Fundus Smooth Muscle Research Articles

Relations between contractile responses and β-adrenoceptors in gastric fundus of diabetic rats

Contractile responses to norepinephrine (NE), and the population of β-adrenoceptors, were determined in gastric fundus smooth muscle from rats with diabetes induced by streptozotocin(STZ), and age-matched controls. Relaxation and/or contraction of fundus strips of controls and diabetics were induced by 10 −5M NE. Responses to NE were mainly relaxation in gastric fundus isolated from controls, and contraction in fundus isolated from diabetics. Contraction was blocked by 10 −8M prazosin and relaxation was blocked by 10 −6 M propranolol. Relaxation by isoproterenol of contraction induced by 10 −6M acetylcholine was significantly less in fundus from diabetics than in that from controls. The number of β-adrenoceptors, measured with [ 125I]iodocyanopindorol as a ligand, was significantly less in gastic fundus membrane isolated from diabetics than in that from controls, but affinity was no different. The level of plasma catecholamine was higher in diabetics than in controls. Results suggest that depression of gastric fundus relaxation and increase of contraction by NE in diabetics could be due to fewer β-adrenoceptor binding sites caused by down-regulation by higher catecholamine level in diabetic rats.

O-073 - Characterization of adrenoceptors of gastric fundus smooth muscle of diabetic rats

O-073 - Characterization of adrenoceptors of gastric fundus smooth muscle of diabetic rats

Evidence for a lower oesophageal sphincter in the guinea-pig

1. In vitro balloon pull-through experiments have been used to identify the guinea-pig lower oesophageal sphincter (LOS). 2 Histologically, the LOS forms a 1–2 mm ring of smooth muscle at the distal termination of the oesophagus, immediately adjacent to the gastric sling muscle. 3. Tetrodotoxin (10 −6M) sensitive, guanethidine (10 −6M) insensitive “on” relaxation of circular LOS muscle strips was evoked by electrical field stimulation (ES). 4. ES evoked atropine (10 −6 M) sensitive “on” contractions of gastric sling and fundus smooth muscle strips. 5. Following cessation of ES a partially atropine-sensitive “off” contraction was observed in all the smooth muscle strips. 6. The predominant response of the LOS to ES was relaxation.

Different binding properties of muscarinic M2-receptor subtypes for agonists and antagonists in porcine gastric smooth muscle and mucosa

The hypothesis that muscarinic receptors on smooth muscle differ from those in epithelial glands was tested by comparing the properties of muscarinic binding sites in porcine fundic smooth muscle with those in mucosal membranes. The binding of agonists and of antagonists was assessed by displacement of [3H]N-methylscopolamine. Pirenzepine (M1-antagonist) labeled low-affinity binding sites in smooth muscle (KD = 229 nM) and in mucosa (KD = 124 nM) consistent with the presence of M2 sites. Carbachol interacted with a high-affinity (KD = 164 nM) and a low-affinity (KD = 18.2 μM) state of binding sites in smooth muscle. Guanyl 5′-yl-imidodiphosphate converted all sites to the low-affinity state. N-ethylmaleimide pretreatment increased the affinity of carbachol and the proportion of high-affinity sites. In clear contrast, only low-affinity sites of carbachol were detectable in mucosa (KD = 17 μM) that were not modulated by N-ethylmaleimide or guanyl 5′-yl-imidodiphosphate. The cardioselective antagonist AF-DX 116 displayed low affinity to mucosal binding sites (KD = 3.4 μM), whereas its affinity to smooth muscle was 503 nM. The antagonist hexahydro-sila-difenidol had a very high affinity (KD = 2.9 nM) to mucosal receptors, whereas its affinity to smooth muscle sites was 88 nM. These data show that muscarinic M2 binding sites in mucosa and smooth muscle can be distinguished by both agonist and antagonist binding experiments, and suggest the existence of different subtypes of M2-binding sites in these tissues.

Target size analysis of neurotensin receptors.

The neurotensin receptors from rat brain synaptosomal membranes differed in subunit structure from those in rat gastric fundus smooth muscle plasma membranes when studied following photoaffinity labelling or after exposure to cross-linking reagents. However, these two receptors were similar in their recognition properties. In this study we compared the target size of the two receptors by radiation inactivation and observed that the receptors in both tissues had similar target sizes (mean values 103,000 and 108,000 daltons). This suggests that the differences in size observed in biochemical studies may reflect changes occurring during the isolation procedures, or, on the other hand, there might be inherent difference in the subunit structure of these receptors.

Aggregation of smooth muscle membranes and its use in the preparation of plasma membrane enriched fraction from gastric fundus smooth muscle.

