Signal transduction pathways associated with contraction during development of the feline gastric antrum

GASTROENTEROLOGY 1997;113:507–513

Signal Transduction Pathways Associated With Contraction During Development of the Feline Gastric Antrum A. CRAIG HILLEMEIER, DAVID E. DEUTSCH, and KHALIL N. BITAR Department of Pediatrics, University of Michigan Medical Center, Ann Arbor, Michigan

Background & Aims: Unlike adult antral cells, feline newborn antral cells are unable to contract in response to agonists in the absence of extracellular calcium or in response to exogenous inositol 1,4,5-triphosphate (IP3 ) after permeabilization. Changes in intracellular pathways that are associated with these differences were examined. Methods: In adult and kitten antrum isolated smooth muscle cell contraction, levels of 1,2diacylglycerol (DAG) and IP3 were assessed in response to cholecystokinin (CCK). Results: CCK-induced contraction was transient in the adult and sustained in the kitten. U73122 blocked contraction in adult antral cells but not kitten antral cells. In adult antral tissue, CCK (1007 mol/L) caused an early transient increase in the level of DAG, whereas in the newborn antrum, CCK (1007 mol/L) caused a sustained increase in the DAG level for up to 4 minutes. IP3 showed an early increase in both age groups. Newborn contraction is associated with an initial increase in IP3 and sustained elevation of DAG levels, whereas in adult antral cells, there is a transient increase in both IP3 and DAG. Conclusions: The relative inaccessibility of intracellular calcium stores in the newborn is associated with age-related differences in signal transduction pathways.

ability to use intracellular calcium stores during contraction.2,3 Gastrointestinal smooth muscle contraction is modulated by specific intracellular pathways that may use different sources of calcium (i.e., intracellular vs. extracellular).4 Because newborn antral circular smooth muscle cells have a relative inability to use intracellular calcium stores, we have examined the hypothesis that the intracellular pathways followed during contraction change during development. In this study we used cholecystokinin (CCK) as an agonist to study the contractile process in antral circular smooth muscle cells of both the kitten and the adult cat. The results show that, in the kitten, CCK-induced contraction is sustained and is associated with prolonged elevation of 1,2-diacylglycerol (DAG) levels, whereas in the adult, the contraction is transient and associated with only a transient increase of DAG and inositol 1,4,5triphosphate (IP3 ). These kinetically different contractile patterns and differences in the levels of second messengers could be the basis for maturational changes that occur in circular smooth muscle cells of the antrum.

Materials and Methods Animals

T

he newborn’s diet is in a period of transition from liquid breast milk to a diet that is a mixture of solids and liquids. The fundus of the stomach is thought to have primarily a storage function, whereas the antrum has responsibility for trituration of solids and control of gastric emptying. The period of dietary transition from a liquid to mixed liquid/solid meal is the first time that the antrum is required to triturate and empty a solid meal, and this places an additional requirement on the developing stomach. Because gastroesophageal reflux during this period has been associated with abnormalities in gastric motor function,1 it is relevant and important to examine the development of gastric motor function during infancy. In circular smooth muscle of the gastrointestinal tract, changes in intracellular calcium levels are fundamental to the contractile process. Previous data have shown that newborn antral circular smooth muscle has a decreased / 5e1f$$0009

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Adult cats of both sexes weighing between 2.5 and 5 kg and newborn kittens between the fourth and seventh days of life were used in this study. According to methods approved by the University of Michigan’s Committee on Use and Care of Animals, the animals were initially anesthetized with a combination of ketamine and xylazine and then killed with an overdose of phenobarbital. The chest and abdomen were opened with a midline incision exposing the esophagus, stomach, and small intestine. The stomach and duodenum were removed together and pinned on a wax block at their in vivo dimensions and orientation. The stomach was opened along the lesser curvature. The mucosa was removed by sharp dissection under a microscope Abbreviations used in this paper: DAG, 1,2-diacylglycerol; DG kinase, sn-1,2-diacylglycerol kinase; EGTA, ethylene glycol-bis(b-aminoethyl ether)-N,N,N*,N*-tetraacetic acid; IP3 , inositol 1,4,5-triphosphate. q 1997 by the American Gastroenterological Association 0016-5085/97/$3.00

