Characterization of neurotensin binding to rat gastric smooth muscle receptor sites

The binding of monoiodo 125I-Trp11-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-Trp11-neurotensin binding to smooth muscle plasma membranes at 25 degrees 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-Trp11-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 IC50 of neurotensin for inhibition of 125I-Trp11-neurotensin binding. At this concentration of Na+, the IC50 for neurotensin was 1 nM, a value close to the Kd of the low affinity site.     

Lactogenic and somatogenic binding sites in intact and detergent-solubilized membrane preparations of female rat liver.

1. Lactation results in decreased glucose and acetate utilization and increased lactate output by sheep adipose tissue. 2. The ability of insulin to stimulate acetate uptake was lost in adipose tissue from lactating sheep, whereas both the response and the sensitivity (ED50) for insulin for stimulation of glucose conversion into products other than lactate were decreased. These impairments were partly restored by prolonged incubation of adipose tissue for 48 h. 3. The ability of insulin to stimulate lactate output was not altered by lactation. 4. Dexamethasone inhibited glucose uptake, lactate output and glycerol output in adipose tissue from both non-lactating and lactating sheep, with an ED50 of about 1 nM. Dexamethasone inhibited acetate uptake by adipose tissue from non-lactating sheep, but this effect was not observed with adipose tissue from lactating sheep. 5. Dexamethasone inhibited the stimulation of glucose uptake at all concentrations of insulin used; the effect varied with insulin concentration and resulted in an accentuation of the insulin dose-response curve. The insulin dose-response curve in the presence of dexamethasone was muted during lactation. 6. The overall effect of these adaptations is to ensure that glucose and acetate utilization by adipose tissue after an insulin surge is diminished during lactation.     

Characterization of insulin binding sites in cultured smooth muscle cells of rat aorta

Insulin receptors could be demonstrated in cultured smooth muscle cells of rat aorta. The specific binding of 125I-insulin was time-, temperature- and pH-dependent. The optimal temperature for our studies was 12 degrees C. At this temperature maximal specific binding was 0.5% of total counts at 120 min incubation. The pH-optimum for the binding process was between 7.5 and 8. Degradation of 125I-insulin at 12 degrees C was 14%, no degradation of binding sites could be measured at this temperature. Dissociation of 125I-insulin was rapid. 50% of the labeled hormone remained associated with the cells. Half-maximal inhibition of 125I-insulin binding was produced by insulin at 4 X 10(-11) mol/l. Scatchard-analysis gave curvilinear plots, that may suggest negative cooperativity. Specificity of binding was studied in competition experiments between 125I-insulin, insulin, proinsulin, insulin-like growth factors and human growth hormone. Half-maximal inhibition of 125I-insulin binding was produced by proinsulin at 2 X 10(-9) mol/l and by insulin-like growth factors at 9 X 10(-9) mol/l. Human growth hormone had no significant effect on the insulin binding.     

Hyperplasia of jejunal smooth muscle in the myenterically denervated rat

Summary Application of a cationic surfactant, benzalkonium chloride, to the serosa of rat jejunum results in an increase in thickness of both longitudinal and circular smooth muscle layers. The increase in thickness is due primarily to an increase in the number of smooth muscle cells (hyperplasia). Little cellular hypertrophy was observed. The time sequence of surfactant-induced effects on the muscle layers was determined. Within 24 h, total destruction of the longitudinal muscle and partial destruction of the circular muscle was evident. The myenteric plexus was also necrotic; however, the submucosal plexus remained intact. By 48 h after surfactant treatment, the smooth muscle cells remaining in the circular muscle layer had begun to divide, as indicated by the presence of mitotic figures and incorporation of ³H-thymidine. A repopulation of the longitudinal muscle layer began at this time, apparently the result of migration of cells arising in the circular muscle layer. By 5 days post-treatment, both muscle layers had regenerated to their original states. The myenteric plexus was totally absent. The denervated smooth muscle cells proceeded to divide until approximately day 15, resulting in hyperplasia of both muscle layers. Between 15 and 105 days, the number of muscle cells in the circular layer progressively declined, eventually returned to the value seen in control tissue. In contrast, the number of smooth muscle cells in the longitudinal layer remained elevated through the period of study (165 days). We hypothesize that the smooth muscle hyperplasia observed after serosal benzalkonium chloride application results from loss of the myenteric nerves.     

