Fundus Muscle Research Articles

Isolation and primary structure of pituitary human galanin, a 30-residue nonamidated neuropeptide.

Galanin (Gal), a 29-amino acid C-terminally amidated neuropeptide, is widely distributed throughout the central and peripheral nervous system. The primary structures of rat and bovine Gals were derived from the cDNA sequences of their precursors. To elucidate the structure of human Gal (hGal), we extracted 280 postmortem pituitaries in trifluoroacetic acid and purified hGal binding activity, by three successive HPLC steps, to homogeneity based on a radioreceptor assay. The primary structure of hGal was determined by automatic Edman degradation to be Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu- Gly-Pro-His-Ala-Val-Gly-Asn-His-Arg-Ser-Phe-Ser-Asp-Lys-Asn-Gly-Leu-Thr- Ser-COOH. The structure was confirmed by plasma desorption time-of-flight mass spectrometry, revealing a mass of 3156.1. Compared to the 29-residue porcine, rat, and bovine Gals, hGal uniquely comprises 30 amino acids possessing an additional nonamidated serine residue as C terminus. The nonamidated carboxylic group at the C terminus was proven by synthesis of amidated and nonamidated hGal and by mass spectrometry after selective methylation of all free carboxylic groups. Synthetic hGal possesses full biological activity on isolated rat fundus muscle strips.

Developmental characteristics of the kitten antrum

The changes in dietary constituents during growth and development from predominantly liquid to mixed solid liquid meals places an increasing burden on the antrum, which is required to triturate solids. The authors hypothesize that concomitant with these changing dietary constituents different pathways are used to mediate contraction in response to acetylcholine. Smooth muscle cells were isolated by collagenase digestion from the fundus and circular muscle layer of the antrum in the adult cat and 1-week-old kitten. The unstimulated cells from the fundus and antrum were larger in the adult than in the kitten. In normal physiological salt solution, all cells contracted in a similar dose dependent manner in response to acetylcholine in both age groups. Fundic muscle cell contraction in response to acetylcholine was unchanged in calcium-free physiological salt solution or in the presence of the calcium channel blocker, methoxyverapamil. In the adult, antral cells contracted 50% less in the absence of extracellular calcium. In the kitten, antral cells did not contract under the same conditions. After permeabilization with saponin, fundic muscle cells from the adult and kitten contracted fully in response to 1,4,5-inositol trisphosphate, whereas contraction of adult antral cells was reduced by 50% and contraction of kitten antral cells was completely blocked. These data suggest that isolated muscle cells from the fundus of both the adult cat and newborn kitten use intracellular calcium stores to contract in response to acetylcholine and 1,4,5-inositol triphosphate. In adult antral cells, acetylcholine-induced contraction requires both influx of extracellular calcium and release of intracellular calcium. In kitten antral cells, intracellular calcium stores are not used or available to mediate contraction in response to acetylcholine.

Mechanical responses to catecholamines in the longitudinal muscle of guinea‐pig gastric fundus

1. In the longitudinal muscle of guinea-pig gastric fundus, adrenaline and phenylephrine (1-30 microM) both produced a slow contraction preceded by a relaxation. The slow contraction was strongly inhibited by prazosin (0.1 microM), but only weakly by yohimbine (1 microM), suggesting main contribution of alpha 1-adrenoceptors. 2. Most of the slow contraction was blocked by meclofenamate or indomethacin (0.1-0.3 microM). Both these drugs also inhibited spontaneously generated muscle tone. In some preparations, obtained from the apical fundus, a small contraction remained in the presence of meclofenamate. 3. During contraction induced by prostaglandin E2, adrenaline produced sustained relaxation and phenylephrine often transient relaxation, in the presence of meclofenamate. The transient relaxation, but not the sustained relaxation, was suppressed by prazosin. 4. In the presence of prostaglandin E2 (5 nM), after treating with phenoxybenzamine (30 microM) for 30 min, isoprenaline and adrenaline produced concentration-dependent relaxation, with IC50 s of 3.9 nM and 64 nM, respectively. Propranolol shifted these concentration-response curves to the right, with apparent pA2 s of 8.15 and 7.34, respectively. 5. It is suggested that in the fundic longitudinal muscle, adrenaline-induced contraction is mediated mainly by an increase in endogenous prostaglandin production through activation of alpha 1-adrenoceptors and that adrenaline produces transient relaxation through alpha 1-adrenoceptors and sustained relaxation through beta-adrenoceptors. The beta-adrenoceptors in the longitudinal muscle are more sensitive to adrenaline and isoprenaline than those in the circular muscle.