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at 47C in a dish containing oxygenated Krebs’ buffer ([in mmol/L]: NaCl, 116.6; NaHCO3 , 21.9; KH2PO4 , 1.2; glucose, 5.4; and MgCl2 , 1.2; pH 7.4) with 1% b-mercaptoethanol, 0.1 mmol/L phenylmethylsulfonyl fluoride, 1 mg/mL pepstatin A, and 5 mg/mL leupeptin. The underlying external circular muscle layer was cut into 0.5-mm-thick slices with a Stadie Riggs tissue slicer (Thomas Scientific Apparatus, Philadelphia, PA), and the outer longitudinal layer was discarded. The slices of circular muscle were placed flat on a wax surface, and tissue squares were made by cutting twice with a 2-mmblade block; the second was cut at right angles to the first.

Dispersion of Smooth Muscle Cells Isolated smooth muscle cells were obtained by enzymatic digestion, as described previously.2,4 Antral tissue squares were digested in HEPES-buffered physiological solution containing 190 U/mL collagenase for 2 hours. The physiological solution contained the following: 115 mmol/L NaCl, 5.8 mmol/L KCl, 12 mmol/L KH2PO4 , 2.5 mmol/L glucose, 25 mmol/L HEPES, 2 mmol/L CaCl2 , and 0.6 mmol/L MgCl2 , with 0.3 mg/mL BME amino acid supplement and 0.1 mg/ mL soybean trypsin inhibitor (final pH 7.4). The solution was gently gassed with 100% O2 . At the end of the digestion period, the tissue was placed over a 500-mm Nitex mesh (TETCO Inc., Elmsford, NY), rinsed in collagenase-free physiological solution to remove any trace of collagenase, and then incubated in collagenase-free physiological solution at 317C that had been gassed with 100% O2 . The cells were allowed to dissociate freely in this physiological solution 10–20 minutes. Throughout the entire procedure, care was taken not to agitate the fluid to avoid cell contraction in response to mechanical stress. When cell contraction studies were performed in an environment in which extracellular calcium was absent, the tissue was handled in the manner described above until the tissue was ready to harvest in a collagenase-free media. At that point, the tissue was placed in a collagenase-free physiological solution that contains 0 calcium and 2 mmol/L ethylene glycolbis(b-aminoethyl ether)-N,N,N*,N*-tetraacetic acid (EGTA). Cells from both the adult and kitten were collected at 20 minutes and subjected to the cell contraction protocol as described. All glassware was prerinsed in a 0.05% silicon solution to prevent the cells from adhering to the glass.

Drugs and Chemicals L-a-1,2-dioctanoylglycerol was obtained from Avanti Polar Lipids (Alabaster, AL), collagenase type II from Worthington Biochemicals (Freehold, NJ), pepstatin A and leupeptin from Boehringer-Mannheim (Indianapolis, IN), sn-1,2-diacylglycerol kinase (DG kinase) from Lipidex Inc. (Westfield, NJ), U73122 from Biomol (Plymouth Meeting, PA), and [g-32P]adenosine triphosphate from New England Nuclear Corp. (Boston, MA). CCK, IP3 , EGTA, and other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO).

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DAG Measurements DAG measurements were determined by incubating the tissue squares with 10 mg of DG kinase in the presence of [32P]adenosine triphosphate to form [32P]phosphatidic acid. The lipids were then extracted with chloroform/methanol (1:2) and separated by thin-layer chromatography in chloroform/methanol/ acetone/acetic acid/water (30:9.8:11.3:9:5.3). The presence of phosphatidic acid was determined by exposure to radiograph film against the thin-layer chromatography plate in a film cassette. A control lane for a known amount of DAG incubated with DG kinase was used both to document the precise location of phosphatidic acid and to ensure the quantitation of phosphatidic acid. The bands determined to contain [32P]phosphatidic acid were scraped into scintillation vials and assayed for radioactivity.

