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Biomedical subjects

C Hillemeier

Publications and source records attributed to C Hillemeier.

At least 19 recordsLinked to original sources
PubMed1996

Protein kinase C mediates spontaneous tone in the cat lower esophageal sphincter.

The intracellular pathways responsible for maintenance of tone in the lower esophageal sphincter (LES) are not well understood. We show that the protein kinase C (PKC) antagonists (1-(5-isoquinolinesulphonyl)-2-methylpiperazine dihydrochloride) and calphostin C reduce spontaneous resting tone in LES muscle strips, whereas the calmodulin antagonist N-(6-aminohexyl-5-chloro-1-naphthalenesulfonamide hydrochloride) has no effect, which suggests that LES tone is maintained by a PKC-mediated mechanism. In addition, U73122, an inhibitor of phosphatidylinositol-4,5-bisphosphate (PIP2)-specific phospholipase C, and D609, an inhibitor of phosphatidylcholine-specific phospholipase C, reduced diacylglycerol formation and LES tone in a concentration-dependent manner. Finally diacylglycerol levels and PKC activity were reduced during relaxation of the LES induced by the inhibitory neurotransmitter vasoactive intestinal peptide. These data suggest that resting LES tone is associated with elevated diacylglycerol levels and PKC activity, which are reduced during relaxation. Diacylglycerol is derived from at least two different sources. Hydrolysis of PIP2 by PIP2-specific phospholipase C produces equimolar amounts of inositol 1,4,5-triphosphate and diacylglycerol, which may interact synergistically to activate PKC and develop tone. Furthermore, PKC-mediated contraction may be augmented by additional diacylglycerol production arising from the hydrolysis of phosphatidylcholine by phosphatidylcholine-specific phospholipase C.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1995

Characterization of actin and myosin in the developing stomach.

During growth and development, dietary intake changes from being predominantly liquid in the newborn period to mixed solid liquid meals. These alterations in diet vary the functional demands placed on the stomach. It has been shown that, during development, smooth muscle of the stomach undergoes changes in the mechanism responsible for the contractile process. In this study, we have investigated the possibility that there are structural changes in two of the major proteins that are responsible for generation of force during smooth muscle contraction: actin and myosin. Actin and myosin were identified in newborn kittens (1 wk old) and adult gastric smooth muscle using one-dimensional SDS-PAGE. Although both the antrum and fundus of the kitten have significantly smaller total amounts of actin and myosin per mg protein than the adult, the ratio of actin to myosin is not significantly different between the age groups. Two different myosin heavy chain (MHC) isoforms, MHC1 (205 kD) and MHC2 (200 kD), were identified in all tissues. The relative amount of MHC1 remained constant during maturation of the stomach. We observed an increase in the amount of MHC2 in the adult, which resulted in a decreased ratio of MHC1 to MHC2 in the adult. We postulate that the decreased quantity of actin and myosin in the kitten stomach and the observed changes in the ratio of the MHC isoforms are related to changes in the gastric motor that occur during growth and development.

Actins↗
PubMed1995

An overview of the effects of dietary fiber on gastrointestinal transit.

This article is a review of the effects that ingested dietary fiber has on gastrointestinal transit time. The various phases of gastrointestinal motility are considered, and the effect of fiber on these various components is explored. Fiber affects each phase of gastrointestinal motility differently; however, the most dramatic effect on decreased transit time and frequency of bowel movements result from the variations it causes in colonic transit time. The mechanisms of defecation in children and the ways in which fiber affects the frequency of bowel movements are emphasized. All types of fiber do not affect gastrointestinal transit in a similar manner, and different preparations of the same fiber either may delay or may increase the time of intestinal transit. In general, fiber is found to increase the frequency of bowel movements and may prove to be of considerable benefit in treating constipation, a common childhood disorder.

Child↗
PubMed1994

Calcium requirements for acetylcholine-induced contraction of cat esophageal circular muscle cells.