Ca binding to the human red cell membrane: characterization of membrane preparations and binding sites.

Inside out and right side out vesicles were used to study the sidedness of Ca binding to the human red cell membrane. It was shown that these vesicles exhibited only a limited permeability to Ca, enabling the independent characterization of Ca binding to the extracellular and cytoplasmic membrane surfaces...     

Calcium binding proteins in smooth muscle plasma membrane

Rat myometrium plasma membrane showed a number of 45Ca-binding proteins as identified by gel electrophoresis. An attempt was made to identify these either by studying the inhibition of this binding by several ions or by studying binding of these proteins to calmodulin, A9 an antibody against skeletal muscle Ca-binding proteins and Stains-all. On the basis of the molecular weight, calmodulin binding and La-sensitivity of Ca-binding, the Ca-binding protein at 137 +/- 2 kDa has been identified as the Ca-pump. This protein as judged from Coomassie blue staining forms a very small percentage of the proteins present in the plasma membrane.     

Characterization of intestinal smooth muscle responses and binding sites for endothelin.

The contractile activity of and binding sites for endothelin-1 (ET-1) were investigated in isolated guinea-pig ileal longitudinal smooth muscle (GPILM). ET-1 produced concentration-dependent contractions of GPILM that either slowly subsided in the continued presence of ET-1 or rapidly subsided following washing of the tissue. The ED50 value for ET-1 contractions was 4.2 +/- 1.3 x 10(-9) M. The removal of extracellular calcium or pretreatment with nifedipine produced a complete inhibition of the contractions to ET-1. The IC50 value of nifedipine for inhibition of ET-1 mediated contractions was 3.0 +/- 0.8 x 10(-8) M. ET-1 produced a marked prolonged homologous desensitization of its contractile response but did not affect the responses mediated by carbachol, histamine, serotonin, substance P, and PLA2. High-affinity binding sites for 125I-labelled ET-1 were identified on microsomal membranes prepared from GPILM with Kd and Bmax values obtained by Scatchard analysis of 3.5 +/- 0.6 x 10(-10) M and 2138 +/- 159 fmol/mg protein, respectively. The binding of 125I-labelled ET-1 to GPILM microsomes was characterized by a rapid association (kob value of 0.077 min-1 at a radioligand concentration of 0.45 nM and an extremely slow dissociation (k1 value of 0.011 min-1; t1/2 value of 793 min). The binding was unaffected by the calcium channel antagonists nifedipine, verapamil, and diltiazem (10(-6) M); the receptor antagonists phenoxybenzamine, atropine, and naloxone (10(-6) M) and propranolol; and the peripheral benzodiazepine receptor antagonists Ro 5-4864 and PK 11195 and psychotomimetic drug phencyclidine (10(-5) M).(ABSTRACT TRUNCATED AT 250 WORDS)     

Galanin and its analogues: A structure-activity relationship studies in rat isolated gastric smooth muscles

Summary Galanin (GAL), a 29-amino-acid-residue neuropeptide, modulates gastric smooth muscles activity by interacting with specific receptors. However due to the lack of specific antagonists in the gastrointestinal (GI) tract the actual level of GAL involvement in GI motility remains largely unknown. In our studies we have performed structure-activity relationship studies of two porcine galanin fragments, two chimeric galanin analogues and several 15-amino-acid-residue galanin analogues modified in positions 2, 3, 4, 6, 8 or 14, investigating their contractile action on rat isolated gastric fundus strips, employed as in vitro assay of peptides activity. Thus we intended to characterize the molecular domains of GAL responsible for binding and activation of GAL receptors in rat gastric smooth muscle cells. The data acquired in the course of our structure-activity relationship studies suggest that both N-and C-terminal fragment of GAL molecule contribute towards the affinity and activity of GAL gastric smooth muscle cell receptors. Moreover, we concluded that positions 2, 3, 4, 6, 8 and 14 in the amino acid sequence of GAL may play important roles in binding and activation of GAL receptors in rat gastric smooth muscle cells. Abbreviations: The symbols of the amino acids, peptides and their derivatives are in accordance with the 1983 Recommendations of the IUPAC-IUB Joint Commission on Biochemical Nomenclature (Eur. J. Biochem. 138, 9 (1984)). Other symbols     