Perinatal changes in bombesin-stimulated muscle contraction in rabbit stomach and colon

Bombesin and its mammalian homologue, gastrin-releasing peptide, stimulate smooth muscle contraction and may promote the growth of gastrointestinal tissues. Isometric contraction of strips from circular muscle of the gastric fundus and longitudinal muscle of the distal colon were used to compare changes in the response to bombesin in newborn and weanling rabbits. There was an age-related qualitative change in gastric muscle from biphasic contractions including phasic and tonic components in the newborn to phasic contractions alone in the weanling. The colon contractions were tonic at both ages. In both tissues there was an age-related fivefold increase in stress in response to maximally effective concentrations of bethanechol (P < 0.05). In contrast, in the stomach age-related decreases in the response to maximally effective concentrations of bombesin were observed, from 2930 ± 179 mN/cm2 (98% of the maximal response to bethanechol) in the newborn to 565 ± 81 mN/cm2 (4% of the maximal response to bethanechol) in the weanling (P < 0.005). In the colon, a twofold increase in response to bombesin was observed, from 446 ± 59 mN/cm2 (82% of the response to bethanechol) in the newborn to 862 ± 11 mN/cm2 (29% of the response to bethanechol) in the weanling (P < 0.05). No age-related changes were observed in the potency of bombesin in either tissue. Neither atropine nor tetrodotoxin altered the contractions in either tissue, suggesting that bombesin interacted directly with myocytes. There was three times as much bombesinlike immunoreactivity in the stomach compared with the colon, but no age-related changes in either tissue. In summary, by the age of weaning the stomach lost the tonic component of contraction and 80% of the efficacy of bombesin-stimulated phasic contraction that had been present in the newborn. The loss of efficacy, absolute in the stomach and relative to bethanechol in the colon, suggest that bombesin may be most important in stimulating motility in the neonatal period.

Effects of Neurotensin on Gastric Smooth Muscle of Dog and Guinea Pig

Mechanical activity was recorded in muscle preparations isolated from different regions of dog and guinea pig stomach. Neurotensin (10−11 to 10−8 mol/liter) produced three types of responses in canine gastric muscle: a small activation of tone infundic strips and in longitudinal strips from corpus and antrum; a transient inhibition of the rhythmic activity in circular strips from corpus, antrum, and outer pylorus; and a strong activation in the strips from inner pylorus. In guinea pig gastric muscle strong tonic responses were observed in circular fundic muscle strips, whereas in all other preparations the responses were negligible. All these effects were insensitive to atropine, dimethpyrindene, and tetrodotoxin. The neurotensin effect on strips from guinea pig stomach was strongly reduced in the presence of indomethacin or cortisone and markedly potentiated in the presence of arachidonic acid or prostaglandin F2α. contrast, the excitatory effect of neurotensin on canine inner pylorus was potentiated during indomethacin treatment.

The motor effect of neuromedin U on rat stomach in vitro

A series of neuromedin U (NmU)-like peptides were found to evoke concentration-response contraction of rat fundic circular muscle in vitro. Isometric contraction of this tissue was induced by rat NmU, porcine NmU-8 and NmU-25, and frog NmU. Rat NmU was significantly more potent than frog NmU. The contractile action of rat NmU upon rat fundic strips was not affected by either atropine or tetrodotoxin indicating a direct effect. Maximal NmU-induced contractions were 10% of the maximal contraction induced by carbachol and ranged between those of the bradykinin and angiotensin II. None of the NmU peptides were active on the circular smooth muscle of the frog stomach or on the small and large intestinal longitudinal smooth muscle of either rat or frog. The results of this study demonstrate that members of the NmU peptide family all induce in vitro contraction of rat gastric circular smooth muscle independent of cholinergic or other neuronal mechanisms. Their activity, however, appears to be both tissue and species specific.