Esophageal circular muscle cells isolated by enzymatic digestion contracted in response to acetylcholine (ACh) and in response to the protein kinase C (PKC) agonist 1,2-dioctanoylglycerol (1,2 DAG). Both responses were blocked by PKC antagonists but not by calmodulin antagonists. Furthermore, specific PKC activity, measured in the particulate fraction of the muscle, increased in response to cholinergic stimulation, suggesting that ACh-induced contraction is mediated by a PKC-dependent pathway. ACh-induced contraction decreased with decreasing extracellular Ca2+ and was blocked in Ca(2+)-free physiological salt solution (PSS). Similarly, contraction by the nonhydrolyzable GTP analogue guanosine 5'-O-(3-thiotriphosphate) was blocked by removal of Ca2+ from the PSS. Diacylglycerol production in response to ACh was reduced when extracellular Ca2+ was reduced from 2 to 0.5 mM and was abolished in Ca(2+)-free PSS. The response to 1,2-DAG, however, did not significantly change as extracellular Ca2+ or cytosolic Ca2+ was reduced to zero. Heparin (10 micrograms/ml), thapsigargin (3 microM), or the Ca2+ ionophore A-23187 (3 microM) had no effect on 1,2-DAG or ACh-induced contraction in permeable cells. The data suggest that contraction in response to ACh is mediated by influx of extracellular Ca2+ and a PKC-dependent pathway. Ca2+ may be required mainly to activate the phospholipases responsible for production of diacylglycerol, since contraction of esophageal muscle cells in response to 1,2-DAG is Ca2+ independent.

Acetylcholine↗
PubMed1994

Differential signal transduction pathways in cat lower esophageal sphincter tone and response to ACh.

Lower esophageal sphincter (LES) basal tone and contraction in response to maximally effective doses (Emax) of acetylcholine (ACh) may be mediated by different intracellular transduction pathways. In the basal state resting tone, inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] formation and levels of diacylglycerol (DAG) (C. Hillemeier, K. N. Bitar, and P. Biancani, unpublished data) are higher in LES circular muscle than in esophageal muscle, which does not maintain tone. In vitro resting tone and spontaneously elevated formation of Ins(1,4,5)P3 in LES circular muscle strips decrease in a dose-dependent manner in response to the phospholipase C antagonist 1-[6-([(17-beta)-3-methoxyestra-1,3, 5(10)-trien-17-yl]amino)hexyl]-1H-pyrrole-2,5-dione (U-73122). Basal Ins(1,4,5)P3 formation, however, is submaximal, since it can be increased by cholinergic stimulation. These data suggest that LES tone is associated with partial activation of phospholipase C. We therefore tested submaximal doses of Ins(1,4,5)P3 and DAG in permeabilized LES muscle cells and found that they act synergistically; their interaction depends on calcium release and is mediated through a protein kinase C (PKC)-dependent pathway. In contrast, we have previously shown that contraction induced by Emax of ACh is mediated through calmodulin-dependent mechanisms (14). To investigate these differences, we tested high and low doses of ACh. Contraction induced by high doses of ACh was inhibited by calmodulin but not by PKC antagonists, as previously reported, but low ACh doses were preferentially inhibited by PKC antagonists. Similarly, low Ins(1,4,5)P3 concentrations activated a PKC-dependent pathway, whereas contraction induced by Emax of Ins(1,4,5)P3 was calmodulin dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

Acute experimental esophagitis impairs signal transduction in cat lower esophageal sphincter circular muscle.