Ca binding to the human red cell membrane: Characterization of membrane preparations and binding sites

Summary Inside out and right side out vesicles were used to study the sidedness of Ca binding to the human red cell membrane. It was shown that these vesicles exhibited only a limited permeability to Ca, enabling the independent characterization of Ca binding to the extracellular and cytoplasmic membrane surfaces. Ca binding was studied in 10 mM Tris HCl at pH 7.4, 22±2°C and was shown to be complete in under 5 min. Scatchard plots were made from Ca binding data obtained at free Ca concentrations in the range of 10^–6 to 10^–3M. Under these conditions inside out vesicles exhibit two independent binding sites for Ca with association constants of 1×10⁵ and 6×10³ M^–1, and right side out vesicles exhibit three independent binding sites with association constants of 2×10⁵, 1.4×10⁴ and 3×10²M^–1. Upon the addition of 0.1M KCl a third, high affinity site was found on inside out vesicles with an association constant of 3×10⁵, (in 0.1 M KCl). Ca binding to inside out vesicles increased nearly linearly with pH in the, range of pH 4 to pH 11, while binding to right side out vesicles remained practically unchanged in the range of pH 7 to pH 9. Progressive increase of the ionic strength of the medium by the addition of K, Mg or Tris decreased Ca binding to inside out vesicles as did the addition of ATP. Comparison of a series of cation competitors for Ca binding sites on inside out vesicles at 0.003 mM Ca showed that La was the most effective competitor of all while Cd was the most effective divalent cation competitor of those tested. Our findings suggest that the effects of low concentrations of Ca at the inner surface of the red cell membrane are mediated primarily through Ca binding to site 1 (and, possibly site 2) of inside out vesicles of which there are approximately 1.6×10⁵ per equivalent cell.     

Galanin and its analogues: A structure-activity relationship studies in rat isolated gastric smooth muscles

Galanin (GAL), a 29-amino-acid-residue neuropeptide, modulatesgastric smooth muscles activity by interacting with specific receptors. However due to the lack of specific antagonists in thegastrointestinal (GI) tract the actual level of GAL involvement in GI motility remains largely unknown. In our studies we have performed structure-activity relationship studies of two porcinegalanin fragments, two chimeric galanin analogues and several 15-amino-acid-residue galanin analogues modified in positions 2, 3, 4, 6, 8 or 14, investigating their contractile action on rat isolated gastric fundus strips, employed as in vitro assay of peptides activity. Thus we intended to characterize the moleculardomains of GAL responsible for binding and activation of GAL receptors in rat gastric smooth muscle cells. The data acquired in the course of our structure-activity relationship studies suggest that both N- and C-terminal fragment of GAL molecule contribute towards the affinity and activity of GAL gastric smooth muscle cell receptors. Moreover, we concluded that positions 2, 3, 4, 6, 8 and 14 in the amino acid sequence of GAL may play important roles in binding and activation of GAL receptors in rat gastric smooth muscle cells.     

Characterization of prolactin binding by membrane preparations from rat liver.

Binding sites for prolactin were identified in a plasma-membrane-enriched fraction isolated from livers of mature female rats. 125I-labelled sheep prolactin prepared by the lactoperoxidase procedure retained the same molecular integrity and binding affinity as the native hormone at physiological pH. The receptors bound prolactin from different species, whereas non-lactogenic hormones were not bound. The binding of 125I-labelled sheep prolactin was activated equally by bivalent and univalent cations, bivalent cations exerting their maximal effect at much lower concentrations. The association of 125I-labelled sheep prolactin with the receptor was a time- and temperature-dependent process. Partial dissociation was detected. The binding of 125I-labelled sheep prolactin was strongly influenced by pH, with an optimum observed at pH 6.5. Receptor activity was destroyed by Pronase and phospholipase C, whereas neuraminidase increased binding. Treatment of the membranes by ribonuclease and deoxyribonuclease did not affect the binding. Binding of 125I-labelled sheep prolactin was inhibited by p-chloromercuribenzoic acid, dithiothreitol and by brief exposure to high temperatures. Scatchard analysis of the binding of 125I-labelled sheep prolactin to receptors indicated that prolactin has a high affinity for its receptor. Binding of prolactin to liver membranes showed some properties different from those observed with mammary cells. Binding by these tissues differed in pH optimum, in effects of ions, and in response to neuraminidase.     