Suppression of steady membrane currents by acetylcholine in single smooth muscle cells of the guinea‐pig gastric fundus.

1. Single smooth muscle cells from the fundus region of the guinea-pig stomach, which showed contractile responses to acetylcholine (ACh) at concentrations greater than or equal to 10(-7) mol/l, were obtained by enzymatic digestion using highly purified collagenase and papain. They were studied by recording membrane currents under voltage clamp with the patch pipette technique in the whole-cell configuration at 25-28 degrees C. 2. By applying voltage jumps from negative holding levels (-70 to -60 mV) to more positive levels, we identified two major activating currents: an initial inward Ca2+ current (ICa) was followed, and partly overlapped, by an outward K+ current (IK). 3. Cholinergic effects on membrane currents were investigated in the range of negative membrane potentials by determining current-voltage relations in the absence of ACh and during its continuous presence in the bathing fluid. 4. ACh induced a decrease in the steady-state conductance which was reversibly blocked by atropine. At physiological external K+ concentration [( K+]o = 6 mmol/l), the reversal potential (Erev) of the current suppressed by ACh (3 x 10(-6) mol/l) was about 20 mV more positive than the calculated K+ equilibrium potential (EK). 5. When [K+]o was increased, Erev was shifted positively; but at each [K+]o, Erev was more positive than EK. 6. Like ACh (10(-6) mol/l), tetraethylammonium (TEA, 1 mmol/l) also suppressed a current with a reversal potential that was, at physiological [K+]o, 20 mV more positive than EK. ACh (10(-5) mol/l) applied in the presence of 1 mmol/l TEA suppressed a pure K+ current (Erev = EK), which was also suppressed by 10 mmol/l TEA. 7. When K+ in the pipette and in the bathing solution was completely replaced by Na+, both ACh (10(-5) mol/l) and TEA (1 mmol/l) caused a reduction of the membrane conductance that appeared to be identical. TEA added to the bathing solution in the presence of ACh did not produce a significant additional conductance decrease. These results did not depend on whether Cl- was present as a charge carrier or not. 8. It is concluded that in fundus muscle of the guinea-pig stomach a major mechanism underlying muscarinic activation is a decrease of a K+ conductance. In addition the results indicate a suppression of a small Na+ conductance which is made up by a population of channels that are also blocked by TEA.

Nitric oxide and vasoactive intestinal polypeptide mediate non-adrenergic, non-cholinergic inhibitory transmission to smooth muscle of the rat gastric fundus

The nitric oxide (NO) synthesis inhibitor N G-monomethyl L-arginine (L-NMMA) reduced NANC-mediated relaxations of isolated strips of the rat gastric fundus elicited by low frequencies or shcrt periods of field stimulation, but D-NMMA had no effect. The inhibitory effect of L-NMMA on NANC-mediated relaxations was partially reversed by L-arginine but was not affected by D-arginine. A VIP antibody abolished the relaxant response to VIP and reduced the responses to stimulation. Residual responses to stimulation in the presence of VIP antibody were further reduced by L-NMMA. The tone of the fundus strip was slightly increased by L-NMMA and slightly reduced by L-arginine. The relaxation produced by VIP was slightly reduced by L-NMMA and enhanced by L-arginine. Relaxations produced by peptide histidine isoleucine, sodium nitroprusside or isoprenaline were not affected by L-NMMA or L-arginine. The results suggest that NO as well as VIP is involved in NANC-mediated relaxations of the rat gastric fundus.

Mechanical, electrical and cyclic nucleotide responses to peptide VIP and inhibitory nerve stimulation in rat stomach.