It has been previously shown that induction of experimental esophagitis in the cat by esophageal perfusion for 30 minutes with 0.1N HCl for 4 consecutive days results in a significant reduction of in vivo lower esophageal sphincter (LES) resting pressure and in vitro spontaneous tone without affecting esophageal response to KCl. It has also been shown that basal LES tone and LES contraction in response to acetylcholine depend on the release of calcium from intercellular stores, whereas esophageal contraction is mediated by extracellular calcium. The present report shows that esophageal acid perfusion impairs the transduction pathway mediating lower esophageal sphincter contraction in response to acetylcholine through release of intracellular calcium because LES strips and single cells no longer contract in response to acetylcholine if calcium is removed from the physiologic salt solution. This suggests that either the intracellular calcium stores or the release mechanisms that mediate maintenance of tone and contraction in response to acetylcholine may be damaged. However, the acid perfusion has no effect on the acetylcholine response in the esophagus, which is mediated by the influx of extracellular calcium. In the LES circular muscle, the injury results in reduced levels of inositol phosphates without affecting resting levels of 5'-cyclic adenosine monophosphate or 5'-cyclic guanosine monophosphate. The reduced levels of 1,4,5-inositol trisphosphate are consistent with impairment in the mechanisms responsible for release of intracellular calcium, although concurrent damage to calcium stores may also occur.

Acute Disease↗
PubMed1991

Regulation of smooth muscle contraction in rabbit internal anal sphincter by protein kinase C and Ins(1,4,5)P3.

We have examined the role of protein kinase C (PKC)-beta II and its functional relationship to inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and intracellular Ca2+ in the contraction of smooth muscle cells from the rabbit internal and sphincter (IAS). PKC-beta (0.1-100 U/ml) and Ins(1,4,5)P3 (10(-9) to 10(-6) M) caused concentration-dependent contraction of IAS smooth muscle cells permeabilized by saponin. The combination of threshold concentrations of Ins(1,4,5)P3 (10(-9) M) and PKC (0.1 U/ml) was more than additive, causing near maximal shortening (28.2 +/- 2.1% decrease in cell length from control). The response to high concentrations of Ins(1,4,5)P3 and PKC used in combination was not greater than the response to either agent alone. The calmodulin antagonist W-7 (10(-9) M) inhibited the maximal contraction induced by Ins(1,4,5)P3 but not contraction caused by PKC, whereas the PKC antagonist H-7 (10(-6) M) inhibited the maximal contraction induced by PKC but not contraction caused by Ins(1,4,5)P3. Threshold doses of the ionophores A23187 (10(-9) M) and ionomycin (0.2 ng/ml) caused little contraction by themselves, but they potentiated the response elicited by a threshold concentration of PKC (0.1 U/ml), inducing maximal contraction. Preincubation of IAS cells with 4 mM Sr2+, which inhibits the release of intracellular Ca2+, abolished the potentiating effect of Ins(1,4,5)P3 and calcium ionophores on PKC, but the calmodulin antagonist W-7 did not. These data suggest that the contractile effect of maximally effective doses of PKC is independent of the effects of Ins(1,4,5)P3. At submaximal concentrations, however, PKC-dependent contraction is potentiated by Ins(1,4,5)P3 or by ionophore-mediated release of intracellular Ca2+ without requiring calmodulin activation.

Anal Canal↗
PubMed1991

Intracellular pathways for contraction in gastroesophageal smooth muscle cells.

Intracellular pathways utilized for contraction in response to acetylcholine (ACh), inositol 1,4,5-trisphosphate (IP3), and KCl were examined in isolated circular smooth cells from the esophagus, lower esophageal sphincter (LES), and fundus. In physiological nutrient salt solution (PSS) intact muscle cells isolated from these three tissues responded in a similar dose-dependent manner to ACh. In all tissues the contractile response to ACh was maximal at 30 s. Contraction of smooth muscle cells from LES and fundus did not change after incubation in Ca(2+)-free medium, but contraction of esophageal cells was abolished. KCl-induced contraction of all three cell types was also abolished after incubation in Ca(2+)-free medium. After permeabilization with saponin in cytosolic (low Ca2+) salt solution, muscle cells from LES and fundus contracted in a dose-dependent manner in response to IP3, whereas cells from esophagus did not contract. Contraction of permeabilized LES and fundic cells in response to ACh was the same as that of intact muscle cells. Response to IP3 was more rapid than response to ACh; it reached 85% of maximum by 5 s and peaked at 15 s. Calmodulin antagonists W-7 and CGS 9343B blocked contraction in response to ACh in intact cells from LES and fundus but had no effect on ACh-induced contraction in esophageal cells. These antagonists blocked IP3-induced contraction in permeabilized cells from LES and fundus. KCl-induced contraction in intact cells from all three tissues was not blocked by either W-7 or CGS 9343B. These data suggest that calmodulin antagonists block contraction mediated by release of intracellular Ca2+ induced by ACh or IP3.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed1990