Measurement of IP3 Antral muscle cell suspensions were exposed to agents and agonists, and the incubation was terminated at the times indicated by addition of one half of the volume of 20% trichloroacetic acid. The resultant precipitate was centrifuged at 3000g for 10 minutes. Trioctylamine-freon was then used to extract the acid from the supernatant. The mass of IP3 was measured by a radioreceptor binding assay using rat cerebellar protein. Briefly, 100-mL aliquots of prepared samples or 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 a buffer of 50 mmol/L Tris-Cl, 5 mmol/L BME, and 1 mmol/L ethylenediaminetetraacetic acid, pH 8.4, and incubated for 30 minutes at 47C. 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 .

Agonist-Induced Contraction of Isolated Muscle Cells The cells were contracted by exposing them to specified concentrations of CCK octapeptide. At the appropriate time intervals, the cells then were fixed in acrolein at a final concentration of 1%. The cell length of 30 consecutive intact cells from each slide were measured through a phase-contrast microscope (Carl Zeiss), a television camera (model WV-1550; Panasonic, Seacaucus, NJ), and a television screen (model WV5410; Panasonic). The camera was connected to a video microscaler (model IV-550; For-A Co., West Newton, MA) that was used to obtain a measurement of cell length. Data were expressed as percent decrease in cell length from control.

Protein Determination Protein values were obtained for tissues after being used in DAG experiments by hydrolyzing the tissue squares with 0.1N NaOH at 607C to solubilize the protein, followed

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by neutralization with HCl. The amount of protein present was determined by colorimetric analysis.

Statistical Analysis Data are expressed as the means { SEM. Statistical differences between means were determined by the Student’s t test. Values of P ° 0.05 were considered the threshold for statistical significance.

Results CCK-Induced Contraction in Adult and Newborn Antral Cells Isolated smooth muscle cells from adult and newborn cats were exposed to increasing doses of CCK for 60 seconds. The responses were similar in the different age groups with the maximum contraction of both being achieved at 1009 mol/L (Figure 1). We then examined the ability of the cells to sustain contraction on continuous exposure to the maximally effective dose of CCK (1009 mol/L). We found that, although the adult cells showed a transient response, returning rapidly to preexposure lengths, the kitten cells remained contracted for at least 8 minutes (Figure 2). DAG Levels in Adult and Newborn Smooth Muscle in Response to CCK

seconds and were analyzed for DAG content (Figure 3). The response was an increase in the DAG level in response to increasing concentrations in both age groups. Tissue squares of the antral circular smooth muscle from adult and newborn cats were exposed to the maximal dose of CCK (1006 mol/L) for increasing times. As shown in Figure 4, tissues from both age groups responded similarly with a sharp initial increase in DAG level produced. However, the increased level of DAG is sustained for at least 8 minutes in the newborn, while decreasing to control levels in the adult within 1 minute. The concentration of an agonist required to generate a maximal increase in DAG levels in tissue squares is considerably higher than the concentration of an agonist that results in maximal contraction of isolated muscle cells. This discrepancy in tissue and isolated smooth muscle cells has been noted previously, whereby isolated cells contract in response to concentrations of an agonist that are considerably lower than needed in tissue squares.5,6 IP3 Levels in Smooth Muscle Cells From the Adult and Newborn Isolated smooth muscle cells from the antra of adults and newborns were exposed to CCK at 1008 mol/ L for 15 seconds (Figure 5). The level of IP3 was assayed

Tissue squares from adults and newborns were then exposed to increasing concentrations of CCK for 30

Figure 1. Shortening of isolated circular smooth muscle cells from adult (h) and kitten (j) antrum in response to increasing doses of CCK. The dose that elicits a maximally effective response in both ages is 1009 mol/L CCK. Each point is the mean of n Å 6 for kittens and n Å 7 for adults, with 30 cells counted for each animal. Error bars represent the SEM.

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Figure 2. Time course of response to the maximally effective dose of CCK (1009 mol/L) in isolated smooth muscle cells from adult (h) and kitten (j) antrum. The response to CCK is transient in adult antral smooth muscle cells, whereas it is sustained in kitten antral smooth muscle cells. Each point is the mean of n Å 4 for kittens and n Å 5 for adults, with 30 cells counted for each animal. Error bars represent the SEM. The responses at 240 and 480 seconds are significantly different (**P õ 0.001) between kitten and adult.

