In addition to developmental differences in the magnitude of smooth muscle contraction, utilization of different intracellular pathways could result in different patterns of contractile activity in the antrum. We have previously shown that isolated adult feline antral smooth muscle cells are able to use both intra- and extracellular calcium stores to contract in response to cholinergic stimulation, whereas newborn antral cells are unable to contract in the absence of extracellular calcium. Further support that newborn circular smooth muscle antral cells are relatively unable to use intracellular calcium stores compared with the adult is given by the observation that permeabilized newborn antral circular smooth muscle cells do not contract in response to exogenous IP3, which releases intracellular calcium stores, whereas permeabilized adult antral cells are able to generate approximately 50% of their normal contraction(1). Similar results have been suggested in neonatal rabbit bladder smooth muscle cells where contraction has been found to be more dependent on sources of extracellular calcium(911), than in mature bladder cells. These data therefore support the possibility that maturational differences in the accessibility of intracellular calcium stores may determine the manner in which newborn antral circular smooth muscle cells are able to respond to agonist induced contraction.

It is known that agonists cause muscle contraction through activation of second messenger systems. One of the mechanisms by which smooth muscle contraction occurs is when an agonist binds to its receptor and activates a phosphoinositidespecific phospholipase C that hydrolyzes phosphatidyl-inositol 4,5-bisphosphate to form the second messengers 1,2-diacylglycerol and IP3. IP3 causes the release of calcium from sensitive intracellular stores, which leads to the activation of calcium-sensitive proteins and causes contraction via the phosphorylation of myosin light chain(12).

It has been shown that IP3 binds to specific receptors that then cause release of calcium from intracellular stores(1315). It is possible that differences in the production of a second messenger such as IP3 could be responsible for the developmental differences seen in utilization of calcium sources for smooth muscle contraction. Studies in rabbit tracheal smooth muscle, which, unlike antral muscle, generates greater force per gram of muscle in the newborn period than in the adult, have shown greater amounts of IP3 in response to agonists during the newborn period(16, 17). These investigations, and others which have examined rat cerebral cortex during the newborn period(1820), have suggested that there is a relative imbalance in the kinases and phosphatases that are responsible for the production and dephosphorylation of IP3. Our data demonstrates that, in response to carbachol (Fig. 4), the increase in IP3 levels is comparable in both adult and newborn kitten antral circular smooth muscle.

We then examined the possibility that contractile differences during development may result from changes in the ability of IP3 to bind with receptors on intracellular calcium stores. The possibility that the number of receptors at the smooth muscle plasma membrane level increases with age has been suggested with both motilin(18) and muscarinic receptors(21). In immature oocytes intracellular calcium stores have been shown to be less sensitive to IP3(22, 23). Comparison of tracheal smooth muscle in newborn and adult rabbits suggests that in both age groups IP3 receptors are characterized by a single binding site that has a similar Bmax and Kd.(3). In this study we used Scatchard analysis, which is a common way of evaluating equlibrium binding data. In our present study, Scatchard analysis of IP3 binding in adult and kitten antral circular smooth muscle cells suggests a single binding site in both age groups with no difference in binding affinity (Bmax). The data also indicate there is a significant difference in receptor density with the kitten antral cells, demonstrating a Kd of less than one-half that found in the adult. These results suggest that IP3-sensitive intracellular calcium stores may be less accessible as a consequence of a decrease in the number of IP3 receptors in the newborn.

The release of calcium from intracellular stores in response to IP3 is not an all or none occurrence, in other words different levels of IP3 are associated with different levels of calcium release from intracellular stores(24). Several hypothesis have been put forth in an attempt to explain this phenomenon including: 1) heterogeneity in IP3 receptor sensitivity(25),2) heterogeneity in IP3 receptor density(26), 3) control of IP3-mediated calcium release by calcium (within and outside intracellular calcium stores)(27), and 4) down-regulation of IP3 receptors(28).

We have previously reported that kitten antral cells show no detectable contraction in response to exogenous IP3, whereas adult cells are able to contract to approximately 50% of their maximum in response to IP3(1). Inasmuch as our data suggest that kitten antral cells are able to produce IP3 in response to carbachol, the lack of detectable contraction in response to IP3 may be due to a deficiency in the IP3-mediated calcium release mechanism. Our data show the IP3 receptor density on newborn antral smooth muscle cells is reduced to approximately 37% that of the adult cells, and it is possible that activation of IP3-sensitive intracellular calcium stores releases a lower amount of calcium in the newborn than in the adult. In the absence of extracellular calcium it is possible that the reduced amount of calcium released from the IP3-sensitive intracellular calcium stores may be insufficient to bring about contraction in newborn antral cells, or that the contraction induced (3-4%) is not significantly different from the absence of contraction.

Calcium stores in smooth muscle can be separated into at least two different types, one releasable by IP3 and the other by caffeine or ryanodine. There is evidence to suggest that there may be spatial and functional differences between different intracellular calcium stores(21, 29). Calcium can be loaded into these intracellular stores via uptake of cytosolic calcium through an ATP-dependent calcium pump, one type of which is known as the sarco(endo)plasmic reticule Ca2+-ATPase family (SERCA ATPase)(30). Thapsigargin is a potent and selective inhibitor of these SERCA-type Ca2+-ATPases(31, 32) and in vascular smooth muscle cells has been found to increase cytosolic calcium by blocking reuptake of calcium by IP3-sensitive stores and not caffeine-sensitive stores, whereas in chromatin cells thapsigargin has been found to block reuptake of calcium by both stores.

In both adult and newborn antral circular smooth muscle cells, thapsigargin caused a dose dependent contraction that peaked early and was maintained at a lower level for at least 20 min. This suggests that even though intracellular calcium stores do not appear to be readily available for utilization by cholinergic agonists or IP3, intracellular calcium sources are present and dynamically functioning in newborn kitten antral smooth muscle cells. It is possible that in these newborn antral cells thapsigargin is releasing calcium from IP3 insentive stores as has been described in other tissues(33).

The ability of the cells to shorten in a dose-dependent fashion to thapsigargin with a pattern of contraction that is stoichiometrically and kinetically similar in both the newborn and adult suggests that both ages can respond similarly to intracellular increases in calcium. Because the binding affinities of IP3 receptors, IP3 production, and the response to thapsigargin is similar in both age groups, it is reasonable to speculate that the relative inaccessibility of IP3-sensitive intracellular calcium stores during the newborn period is due to a lower IP3 receptor density or other differences in the ability of the IP3-sensitive calcium stores to respond to IP3.