Failure to detect specific binding sites for prostaglandin F-2 alpha in membrane preparations from rat endometrium

Membrane preparations from endometria of rats in different physiological states (e.g. pseudopregnancy, ovariectomized animals receiving progesterone + oestradiol or oestradiol alone) were studied for [3H]PGF-2 alpha binding by methods which detected PGF-2 alpha binding in ovary preparations and PGE binding in the same endometrial preparations. There was no evidence of high-affinity binding sites for [3H]PGF-2 alpha. Saturable [3H]PGF-2 alpha binding that increased with the onset of uterine sensitivity was detected but this binding does not fulfil all the criteria required for a PGF-2 alpha receptor and is probably due to binding to PG metabolizing enzymes in our preparations, or to binding of [3H]PGF-2 alpha to PGE binding sites. The failure to detect specific PGF-2 alpha binding sites seems to reflect a true absence of these sites in the rat endometrium.     

Ultrastructural localization of apamin binding sites in guinea pig intestinal smooth muscle

Binding sites of the neurotoxin apamin were located by immunocytochemical analysis using apamin antibodies. It was found that these bindings sites were located on the plasma membrane of smooth muscle cells in the longitudinal muscle of the cecum and at the nerve terminals in the guinea pig.A. V. Palladin Institute of Biochemistry, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 17, No. 6, pp. 824–827, November–December, 1985.     

Calcium channel antagonist binding and pharmacology in rat uterine smooth muscle

The calcium channel antagonists altered Ca-dependence of high K+-contractions of the estrogen dominant rat myometrium with the following pA2 values: PN-200-110, 10.63; nitrendipine, 9.56; nifedipine, 9.41; D-600, 9.05; and diltiazem, 7.57. Specific binding of 3H-nitrendipine occurred to the isolated plasma membrane vesicles with Kd of 0.1 to 0.3 nM and was inhibited by PN-200-110, nitrendipine, nifedipine and D-600, and slightly activated by diltiazem. The binding studies and the contractility studies were in excellent agreement for the three dihydropyridines, but not for D-600 and diltiazem.     

Characterization of 3H-AVP binding sites in particulate preparations of rat brain

Specific binding sites for vasopressin (AVP) were located in subcellular particulate fractions of rat brain with tritiated vasopressin of high specific activity, 22.5 Ci/mmol. Rat brain tissue was dissected, placed in cold 0.32 M sucrose containing proteolytic inhibitors, homogenized and fractionated into a crude nuclear fraction (1K pellet), crude mitochondrial fractions (12K pellet), and plasma membranes and microsomes (100K pellet). Specific binding of vasopressin was found in the 12K and 100K pellets in the presence of a divalent metal ion with Ni greater than Co greater than Mg greater than Mn greater than no metal ion at pH 7.4 in 50 mM Tris-Maleate buffer. Maximum specific binding of 16 nM AVP was located in the 100K anterior cortex fraction which bound 350 fmoles/mg protein; striatum, midbrain/thalamus, cerebellum, and medulla oblongata and pons bound specifically about 200 fmoles/mg protein and frontal poles and parietal cortex about 100 fmoles/mg protein in the 100K pellet. In all of the brain regions studied, except hippocampus and septum, the 100K pellet bound specifically 2 to 4 times more 3H-AVP than the 12K pellet. In the hippocampus with 16 nM AVP, the 12K pellet bound specifically 150 fmoles/mg protein; the septum, 75 fmoles/mg protein. Little or no binding to the 100K pellet was present in these regions. Bound AVP could be dissociated rapidly from the membranes by the addition of EDTA. The 12K hippocampal pellet was further fractionated into myelin, mitochondria, and synaptosomes; purification was confirmed by marker enzyme assays.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autoradiographic demonstration of gastrin binding sites in rat gastric mucosa