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Figure 3. CCK-induced increases in levels of DAG as measured in tissue squares from circular smooth muscle of adult cat (h) and kitten (j) antrum. The values are expressed as the percentage of measurements in control tissue. Increasing doses of CCK result in increased levels of DAG in both ages. The DAG level in response to CCK was measured at 30 seconds and is shown for the adult antrum and kitten. Each point is the mean of n Å 5 for adults and n Å 6 for kittens. Error bars represent the SEM. The value for the kitten is significantly greater at 1006 mol/L (*P õ 0.05).

Figure 4. Time course of CCK-induced increase in DAG levels in tissue squares from circular smooth muscle of the adult cat (h) and kitten (j) antrum. The values are expressed as the percentage of measurements in control tissue. Stimulation with CCK at 1006 mol/L in the adult tissue results in a transient increase in DAG levels, whereas stimulation in kitten tissue results in a sustained level of DAG. Each point is the mean of n Å 6 for adults and n Å 5 for kittens. Error bars represent the SEM. The levels at 30 seconds (*P õ 0.01), 60 seconds, and 8 minutes (**P õ 0.001) are significantly greater in the kitten than the adult.

and was found to be comparable in the cells of both age groups based on the percent of IP3 found in unexposed cells. At 4 minutes, the level of IP3 was at basal levels in both the adult and newborn. The Effect of 0 Ca2/ on the CCK-Induced Contraction of Antral Smooth Muscle Cells To examine their response to CCK in the absence of extracellular calcium, isolated cells were preincubated in HEPES buffer, which did not contain calcium and did contain 2 mmol/L EGTA. The cells were then exposed to the maximally effective dose of CCK (1009 mol/L) for 30 and 60 seconds (Figure 6). The removal of extracellular calcium completely blocked the CCK-induced contraction in the newborn cells but only inhibited the response in the adult cells by 26.4% at 30 seconds and 53.5% at 60 seconds. The Effect of U73122 on the CCK-Induced Contraction of Antral Smooth Muscle Cells Isolated smooth muscle cells from the antra of adults and newborns were preincubated for 60 seconds / 5e1f$$0009

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Figure 5. IP3 levels in isolated circular smooth muscle cells from adult cat (h) and kitten (j) antrum. The values are expressed as the percentage of measurements in control cells after exposure to 1008 mol/L CCK. The resultant increased IP3 levels in kitten (n Å 3) and adult (n Å 5) at 15 seconds were comparable. The levels are not significantly different from basal at 4 minutes in either age group (kittens, n Å 3; adults, n Å 5).

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Figure 6. The response of isolated antral smooth muscle cells from adults (h) and kittens (j) to the maximally effective dose of CCK (1009 mol/L) after omission of extracellular calcium from the medium and addition of 2 mmol/L EGTA to the medium. The removal of extracellular calcium completely blocks the contractile response in isolated kitten antral cells, while causing an inhibition of 26.4% at 30 seconds and 53.5% at 60 seconds in adult antral cells. The inhibition is calculated as the percent reduction of the contractile response compared with CCK-induced contraction under control conditions and is significantly greater in the kitten than the adult at 30 seconds (*P õ 0.0005) and 60 seconds (**P õ 0.005). Each bar is the mean of n Å 4 for adults and n Å 3 for kittens, with 30 cells counted per animal. Error bars represent the SEM.

with the phosphatidylinositol 4,5-bisphosphate phospholipase inhibitor U73122 at 1008 mol/L and at 1006 mol/L, and then were exposed to the maximally effective dose of CCK (1009 mol/L) for 15 and 60 seconds (Figure 7). The response is the reverse of that observed in the absence of calcium in that the adult cells were inhibited in a dose-dependent manner, whereas the newborn cells were affected minimally.