Microsomal membranes isolated from rat gastric fundus smooth muscle by differential centrifugation aggregate substantially in the presence of the divalent metal ion Mg2+ or Ca2+. The magnitude of cation-induced membrane aggregation is higher for Ca2+ than for Mg2+, but the ion concentration required for half-maximum membrane aggregation (K0.5 value) is similar for Mg2+ and Ca2+. Cation-induced membrane aggregation is suppressed by high ionic strength and low pH of the medium. Cation-induced membrane aggregation of mitochondrial membrane and plasma membrane enriched fractions differ in the rate of aggregate formation, metal ion concentration dependence, and pH dependence. Such different properties of membrane aggregation were used to prepare a plasma membrane enriched fraction by conventional differential centrifugation. Subfractionation of the heterogeneous microsomal membranes by free-flow electrophoresis indicated that smooth muscle plasma membranes showed a higher electrophoretic mobility than the intracellular membranes. These results suggest that ionic interactions on the cell membrane surfaces differ from those on the intracellular membrane surfaces and that induction of membrane aggregation by Ca2+ or Mg2+ is a useful procedure for an effective and rapid preparation of plasma membrane enriched fraction from smooth muscle.

Demarcation of Ca2+ transport processes in guinea pig stomach smooth muscle.

Microsomal fractions were isolated from gastric antrum and fundus smooth muscle of guinea pigs. Ca2+ uptake into and Ca2+ release from the membrane vesicles were studied by a rapid filtration method, and Ca2+ transport properties of the different regions of the stomach were compared. ATP-dependent Ca2+ uptake was similar in microsomes isolated from both regions. This uptake was increased by oxalate and was not affected by NaN3. Oxalate affected Ca2+ permeability of both antrum and fundus microsome vesicles similarly. Fundus microsome vesicles preincubated in 100 mM NaCl and then diluted to 1/20 concentration with Na+-free medium had significantly higher ATP-independent Ca2+ uptake than vesicles preincubated in 100 mM KCl and treated the same way. This was not true for antrum vesicles. Monensin abolished Na+-dependent Ca2+ uptake, and NaCl enhanced Ca2+ efflux from fundus microsome vesicles. The halflife values of Ca2+ loss from fundus vesicles in the presence of NaCl were significantly smaller than those in the presence of KCl. The release of Ca2+ from the vesicles within the first 3 min was accelerated by NaCl to three times that by KCl. However, NaCl had no effect on Ca2+ release from antrum microsome vesicles. Results suggest two distinct mechanisms of stomach membrane Ca2+ transport: (1) ATP-dependent Ca2+ uptake and (2) Na+-Ca2+ exchange; the latter in the fundus only.

Responsiveness of rat vas deferens and stomach smooth muscles after treatment with indomethacin.

The contractile responses of rat vas deferens and longitudinal stomach fundus strips to stimulants and relaxants were studied after treatment with indomethacin (Ind) injected i.p. at doses of 15 mg/kg for 24 hr and 25 mg/kg for 48 hr. The treatment with Ind reduced the amount of both spontaneously and electrically-evoked release of a prostaglandin (PG)-like substance from the isolated n. hypogastricus-vas deferens preparations. A considerable decrease of the responsiveness to exogeneous noradrenaline occurred in vas deferens isolated from Ind-pretreated rats. The EC50 values of noradrenaline increased as compared to the control value. The contractile responses of the isolated longitudinal stomach fundus strips to acetylcholine, serotonin, PGE2, PGF2 and arachidonic acid were reduced while the relaxant action of noradrenaline and papaverine was potentiated after Ind treatment. It is assumed that endogeneous PGs participate in the regulation of smooth muscle activity through potentiation of the postjunctional excitatory action of noradrenaline in vas deferens and through interaction with the transmembrane ion transport in stomach fundus smooth muscles.

Covalent labeling of neurotensin receptors in rat gastric fundus plasma membranes

Neurotensin receptors from plasma membranes of rat gastric fundus smooth muscle were specifically and covalently labeled either by using the photoreactive analogue 125I-labeled azidobenzoyl (Trp 11)-neurotensin or by cross-linking (monoiodo-Tyr 3)neurotensin to the membrane preparation by means of disuccinimidyl suberate. Analysis of plasma membranes by sodium dodecylsulfate-polyacrylamide gel electrophoresis and autoradiography revealed that the same protein band with an apparent molecular weight of 110,000 was specifically labeled by both methods. This band consisted of a single chain protein since its apparent size was found to be the same with or without reduction of membrane samples before electrophoresis. Only neurotensin and its biologically active analogues were able to protect plasma membranes against specific labeling of the protein band of molecular weight 110,000. Comparison of these results with those obtained from rat brain synaptic membranes shows that although rat central and peripheral neurotensin receptors exhibit similar specificities towards a series of neurotensin analogues, their subunit structures are different.