Mechanical properties of obstructed colon in a Hirschsprung's model.

Hirschsprung's disease, induced by aganglionosis of the distal intestinal tract, results in significant morbidity in affected children. This study uses a murine model of congenital aganglionosis in the distal colon to determine the effects of this type of obstruction on the proximal colon. In lethal spotted mice (Ls/Ls) there is a greater increase in the thickness of circular muscle (in the obstructed colon immediately proximal to the aganglionic region) than longitudinal muscle compared with controls. The active forces generated in vitro, as determined by length-tension curves of the circular muscle, are larger than in control littermates, and isolated muscle cells from the area proximal to the obstruction are larger and shorten to a significantly greater degree in response to acetylcholine. These data are consistent with increased muscle mass subsequent to hypertrophy, although simultaneously occurring hyperplasia cannot be excluded. However, the increased forces that developed are not entirely explained by increased muscle mass. When forces are normalized per cross-sectional unit of circular muscle present in the obstructed rings, stress is considerably higher in the rings proximal to the obstruction than in matched rings from obstruction-free littermates. Pressure-diameter relationships calculated from in vitro data show that the Ls/Ls colon is capable of exerting higher intraluminal pressures than the control colon. The increases in circular muscle thickness, forces, and stress are similar to those observed in other models of obstruction. The increased pressure-diameter relationships observed in the colon are unlike an experimental model of obstruction in the ureter and may reflect longstanding obstruction or a difference in response of colonic muscle to obstruction. It is possible that these mechanical changes may affect the course of Hirschsprung's disease.

Animals↗
PubMed1990

Differential regulation of smooth muscle contraction in rabbit internal anal sphincter by substance P and bombesin.

Substance P and bombesin induce contraction of isolated IAS smooth muscle cells by different intracellular mechanisms. The cells contracted in a dose dependent manner to both peptides. The kinetics of contraction were different. Substance P induced contraction peaked at 30 seconds and declined in a time dependent manner while bombesin induced contraction peaked at 30 seconds and was maintained for up to 8 minutes. The absence of extracellular calcium in the medium (0 calcium and 2 mM EGTA) had no affect on substance P induced contraction while it blocked bombesin induced contraction. Substance P induced contraction was blocked by the calmodulin antagonist W7 (10(-9)M) and was not affected by the PKC antagonist H7 (10(-6)M). Bombesin induced contraction was blocked by the PKC antagonist H7 and was not affected by the calmodulin antagonist W7. Our data indicate that substance P induces a transient contraction utilizing intracellular calcium and a calmodulin dependent pathway, while bombesin induces a sustained contraction utilizing calcium from extracellular sources and a calmodulin independent pathway.

Anal Canal↗
PubMed1990

Vitamin A gastrointestinal absorption in persons with Down's syndrome.