Discussion The circular smooth muscle of the gastric antrum is essential to the trituration of solids and the initiation of a peristaltic sequence that promotes gastric emptying.7 We have shown previously that isolated smooth muscle cells from the circular smooth muscle of the newborn antrum are unable to use intracellular calcium stores in support of contraction, whereas adult antral cells are able to use intracellular calcium stores.2 The finding that CCK does not induce isolated antral smooth muscle contraction in the kitten in the absence of extracellular calcium (Figure 6) but is able to bring about contraction in the adult is consistent with these previous observations and suggests that CCK-induced contraction may follow distinct intracellular pathways in the adult and kitten. / 5e1f$$0009

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Agonist-induced contraction in smooth muscle can occur when specific surface receptors are bound-activating phospholipases that result in the hydrolysis of membrane phospholipids and the generation of second messengers that bring about contraction. CCK has been shown to have both myogenic8 and neurogenic9 effects on gastric smooth muscle contraction and is able to activate specific phospholipases with the cell membrane. The activation of phospholipase C can result in the hydrolysis of phosphatidylinositol phosphate to form the intracellular messengers 1,4,5-IP3 , which releases calcium from intracellular sources, and 1,2-diacylglycerol DAG, which is an endogenous activator of protein kinase C.10,11 Hydrolysis of phophatidylcholine can produce DAG and phosphocholine without concurrent production of IP3 . Phosphatidylcholine hydrolysis can occur either by stimulation by a phosphatidylcholine-specific phospholipase C12,13 or by phospholipase D.14 Smooth muscle from the antrum of the adult cat shows a contractile response that is associated with a transient increase in the level of DAG and an early increase in the level of 1,4,5-IP3 that is compatible with phosphatidylinositol phosphate hydrolysis by phospholipase C. In the kitten, the sustained contraction observed in response to CCK is associated with sustained levels of 1,2-DAG but only an early transient increase in 1,4,5-IP3 . This suggests that there may be a difference in the manner in which the two age groups maintain intracellular DAG levels. One possibility is that the substrate used (i.e., phosphatidylinositol phosphate or phospholipase C) for production of DAG is different in the adult and newborn. It is possible that adult antral cells use a pathway that primarily depends on phosphatidylinositol phosphate hydrolysis by phospholipase C, whereas kitten antral cells initially use both this pathway and DAG production directly from phosphatidylcholine hydrolysis by either phospholipase D or phosphatidylcholine–specific phospholipase C. We have further investigated this possibility by examining contraction in the presence of U73122 that has been shown to inhibit phosphatidylinositol phosphate hydrolysis. U73122 is an amphipathic cation that reversibly competes with calcium for the binding site on phospholipase C that regulates expression of the phospholipase activity.15 It has also been suggested that U73122 reduces the pool of phosphoinositides available for hydrolysis.16 U73122 has been shown to inhibit agonist-induced IP3 production in polymorphonuclear neutrophils17 and human amnion cells.15 U73122 (1006 mol/ L) blocks virtually the entire contractile response to CCK in the adult but not in kitten antral smooth cells (Figure 7). This would further support the hypothesis that phosphatidylinositol phosphate hydrolysis is a primary mechWBS-Gastro

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Figure 7. Response of isolated smooth muscle cells from adults (h) and kittens (j) to maximally effective doses of CCK (1009 mol/L) in the presence of (A ) U73122 at 1008 mol/L and (B ) U73122 at 1006 mol/L. U73122 appeared to have a dose-dependent inhibitory effect on contraction in adult antral cells but not in kitten antral cells. The inhibition is calculated as the percentage reduction of the contractile response compared with CCK-induced contraction under control conditions and is significantly greater in adults than in kittens at 15 and 60 seconds for both 1008 mol/L and 1006 mol/L U73122 (*P õ 0.005). Each bar is the mean of n Å 4 for adults and n Å 3 for kittens, with 30 cells counted per each animal. Error bars represent the SEM.

anism responsible for contraction in the adult, whereas the relatively smaller inhibition of contraction observed in the kitten suggests that an additional pathway such as phosphatidylcholine hydrolysis by phospholipase D or phosphatidylcholine–specific phospholipase C is operative in the initial stages of contraction. The sustained contraction observed in the kitten may be associated with DAG production from phosphatidylcholine-specific hydrolysis. Our techniques of measuring DAG measures pool size but does not measure new production of DAG. It is therefore possible that the kitten and adult do not use a different substrate for DAG production and that the differences in levels of DAG are related to different levels of degradation associated with maturation (i.e., the kitten degrades DAG less rapidly than the adult). Although little is known about the developmental regulation of these processes, DAG can be inactivated by several different mechanisms, including DAG kinase, which forms phosphatidic acid that can be used for the resynthesis of phosphoinositides,18 DAG lipase, which would leave the glycerol back bone and free fatty acids,19 or a transferase reaction that creates a triglyceride or phophatidyl choline.20 The observation that there is not an increase in IP3 levels observed in association with sustained contraction in the newborn antrum is consistent with our previous / 5e1f$$0009