A Mg 2+-independent high-affinity Ca 2+-stimulated adenosine triphosphatase in the plasma membrane of rat stomach smooth muscle. Subcellular distribution and inhibition by Mg 2+

Plasma membrane enriched fraction isolated from the fundus smooth muscle of rat stomach displayed Ca 2+-stimulated ATPase activity in the absence of Mg 2+. The Ca 2+ dependence of such an ATPase activity can be resolved into two hyperbolic components with a high affinity ( K m = 0.4 μM ) and a low affinity ( K m = 0.6 mM ) for Ca 2+. Distribution of these high-affinity and low-affinity Ca 2+-ATPase activities parallels those of several plasma membrane marker enzyme activities but not those of endoplasmic reticulum and mitochondrial membrane marker enzyme activities. Mg 2+ also stimulates the ATPase in the absence of Ca 2+. Unlike the Mg 2+-ATPase and low-affinity Ca 2+-ATPase, the plasmalemmal high-affinity Ca 2+-ATPase is not sensitive to the inhibitory effect of sodium azide or Triton X-100 treatment. The high-affinity Ca 2+-ATPase is noncompetitively inhibited by Mg 2+ with respect to Ca 2+ stimulation. Such an inhibitory effect of Mg 2+ is potentiated by Triton X-100 treatment of the membrane fraction. Calmodulin has little effect on the high-affinity Ca 2+-ATPase activity of the plasma membrane enriched fraction with or without EDTA pretreatment. Findings of this novel, Mg 2+-independent, high-affinity Ca 2+-ATPase activity in the rat stomach smooth muscle plasma membrane are discussed with those of Mg 2+-dependent, high-affinity Ca 2+-ATPase activities previously reported in other smooth muscle plasma membrane preparations in relation to the plasma membrane Ca 2+-pump.

Characterization of neurotensin binding to rat gastric smooth muscle receptor sites

The binding of monoiodo 125I-Trp 11-neurotensin to purified rat gastric fundus smooth muscle plasma membranes was characterized. Specific binding of ligand in subcellular fractions from rat fundus smooth muscle showed a distribution that paralleled that of several plasma membrane marker enzymes. 125I-Trp 11-neurotensin binding to smooth muscle plasma membranes at 25°C was maximal at 30 min, reversible and saturable. Scatchard analysis of equilibrium data indicated the existence of two classes of binding sites with dissociation constants (Kd) of 56 pmol and 1.92 nM, and corresponding binding capacities (Bmax) of 6.6 fmol/mg and 11.4 fmol/mg of membrane protein. Analogues and fragments of neurotensin competed for 125I-Trp 11-neurotensin binding with a rank order of potency similar to that previously reported for their contracting effect in rat fundus strips. Na + decreased in a concentration dependent manner the binding of labelled ligand to the high affinity site. At 100 mM, Na + induced a 6-fold increase in the IC 50 of neurotensin for inhibition of 125I-Trp 11-neurotensin binding. At this concentration of Na +, the IC 50 for neurotensin was 1 nM, a value close to the Kd of the low affinity site.

Subcellular distribution of [3H]nitrendipine binding in smooth muscle.

Distribution of specific binding sites for [3H]nitrendipine was studied in subcellular fractions isolated from rat gastric fundus smooth muscle and from rat myometrium. There was an excellent correlation between the distribution of [3H]nitrendipine binding determined at the nitrendipine concentrations of 0.138 and 1.38 nM, and the distribution of the plasma membrane markers K+-activated ouabain-sensitive p-nitrophenylphosphatase, 5'-nucleotidase, phosphodiesterase I, and Mg-ATPase, but not between the mitochondrial markers cytochrome c, oxidase, succinate-dependent cytochrome c reductase, or rotenone-insensitive NADH-dependent cytochrome c reductase or the putative endoplasmic reticulum marker NADPH-dependent cytochrome c reductase. The binding occurred with high affinity and with a similar (0.097-0.146 nM) equilibrium dissociation constant to all the fractions, even though the density of binding sites varied and was highest in the plasma membrane marker-enriched fractions. The maximal binding in the plasma membrane-enriched fraction from the rat gastric fundus smooth muscle was 0.43 +/- 0.04 pmol/mg, and in that from rat myometrium was 0.72 +/- 0.09 pmol/mg. Thus in the two smooth muscles studied the plasma membrane is the locus of the high affinity nitrendipine binding.