The total daily vitamin A intake, physical signs associated with vitamin A deficiency, and the response to a vitamin A challenge were studied in 33 patients with Down's syndrome and in 14 intellectually normal persons (comparison group). The evaluation of detailed dietary histories revealed that the study subjects' mean daily vitamin A intake was similar to the recommended daily allowance for adults and that there was no significant difference of the mean daily vitamin A intake between study and comparison groups (5029 IU and 5706 IU, respectively). Subjects with Down's syndrome had more symptoms usually seen in patients with hypovitaminosis A than the persons in the comparison group. The baseline serum vitamin A levels in the Down's syndrome and comparison groups were within the normal range (106.0 micrograms/dl and 136.5 micrograms/dl, respectively). The vitamin A absorption curve of persons with Down's syndrome paralleled that of normal individuals and no significant difference of vitamin A levels between study and comparison groups was observed except for the 6-h values. Also, the absorption differential (delta = 3-h value + 6-h value/2-O-h value) did not show a significant difference between the two groups. Thus, these investigations do not support previous reports of significantly decreased vitamin A absorption in individuals with Down's syndrome.

Adolescent↗
PubMed1990

Membrane currents and cholinergic regulation of K+ current in esophageal smooth muscle cells.

The tight-seal whole cell recording technique with patch pipettes was used to study membrane currents of smooth muscle cells freshly dissociated from the esophagus of cats. Under voltage clamp with K+ in the pipette, depolarizing commands elicited an initial inward current followed by a transient outward current that peaked and then declined to reveal spontaneous outward currents (SOCs). SOCs were evident at -60 mV and more positive potentials. The reversal of SOCs at the K+ equilibrium potential and their suppression by tetraethylammonium chloride lead to the conclusion that they represent the activity of K+ channels. Acetylcholine (ACh) caused reversible contraction of these cells and had two successive effects on membrane currents, causing transient activation of K+ current followed by suppression of SOCs. Both of these effects were blocked by atropine. Consistent with these observations, in current clamp, ACh caused a transient hyperpolarization followed by depolarization. The inward current activated by depolarization was blocked by external Cd2+, consistent with the inward current being a voltage-activated calcium current. Two types of Ca2+ current could be distinguished on the basis of voltage-activation range, time course of inactivation and "run-down" during whole cell recording.

Acetylcholine↗
PubMed1990

VIP-induced alterations in cAMP and inositol phosphates in the lower esophageal sphincter.

Adenosine 3',5'-cyclic monophosphate (cAMP), guanosine 3',5'-cyclic monophosphate (cGMP), and inositol phosphate (IP) levels were measured in thin tissue samples from the circular smooth muscle of the cat lower esophageal sphincter (LES) at 37 degrees C during vasoactive intestinal peptide (VIP)-induced relaxation. On exposure of in vitro LES circular muscle strips to 10(-6) M VIP at the same temperature, relaxation of spontaneous resting tone begins within 3-6 s, is half maximal at 30 s, and maximal at 1 min. VIP-induced changes in cAMP, cGMP, and IP metabolite levels were measured at 5 and 30 s after the addition of 10(-6) M VIP. At 5 s cAMP levels increased significantly with respect to time-matched unstimulated controls, whereas inositol 1,4,5-trisphosphate (1,4,5-IP3) decreased and these changes remained constant at 30 s. cGMP levels were unchanged at either 5 or 30 s after exposure to 10(-6) M VIP. These data suggest that VIP-induced relaxation is temporally linked to decreased 1,4,5-IP3 as well as increased cAMP levels.

Animals↗
PubMed1989

Role of peptide histidine isoleucine in relaxation of cat lower esophageal sphincter.