1. Effects of apamin on electrical and mechanical activities and cyclic nucleotide accumulation in response to vasoactive intestinal peptide (VIP) and intramural nerve stimulation were investigated in isolated circular strips of the rat stomach in the presence of atropine and guanethidine. 2. Circular muscles generated rhythmic contractions and slow waves in the antrum but not in the fundus. Intramural nerve stimulation and VIP caused frequency- and dose-dependent relaxation of fundic strips and inhibition of spontaneous contractions of antral strips. Apamin partly reduced the responses to intramural nerve stimulation but not those to VIP. 3. In the antrum, apamin reduced inhibitory junction potentials (IJPs) evoked at the nadir of slow waves but not at their zenith. In the fundus, apamin partly decreased the amplitude of IJPs. Repetitive nerve stimulation was associated with an apamin-sensitive hyperpolarization and apamin-resistant decrease in the slow wave amplitude in the antrum. 4. VIP caused a dose-dependent hyperpolarization of fundic circular muscle membrane. In the antrum, VIP inhibited spike potentials superimposed on slow waves and it decreased the slow wave amplitude in about half of the preparations. These electrical responses to VIP were resistant to apamin. 5. Intramural nerve stimulation evoked an apamin-resistant output of VIP from muscle strips, which no longer occurred after tetrodotoxin or removal of extracellular Ca2+. 6. Intramural nerve stimulation and VIP elicited apamin-resistant increases in cyclic AMP and cyclic GMP accumulations. The effects of VIP on cyclic AMP were greater than those on cyclic GMP. The effects of intramural nerve stimulation on cyclic GMP were faster in onset than those of cyclic AMP. 7. It is suggested that VIP is a neurotransmitter of the intramural inhibitory nerves concerned in the apamin-resistant relaxation of the rat stomach.

Effects of the dopamine receptor agonists, fenoldopam and quinpirole, in the rat stomach

The effects of the DA 1-receptor agonist, fenoldopam, and the DA 2-receptor agonist, quinpirole, were studied with the longitudinal muscle of rat gastric fundus and circular muscle of rat gastric corpus, as there are contrasting reports about the receptors involved in the inhibitory effect of dopamine in these tissues. Quinpirole had no effect on basal tone in the longitudinal muscle of the rat gastric fundus and did not inhibit the sustained contractions induced by electrical field stimulation or by methacholine. Fenoldopam had no effect on the tone increased by methacholine but slightly potentiated the electrically induced contraction at the highest concentrations; it concentration dependently (10 −7−3 · 10 −5 M) increased the basal tone. The contractile effect of fenoldopam was clearly antagonized by rauwolscine 10 −6 M, yohimbine 10 −6 M and phentolamine 3 · 10 −6 M plus propranolol 10 −5 M. The 5-HT receptor antagonist, methysergide, antagonized the fenoldopam-induced contractions in a non-competitive way. Fenoldopam and quinpirole had no effect on contractions induced in the circular muscle of the rat gastric corpus by methacholine or electrical field stimulation. They induced some contraction at basal tone, at their highest concentrations. As fenoldopam and quinpirole did not mimic the inhibitory effect observed with dopamine in the same models, no evidence was found for the presence of inhibitory dopamine receptors in rat gastric muscle. The contractile effect of fenoldopam in the longitudinal muscle of the fundus is probably due to an interaction with 5-HT receptors.

Isolation of Endopeptidase-24.11 (EC 3.4.24.11, "Enkephalinase") From the Pig Stomach

Abstract The purpose of this investigation was to isolate the cell-surface enzyme endopeptidase-24.11 from the stomach wall of the pig and to examine the hydrolysis of the gastric neuropeptides. Endopeptidase-24.11 was isolated from gastric membranes by immuno-adsorbent chromatography using a monoclonal antibody to porcine kidney endopeptidase-24.11. The enzyme was purified with a yield of 1.2 μg/g wet wt of fundic muscle. A single polypeptide chain of apparent subunit molecular weight of 90,000 was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gastric endopeptidase-24.11 hydrolyzed substance P, gastrin-releasing peptide 10, [Leu 5 ] enkephalin, and [Met 5 ] enkephalin by cleavage of peptide bonds on the N-terminal side of hydrophobic amino acids. The enzymatic activity was inhibited completely by phosphoramidon (10 −6 M) and strongly by 1,10-phenanthroline (10 −3 M), but was unaffected by captopril (10 −5 M).