Contraction and [3H]QNB binding in collagenase isolated fundic smooth muscle cells.

Smooth muscle cells from the guinea pig gastric fundus were isolated by successive collagenase digestions. Tritiated quinuclidinyl benzilate [( 3H]QNB) was used to study the binding characteristics of the muscarinic cholinergic receptors on these cells. Each cell bound 8.3 X 10(-19) mol of QNB, and a concentration of QNB of 0.19 nM was required to label one-half of the binding sites. This suggests a concentration of about 500,000 muscarinic cholinergic receptors per smooth muscle cell. The muscarinic cholinergic receptor antagonists atropine and scopolamine inhibited QNB binding with a 50% inhibiting concentration (IC50) in the nanomolar range, whereas the agonists acetylcholine (ACh), oxotremorine, and carbamylcholine had IC50S in the micromolar range. Hill coefficients (nH) for antagonists approached unity, but agonists displayed fractional nH. Exposure of cells to cholinergic muscarinic agonists resulted in dose-dependent decreases in cell length. The concentration of agonist required to induce half-maximal contractions (ED50) was 8.3 X 10(-12) M for ACh and 6.3 X 10(-13) M for oxotremorine. Atropine (10(-9) M) decreased the sensitivity to ACh, increasing the ED50 for ACh-induced contractions to 1.2 X 10(-10) M. These results suggest the existence of muscarinic receptor heterogeneity for cholinergic agonists but not for antagonists.

Effects of atropine on secretion and motility in isolated gastric mucosa and attached muscularis externa from ferret and cat.

1. A combined in vitro preparation of gastric mucosa and adjacent muscle from young ferrets and kittens has been used to study the effects of atropine on acid secretion and motility produced by acetylcholine (ACh) and pentagastrin.2. The minimal dose of atropine required to abolish a maximum secretory response to ACh also prevented the associated motility response. This dose of atropine also blocked the motility response to pentagastrin, but was without influence on the secretory effect of this agent. A 10(3) times larger dose of atropine reduced the secretory effect of pentagastrin by half, probably not by anti-muscarinic effect. The results exclude the possibility that the acid secretory response to pentagastrin necessarily involves a cholinergic receptor.3. The results support the view that the response of the fundic smooth muscle to pentagastrin depends on the excitation of cholinergic nerves.4. No evidence has been found of any cholinergic component in the acid secretory response to pentagastrin. In assessing the significance of this result, however, it must be remembered that the Auerbach plexus has been removed over the major part of the mucosa, and the Meissner plexus deprived of input and probably damaged.5. The results are compatible with the hypothesis that the depressant effect of atropine on acid secretion produced by gastrin and its derivatives is due to the elimination of a cholinergic potentiating influence arising in the intramural plexuses. The residual Meissner plexus elements in this in vitro preparation appear inadequate to sustain this effect.

Evidence for a visceral smooth muscle abnormality in Okamoto spontaneous hypertension.

1. In order to discover whether the changes in reactivity are related to the primary cause of hypertension in spontaneously hypertensive rats (SHR) or are just an adaptation induced by the high arterial blood pressure we tested the contractile response of a visceral smooth muscle from such rats. 2. Longitudinal strips of the fundus from 20 week old male and female SHR and Wistar normotensive (NW) rats were used. Dose-response curves to Ba2+ in SHR strips were displaced to the right as compared to NW rats. Maximal responses were identical. Male SHR fundus strips contracted much more with Sr2+ (SHR: 42+/-3% of maximum response to Ba2+, n=10; NW: 19+/-4%, n=10, P less than 0.01) than NW strips. There was no difference in the response to both BaCl2 and SrCl2 between female SHR and NW fundus strips, and MnCl2 and LaCl3 were relaxant in all cases. 3. Dose-response curves to Ca2+ of depolarized SHR and NW fundus strips were obtained and the effect of diazoxide on Ca2+ contractions was observed. The contractile action of Ca2+ in depolarized preparations was enhanced in both male and female SHR strips. The effect of diazoxide was more marked in SHR strips than in NW fundus strips. 4. SHR fundus smooth muscle shows the same modification of reactivity to Ba2+, Sr2+, Ca2+ and diazoxide that was previously described in arterial smooth muscle. This indicates that the cellular modification responsible for the increase of vascular tonus in SHR is not an adaptive reaction to high blood pressure. The differences between female SHR and male SHR responses are not unexpected, considering the natural evolution of hypertension in Okamoto rats which is milder in the female.