Vasoactive intestinal peptide (VIP) is a candidate as an inhibitory neurotransmitter mediating relaxation of the lower esophageal sphincter (LES) because VIP antiserum reduces LES relaxation in response to neural stimulation. Vasoactive intestinal peptide antiserum, however, does not completely block LES relaxation. Thus it is possible that other neurotransmitters may be involved. Peptide histidine isoleucine has structural homologies with VIP, is synthesized with VIP from a common precursor protein, coexists in some nerve cells, and is coproduced with VIP in some tumors. In numerous organ systems VIP and peptide histidine isoleucine (PHI) produce similar effects, with PHI being less potent than VIP by approximately one log number. In the LES both VIP and PHI produce tetrodotoxin-resistant dose-dependent relaxation, with PHI being almost equipotent with VIP. We therefore tested the hypothesis that PHI may be a second neurotransmitter, partly responsible for relaxation of the cat LES, by using a highly specific rabbit PHI antiserum that exhibits minimal cross-binding with VIP, secretin, and glucagon. In 3 animals, LES and brain tissue were extracted in 0.1 N HCl and assayed with a PHI radioimmunoassay. The antiserum cross-reacted with cat brain and LES showing PHI concentrations greater than 100 ng/g, with the LES containing equal or greater concentrations of PHI than brain tissue. In other animals consecutive LES circular muscle strips were cut, mounted in 1-ml muscle chambers, and stimulated with 6-s square-wave trains of 0.1-, 0.2-, 0.4-, and 0.8-ms pulses at 1, 2, and 5 Hz. These parameters produced relaxation that was completely blocked by tetrodotoxin, and reduced by VIP antiserum, but not affected by adrenergic or cholinergic receptor antagonists. Some strips were incubated in 5% or 10% PHI antiserum, whereas others were incubated in the same concentration of preimmunization serum from the same animal. Incubation in normal serum did not significantly affect relaxation, whereas in the antiserum-treated strips, LES relaxation was reduced by a significant amount (20%-30%) at all parameters of stimulation tested. Incubation in antiserum however had no effect on relaxation induced by VIP (10(-8)-10(-6) M). These data suggest that PHI may play a role in LES relaxation induced by electrical stimulation.

Animals↗
PubMed1989

Developmental differences in neurohumoral modulation of the cat lower esophageal sphincter.

It is possible that age-related differences in gastroesophageal function during infancy may predispose the infant to abnormalities. We have therefore evaluated developmental differences in neurohumoral modulation of lower esophageal sphincter function in the cat. These studies were performed in an in vitro setting. In the adult cat, lower esophageal sphincter smooth muscle and postganglionic inhibitory neurons are more sensitive to muscarinic agonists than in the kitten. The circular smooth muscle of the adult lower esophageal sphincter is also more responsive to neurally mediated relaxation, brought about by electrical stimulation. However, there is no difference in response to exogenous vasoactive intestinal peptide between the two age groups. These data suggest that developmental differences in neurohumoral modulation of the lower esophageal sphincter occur in the cat.

Animals↗
PubMed1987

Contraction mediated by Ca2+ influx in esophageal muscle and by Ca2+ release in the LES.

Single cells and whole tissue specimens were used in this study to determine sources of Ca2+ utilized for contraction of the circular muscle layer of esophagus and lower esophageal sphincter (LES) of the cat. In vitro circular muscle specimens from the cat esophagus respond to electrical stimulation with phasic contractions at the end of the stimulus, whereas the LES spontaneously maintains tonic contraction and relaxes during stimulation. In Ca2+-free buffer, esophageal contractions rapidly decline and disappear within 10 min after the removal of Ca2+, while LES tone is only partially reduced. Similarly, incubation in a solution containing the Ca2+ influx blocker, La3+, abolishes esophageal contraction but only partially decreases LES tone. Conversely, strontium and caffeine substantially reduce LES tone without affecting the amplitude of esophageal contractions. In single muscle cells isolated by enzymatic digestion from the LES and the body of the esophagus, blockade of extracellular Ca2+ influx by methoxyverapamil (D 600) or ethyleneglycol-bis-(beta-aminoethylether) N,N'-tetraacetic acid abolished esophageal contraction in response to acetylcholine without affecting LES cells, and conversely, strontium abolished LES contraction without affecting esophageal cells. These data are consistent with the view that extracellular Ca2+ is required to initiate esophageal phasic contraction, while the LES has the ability to utilize intracellular Ca2+ to maintain resting tone and to contract in response to acetylcholine.

Acetylcholine↗