Isolation of endopeptidase-24.11 (EC 3.4.24.11, “enkephalinase”) from the pig stomach: Hydrolysis of substance P, gastrin-releasing peptide 10, [Leu5] enkephalin, and [Met5] enkephalin

The purpose of this investigation was to isolate the cell-surface enzyme endopeptidase-24.11 from the stomach wall of the pig and to examine the hydrolysis of the gastric neuropeptides. Endopeptidase-24.11 was isolated from gastric membranes by immuno-adsorbent chromatography using a monoclonal antibody to porcine kidney endopeptidase-24.11. The enzyme was purified with a yield of 1.2 μg/g wet wt of fundic muscle. A single polypeptide chain of apparent subunit molecular weight of 90,000 was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gastric endopeptidase-24.11 hydrolyzed substance P, gastrin-releasing peptide 10, [Leu5] enkephalin, and [Met5] enkephalin by cleavage of peptide bonds on the N-terminal side of hydrophobic amino acids. The enzymatic activity was inhibited completely by phosphoramidon (10−6 M) and strongly by 1,10-phenanthroline (10−3 M), but was unaffected by captopril (10−5 M).

Role of adenine compounds in autonomic neurotransmission

Clearly adenine compounds exert numerous effects throughout the autonomic nervous system. The responses of various peripheral tissues to purines are summarized in Table 2. The evidence supporting a possible excitatory neurotransmitter function for ATP is very good in the vas deferens and good in both the bladder detrusor and certain blood vessels. ATP may also be an excitatory neurotransmitter in the colon, hepatocytes and frog atrium. These responses appear to be mediated by P2x-purinoceptors. There is good evidence supporting a role for ATP as an inhibitory neurotransmitter in the taenia coli and duodenum, and some support in the anal sphincter and possibly the rabbit portal vein; these responses appear to be mediated by P2y-purinoceptors. There is good evidence against ATP being an inhibitory neurotransmitter in the stomach fundic muscle and ileum. ATP (or more likely its metabolite adenosine) may act as an inhibitory neurotransmitter by interacting with postsynaptic P1-purinoceptors in cultured sympathetic neurones and also in the parasympathetic vesicle ganglion of the cat. It seems likely that ATP released from heart, platelets or vascular endothelium could be an endogenous relaxant of blood vessels through its actions on the endothelium. Although the addition of exogenous adenosine affects many tissues, evidence supporting modulatory functions for endogenous extracellular adenosine has only been clearly demonstrated in the ileum, gallbladder, vas deferens, fallopian tubes, kidney, blood vessels, carotid sinus, heart and adipose tissue. Both ATP and adenosine, released during periods of hypoxia or ischemia, could exert negative inotropic, chronotropic and dromotropic actions in the heart. In many cases, the potential sources of extracellular purines have not been established. This is particularly important when attempting to establish a neurotransmitter function for ATP in a tissue. For instance, the one outstanding piece of evidence required to confirm that ATP is an excitatory neurotransmitter released from sympathetic nerves in blood vessels is the unequivocal demonstration that it is, in fact, released from the sympathetic nerves when they are stimulated. To date, only the release of radiolabeled metabolites of ATP, possibly from post- rather than presynaptic sites, has been detected. Studies of the release of ATP are complicated by its rapid degradation extracellularly by ecto-ATPase. Unfortunately, there are no specific inhibitors of ecto-ATPase available at present, but one hopes that a suitable inhibitor will be developed shortly.(ABSTRACT TRUNCATED AT 400 WORDS)

Localization of specific binding sites for bombesin in the canine gastrointestinal tract

The goal of these studies was to determine the tissue and cell types possessing specific binding sites for bombesin/gastrin-releasing peptide in the canine gastrointestinal tract. Monoiodinated, biologically active (Tyr-4)-bombesin 14 (100 pM) was applied to sections of canine gut and localized using quantitative autoradiography. The highest density of bombesin/gastrin-releasing peptide specific binding sites occurred over endocrine cells in the antral mucosa. Specific binding sites were also found on the circular muscle layer of the gastric fundus, gastric antrum, and ileum, on longitudinal muscle of the gastric fundus and antrum, and on neuronal elements in the myenteric plexus in the gastric fundus, antrum, and small intestine. No evidence for specific binding of 125I-(Tyr-4)-bombesin 14 was found in sections of canine esophagus, gastric cardia, gallbladder, pancreas, or colon. These results suggest sites of direct action of bombesin and endogenous gastrin-releasing peptide for gastrin release and gastrointestinal motility.