The location of 125I-iodotyrosyl gastrin I binding sites in rat gastric mucosa was studied. Peptide specificity was demonstrated by competitive binding studies through the addition of a large dose of cold human gastrin I or cholecystokinin-octapeptide. Autoradiography of the stomach tissue was carried out by freeze-drying, embedding in Epon, wet-sectioning with ethylene glycol, and dry-mounting the emulsion film by means of the wire-loop method to prevent loss of the labeled substance. Specific binding sites for gastrin were found on parietal and chief cells, whereas few binding sites were seen on the surface mucous or mucous neck cells. Binding sites on the parietal cells were dispersed in the cytoplasm, while those on the chief cells were found near the basal plasma membrane.     

Migration of vascular smooth muscle cells by phospholipase A2 via specific binding sites.

Pancreatic-type group I phospholipase A2 (PLA2-I), EC 3.1.1.4, long thought to act as a digestive enzyme, has a specific binding site in several types of tissues and cells and these sites promote PLA2-I-stimulated DNA synthesis. In this study we report a PLA2-I action on the migration of rat embryonic thoracic aorta smooth muscle cells (A7r5). A7r5 cells had a single class of PLA2-I binding site with an equilibrium binding constant (Kd) value of 1.7 nM and a maximum binding capacity (Bmax) of 40,000 sites/cell. The migration activity of PLA2-I for A7r5 cells was examined using modified Boyden chambers. PLA2-I stimulated the migration dose-dependently, and the ED50 value was about 1 nM, which was almost the same as the Kd value for PLA2-I binding. Checkerboard analysis showed that the response of A7r5 cells to PLA2-I was chemokinetic, but not chemotactic. These findings reveal a new aspect of PLA2-I in the modulation of vascular function.     

Nitrobenzylthionionosine binding sites in the erythrocyte membrane.

Nitrobenzylthioinosine binds tightly, but reversibly, to sites in the human erythrocyte membrane; occupancy of these sites blocks the transport of uridine and of other nucleosides. This report described the inhibition of nitrobenzylthioinosine binding at these sites by substrates of the uridine transport mechanism and by compounds related to nitrobenzylthioinosine. For some of these compounds dissociation constants for binding at the nitrobenzylthioinosine sites were determined, assumming competition with nitrobenzylthioinosine. Deoxycytidine, a substrate for the uridine transport mechanism, did not inhibit binding of nitrobenzylthioinosine, suggesting that binding sites for the latter are distinct from nucleoside sites directly involved in transport.     

Identification of functional binding sites for progesterone in rat Leydig cell plasma membrane.

Steroid hormones influence cell functions by binding to intracellular receptors and then acting within the nucleus. There is now evidence that steroids affect cell functions also via interaction with plasma membrane receptors in a number of different cell types. In this regard, progesterone appears to be one of the most active steroids. In this paper, we evaluate the effects of progesterone on rat Leydig cell functions, determining variations of ion homeostasis and testosterone production. This steroid was able to effect a depolarization of the plasma membrane that was due to an influx of sodium (Na+) from the external medium since it was absent when extracellular Na+ was iso-osmotically substituted with choline chloride or sucrose. The determination of intracellular sodium concentration ([Na+]i) with the Na+ -sensitive fluorescent dye sodium-benzofuran-isophtalate (SBFI) confirmed these observations. Progesterone did not modify Leydig cell intracellular calcium concentration ([Ca2+]i) at any dose tested. Furthermore, using a cell impermeant progesterone conjugate, we demonstrated that progesterone was able to stimulate Leydig cell steroidogenesis in a dose-dependent manner. The exclusion of calcium (Ca2+) from the extracellular medium did not modify the depolarizing action of progesterone and its steroidogenetic effect while in Na+ -free medium (sucrose supplemented) progesterone-stimulated effects were completely blunted. Finally, using fluorescence microscopy with a fluorescein isothiocyanate-coupled cell impermeant progesterone conjugate, we identified plasma membrane binding sites for progesterone in rat Leydig cells. These results suggest that rat Leydig cells possess progesterone receptors located on the plasma membrane, which when occupied achieves a plasma membrane depolarization, dependent on an influx of Na+ from the external medium, and the subsequent activation of steroidogenesis.