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observation that the presence of exogenous IP3 does not result in the contraction of permeabilized isolated newborn antral smooth muscle cells. This inability to access intracellular calcium sources in the newborn kitten results in utilization of alternative pathways of signal transduction in response to contractile agonists. It is possible that these alternative pathways may result in contractile activity that is quantitatively and qualitatively different from that observed in the adult antrum. Maturation of intracellular pathways has been most extensively studied in the central nervous system where it has been shown that regional quantitative differences in agonist-induced stimulation of phosphoinositide metabolism often occur.21 The location of these regional differences has been noted to shift during development, and at times, they may coincide with brain growth.22 There are data to suggest that IP3 and protein kinase C in the central nervous system act individually or complimentarily rather than synergistically and that their expression is regulated by different mechanisms during development. In the cat, visual cortex and hippocampus high levels of [3H]phorbol dibutyrate (labeling protein kinase C) sites appear much earlier than do [3H]IP3 sites.23 Initial cell length, maximally effective contractile response, and dose dependency were similar in the kitten and adult cat in response to CCK. However, the kinetics and calcium requirements of the contractile response in WBS-Gastro

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kitten antral cells are quite different than in adult antral cells. The inability of the kitten antrum to respond with a transient contraction to CCK may provide the explanation for the sustained contraction (antropyloric spasm) that can be observed in the antrum of human infants who have significant emesis.24,25 We conclude that the relative inability of the newborn antrum to use intracellular calcium stores results in qualitatively different signal transduction events than in the adult. These different pathways are associated with a kinetically different contraction that may have an effect on the ability of the antrum to participate in gastric motor function such as trituration or antropyloric peristalsis. In the infant, the transition from a predominantly liquid to a mixed solid-liquid meal increases the burden on the gastric antrum at a time when there are a large number of clinical problems in the infant relating to upper gastrointestinal motor function. Symptoms such as gastroesophageal reflux or vomiting are common during the first year of life26 and may be related to other medical conditions, such as respiratory problems and/or failure to thrive.27,28 Many of these infants have been shown to have delayed patterns of gastric emptying.1 It remains to be seen if the maturational changes that occur in circular smooth muscle cells of our animal model are relevant to the human condition.

References 1. Hillemeier AC, Lange R, McCallum R, Seashore J, Gryboski JD. Delayed gastric emptying in infants with gastroesophageal reflux. J Pediatr 1981;98:190–193. 2. Hillemeier AC, Bitar KN, Biancani P. Developmental characteristics of the kitten antrum. Gastroenterology 1991;101:339–343. 3. Zitterman J, Ryan JP. Development of gastric antral smooth muscle contractility in newborn rabbits. Am J Physiol 1990;258: G571–G575. 4. Bitar KN, Hillemeier C, Biancani P. Differential regulation of smooth muscle contraction in rabbit internal anal sphincter by substance P and bombesin. Life Sci 1990;47:2429–2434. 5. Bitar KN, Zfass AM, Makhlouf GM. Interaction of acetylcholine and cholecystokinin with dispersed smooth muscle cells. Am J Physiol 1979;237:E172–E176. 6. Fisher RS, Lipshutz W, Cohen S. The hormonal regulation of pyloric sphincter function. J Clin Invest 1973;52:1289–1296. 7. Dent J, Sun WN, Anvari M. Modulation of pumping function of gastric body and antropyloric contractions. Dig Dis Sci 1994;39: 28S–31S. 8. Rakovska A, Milenov K, Henklein P. Effects of a new cholecystokinin antagonist (GE-410) on the smooth muscle of the guinea pig ileum. Life Sci 1990;47:1037–1041. 9. Mantyh CR, Pappas TN, Vigna SR. Localization of cholecystokinin A and cholecystokinin B/gastrin receptors in the canine upper gastrointestinal tract. Gastroenterology 1994;107:1019–1030.