Prejunctional inhibition of vasoactive intestinal peptide release.

The role of vasoactive intestinal peptide (VIP) and its homologue, peptide histidine isoleucine (PHI), as neurotransmitters of inhibitory motor nerves of the gut, were examined in strips of guinea pig taenia coli and gastric fundic muscle. The stoichiometry of VIP release and muscle relaxation was determined in the presence and absence of the bee venom peptide, apamin, and the existence of prejunctional VIP/PHI receptors capable of regulating VIP/PHI release was explored. In both types of muscle, relaxation induced by field stimulation was proportional to the amount of VIP released. Apamin inhibited relaxation and VIP release in a dose-dependent manner: maximal relaxation was inhibited by 85-96% at 10(-7)-10(-6) M apamin. Analysis of residual responses showed that apamin did not affect the stoichiometry of VIP release and muscle relaxation. Because apamin had no effect on basal tone or on relaxation induced by exogenous VIP, its effect on neurally induced relaxation was attributed to inhibition of VIP release. Both secretin and PHI inhibited neurally induced VIP release in the two types of muscle. At the optimal concentration of 10(-7) M, secretin inhibited VIP release by 52%, whereas the closer neural homologue, PHI, abolished VIP release. The dose-dependent inhibition of VIP release by PHI, which is cosynthesized and coreleased with VIP, indicates the existence of prejunctional inhibitory VIP/PHI autoreceptors capable of regulating VIP/PHI release.

Relaxation of rat gastrointestinal smooth muscle by parathyroid hormone.

In this study we investigated whether parathyroid hormone (PTH) can produce relaxation of gastrointestinal (GI) smooth muscle as it has been reported to do for vascular and uterine smooth muscle. Muscle tissue preparations from rat stomach, duodenum, ileum, or colon were mounted in a 37 degrees C tissue bath and perfused with oxygenated medium. Changes in isometric tension were recorded with a force-displacement transducer connected to a polygraph. Decreases in either resting tension or agonist-induced tension (0.5-1.0 microM acetylcholine or carbachol) were observed within 1-2 min of PTH addition and were reversible upon removal of the peptide. All GI regions tested were responsive to PTH. Synthetic rPTH-(1-34) (0.1-100 nM) produced a dose-dependent relaxation of both fundic (ED50 = 5.2 nM) and colonic (ED50 = 2.5 nM) muscle strips. At 100 nM, a 90% decrease in fundic tension and a 70% decrease in colonic tension were seen. At 100 nM, bPTH-(1-34), but not bPTH-(7-34) or rat calcitonin gene-related peptide, also was effective in relaxing fundic or colonic muscle. Similarly, in the fundic muscle, 500 nM bPTH-(3-34) alone was ineffective, but it inhibited the effect of 5 nM rPTH-(1-34) when both peptides were tested in combination. Likewise, 100 nM bPTH-(3-34) also inhibited the relaxation induced by 5, 10, or 100 nM bPTH-(1-34). The results show PTH to be highly effective in nanomolar concentrations in causing relaxation of GI smooth muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

Muscarinic receptor subtypes in smooth muscle and mucosa of the hog gastric fundus differ pharmacologically

Muscarinic receptor subtypes in smooth muscle and mucosa of the hog gastric fundus differ pharmacologically

Properties of potassium activatedp-nitrophenyl phosphatase of plasma membranes isolated from rat stomach muscle

The plasma membrane-enriched fraction isolated from smooth muscle of rat gastric fundus was found to contain a substantial level of potassium-stimulated p-nitrophenylphosphatase activity (K-pNPPase), and its subcellular distribution closely resembled that of other plasma membrane enzyme markers. The kinetic profile of K-pNPPase and its sensitivity toward ouabain and vanadate confirmed the identification of this activity with the partial reaction of the sodium pump. The specific activity of K-pNPPase and its sensitivity to ouabain was significantly increased in the presence of saponin, indicating that part of this activity is latent when assayed on native membrane preparation. K-pNNPase was sensitive to the presence of calcium ions in the assay medium. The Ca2+-inhibition of K-pNNPase was accompanied by increased sensitivity of the enzyme to ouabain. On the other hand, calmodulin and Ca antagonists had no effect on K-pNPPase activity nor its sensitivity to calcium.

Subcellular origin of the oxalate- or inorganic phosphate-stimulated Ca 2+ transport by smooth muscle microsomes: revisitation of the old problem by a new approach using saponin

Saponin, a cell-skinning reagent which perforates the cell membrane via its specific interaction with plasmalemmal cholesterol, was used to identify the subcellular origin of ATP-dependent Ca 2+ accumulation in the presence and absence of inorganic phosphate and oxalate by microsomal fractions isolated from rat vas deferens and dog aorta. The purified plasma membranes from rat gastric fundus muscle, which elicit the stimulation of ATP-dependent Ca 2+ accumulation by inorganic phosphate but not by oxalate, were used as a control reference. Saponin at concentrations effective for skinning smooth muscle fibres (10–50 μg/ml) inhibited Ca 2+ binding in the absence of ATP to a similar extent in all fractions, but the inhibition of ATP-dependent Ca 2+ accumulation was more pronounced in dog aorta microsomes and rat gastric fundus muscle plasma membranes than in rat vas deferens microsomes. The resistance of phosphate- and oxalate-stimulated ATP-dependent Ca 2+ accumulation to inhibition by saponin was much greater in rat vas deferens than in dog aorta microsomes. Our results suggest that phosphate- and oxalate-stimulated ATP-dependent Ca 2+ accumulation also occurs in plasma membrane vesicles isolated from smooth muscle and is by no means an unique property of endoplasmic reticulum.

Excitatory neurotensin receptors on the smooth muscle of the rat fundus: possible implications in gastric motility.

Picomolar concentrations of neurotensin caused concentration-dependent contractions of the longitudinal musculature of the fundus of the rat stomach. The EC50 of neurotensin was approximately 1.5 nM. On a molar basis neurotensin was about 5-10 times more potent than 5-hydroxytryptamine (5-HT) and approximately 80 times as active as acetylcholine in producing similar contractions. Studies with structurally related peptides indicated that whereas the carboxy terminal portion of neurotensin was essential for biological activity, a substantial part of its amino terminus end could be removed without affecting its potency. The EC50 for the neurotensin fragment 8-13 was identical to that of neurotensin, however its 1-8 or 1-11 fragments were completely inactive. Tetrodotoxin did not modify the potency of neurotensin or structurally related analogues suggesting that the neurotensin receptor is probably located on the smooth muscle membrane. In addition, the potency of neurotensin in contracting the fundus was not modified by pretreatment with atropine, methysergide or diphenhydramine. Fade to the contractile response of neurotensin was followed by the development of tachyphylaxis; desensitization was concentration-dependent and characterized by a shift in the agonist concentration-response curve to the right and downwards. Desensitization with a priming concentration of neurotensin (approx. EC50) caused a substantial blockade of its excitability. There was cross-desensitization between neurotensin and the contractile activity of neurotensin 8-13 or xenopsin, but not with angiotensin II, bradykinin, substance P, acetylcholine, 5-HT or histamine. Pretreatment of the fundus strip with verapamil 0.3-1 microM antagonized in a concentration-dependent fashion the neurotensin-induced contractions but not the muscular contractions caused by acetylcholine. It is concluded that neurotensin activates a specific excitatory receptor probably located on the cell membrane of the smooth muscles of the rat fundus. In addition, we suggest that this receptor is somehow related to a voltage-dependent calcium channel, sensitive to verapamil.