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10. Hokin LE. Receptors and phosphoinositide-generated second messengers. Annu Rev Biochem 1985;54:205–235. 11. Berridge MJ. Inositol trisphosphate and diacylglycerol: two interacting second messengers. Annu Rev Biochem 1987;56:159– 193. 12. Billah MM, Anthes JC. The regulation and cellular functions of phosphatidylcholine hydrolysis. Biochem J 1990;269:281–291. 13. Dennis EA, Rhee SG, Billah MM, Hannun YA. Role of phospholipases in generating lipid second messengers in signal transduction. FASEB J 1991;5:2068–2077. 14. Murthy KS, Makhlouf GM. Agonist-induced translocation of Ca2/independent PKC-e mediates sustained contraction in intestinal smooth muscle (abstr). Gastroenterology 1995;108:A992. 15. Bleasdale JE, Bundy GL, Bunting S, Fitzpatric FA, Huff RM, Sun FF, Pike JE. Inhibition of phospholipase C–dependent processes by U-73122. New York: Raven, 1989. 16. Vickers JD. U73122 affects the equilibria between the phosphoinositides as well as phospholipase C activity in unstimulated and thrombin-stimulated human and rabbit platelets. J Pharmacol Exp Ther 1993;266:1156–1163. 17. Smith RJ, Sam LM, Justen JM, Bundy GL, Bala GA, Bleasdale JE. Receptor-coupled signal transduction in human polymorphonuclear neutrophils: effects of a novel inhibitor of phospholipase c–dependent processes on cell responsiveness. J Pharmacol Exp Ther 1990;253:688–697. 18. Kanoh H, Sakane F, Shin-Ichi I, Wada I. Diacylglycerol kinase and phosphatidic acid phosphatase-enzymes metabolizing lipid second messengers. Cell Signal 1993;5:495–503. 19. Hee-Cheong M, Severson DL. Metabolism of dioctanoylglycerol by isolated cardiac myocytes. J Mol Cell Cardiol 1989;21:829– 837. 20. Florin-Christensen J, Florin-Christensen M, Delfino JM, Rasmussen H. New patterns of diacylglycerol metabolism in intact cells. Biochem J 1993;289:783–788. 21. Balduini W, Candura SM, Costa LG. Regional development of carbachol-, norepinephrine-, and serotonin-stimulated phosphoinositide metabolism in rat brain. Brain Res Dev Brain Res 1991; 62:115–120. 22. Tan XX, Costa LG. Postnatal development of muscarinic receptorstimulated phosphoinositide metabolism in mouse cerebral cortex: sensitivity to ethanol. Brain Res Dev Brain Res 1995;86: 348–353. 23. Jia WW, Liu Y, Cynader M. Postnatal development of inositol 1,4,5-triphosphate receptors: a disparity with protein kinase C. Brain Res Dev Brain Res 1995;85:109–118. 24. Swischuk LE, Tyson KR. ‘‘Burned-out’’ pyloric stenosis: an elusive gastric outlet obstruction. Radiology 1975;117:373–379. 25. Swischuk LE, Hayden CKJ, Tyson KR. Short segment pyloric narrowing. Pylorospasm or pyloric stenosis? Pediatr Radiol 1981; 10:201–205. 26. Hillemeier AC. Gastroesophageal reflux; diagnostic and therapeutic approaches. Pediatr Clin North Am 1996;43:197–212. 27. Burton DM, Pransky SM, Katz RM, Kerarns DB, Seid AB. Pediatric airway manifestations of gastroesophageal reflux. Ann Otol Rhinol Laryngol 1992;101:742–749. 28. Chen PH, Chang MH, Hsu SC. Gastroesophageal reflux in children with chronic recurrent bronchopulmonary infection. J Pediatr Gastroenterol Nutr 1991;16:16–22. Received April 4, 1996. Accepted April 10, 1997. Address requests for reprints to: A. Craig Hillemeier, M.D., A520 MSRB 1 Box 0658, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0658. Fax: (313) 763-5739. Supported by grants R01 HD20054 and R01 DK42876 from the National Institutes of Health.

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