scholarly journals Comparative localization of inositol 1,4,5-trisphosphate and ryanodine receptors in intestinal smooth muscle: an analytical subfractionation study

1994 ◽  
Vol 297 (2) ◽  
pp. 415-423 ◽  
Author(s):  
M Wibo ◽  
T Godfraind
Keyword(s):  
Smooth Muscle ◽  
Binding Sites ◽  
Gradient Centrifugation ◽  
Ryanodine Receptors ◽  
Intestinal Smooth Muscle ◽  
Affinity Binding ◽  
Stoichiometric Ratio ◽  
Cell Fractionation ◽  
High Affinity Binding ◽  
High Affinity

[3H]Ins(1,4,5)P3- and [3H]ryanodine-binding sites were characterized in membrane fractions from guinea-pig intestinal smooth muscle (longitudinal layer) and their subcellular localization was investigated by analytical cell-fractionation techniques. Fractions collected at low centrifugal fields (N and M fractions) contained predominantly low-affinity [3H]Ins(1,4,5)P3-binding sites (KD 80 nM), whereas microsomal (P) fractions contained only high-affinity binding sites (KD 5 nM). Total sedimentable high-affinity binding sites of [3H]Ins(1,4,5)P3 were 9-10-fold more numerous than those of [3H]ryanodine. Both high-affinity binding sites were purified in microsomal fractions, and their sub-microsomal distribution patterns after isopycnic density-gradient centrifugation were similar to those of presumed endoplasmic reticulum (ER) constituents, indicating that Ins(1,4,5)P3 and ryanodine receptors were localized primarily in ER and probably associated with rough as well as smooth ER. However, the stoichiometric ratio of Ins(1,4,5)P3 to ryanodine receptors was distinctly higher in high-density RNA-rich subfractions than in low-density RNA-poor subfractions, suggesting that Ins(1,4,5)P3 receptors were somewhat concentrated in the ribosome-coated portions of ER. The low overall stoichiometric ratio of ryanodine to Ins(1,4,5)P3 receptors in intestinal smooth muscle (1:9-10) might explain, at least partly, the existence of a Ca(2+)-storage compartment devoid of ryanodine-sensitive Ca2+ channels, but equipped with Ins(1,4,5)P3-sensitive channels, in saponin-permeabilized smooth-muscle cells [Iino, Kobayashi and Endo (1988) Biochem. Biophys. Res. Commun. 152, 417-422].

Characterization of intestinal smooth muscle responses and binding sites for endothelin

1992 ◽  
Vol 70 (3) ◽  
pp. 377-384 ◽  
Author(s):  
Gordon T. Bolger ◽  
Francine Liard ◽  
Michel Garneau ◽  
Jorge Jaramillo
Keyword(s):  
Smooth Muscle ◽  
Binding Sites ◽  
Contractile Activity ◽  
Benzodiazepine Receptor ◽  
Intestinal Smooth Muscle ◽  
Scatchard Analysis ◽  
Affinity Binding ◽  
Receptor Antagonists ◽  
High Affinity Binding ◽  
High Affinity

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 × 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 × 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 × 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 (kl 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). Incubation of GPILM with ET-1 (2 × 10−8 M) for 10 min followed by washing of the tissue for 1 h resulted in a significant (p < 0.05 unpaired Student's t-test) reduction (33%) of 125I-labelled ET-1 binding that partially recovered following 2 h of washing the tissue. These results demonstrate that ET-1 is an intestinal smooth muscle spasmogen that produces its pharmacologic effects by a mechanism(s) that is not shared by other major intestinal neurotransmitters. Furthermore, intestinal smooth muscle contains specific high-affinity binding sites that likely mediate the contractile responses to ET-1.Key words: intestine, smooth muscle, endothelin, calcium channels, contraction.

High-affinity binding sites for [3H]saxitoxin are associated with voltage-dependent sodium channels in portal vein smooth muscle

1990 ◽  
Vol 184 (2-3) ◽  
pp. 315-319 ◽  
Author(s):  
Jean Mironneau ◽  
Cécile Martin ◽  
Serge Arnaudeau ◽  
Khadija Jmari ◽  
Lala Rakotoarisoa ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Portal Vein ◽  
Sodium Channels ◽  
Binding Sites ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Voltage Dependent

Postnatal changes in the substance P receptor on rabbit gastric smooth muscle

1992 ◽  
Vol 262 (2) ◽  
pp. G291-G297
Author(s):  
P. E. Hyman ◽  
S. Kimura ◽  
T. Tomomasa ◽  
Q. X. Yuan ◽  
W. J. Snape ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Substance P ◽  
Binding Sites ◽  
Tissue Homogenate ◽  
Neurokinin A ◽  
Affinity Binding ◽  
Binding Studies ◽  
High Affinity Binding ◽  
High Affinity ◽  
Gastric Smooth Muscle

We used radioligand binding to tissue homogenates and isometric contraction of muscle strips to characterize the substance P (SP) receptor on gastric smooth muscle from 1- (newborn) and 7-day-old and 4- and 11-wk-old (weanling) rabbits. Scatchard analysis for newborns was curvilinear, suggesting the presence of multiple binding sites. In newborns the dissociation constant (Kd) of high-affinity binding site was 2.2 +/- 0.3 nM, and the maximum binding (Bmax) was 0.57 +/- 0.06 pmol/mg DNA. The number of high-affinity binding sites decreased with age, disappearing by 11 wk. The Kd for the low-affinity site was more than two orders of magnitude greater than that of the high-affinity site. In competitive binding studies with [3H]SP, the order of potency for the neurokinins was SP much greater than neurokinin A (NKA) greater than neurokinin B (NKB), suggesting that the high-affinity binding sites were NK-1 receptors. [125I]NKA is also bound to newborn tissue homogenate with high affinity. With [125I]NKA the order was NKA greater than SP greater than NKB, suggesting that NK-2 receptors were also present. In contraction studies, atropine and tetrodotoxin had no effect on tachykinin-stimulated contraction, suggesting solely myogenic tachykinin effects on this tissue. In newborn rabbits, the potency and efficacy of SP and NKA were similar. The half-maximal effective dose (ED50) of SP was nearly two orders of magnitude less in newborn rabbits than in weanlings; the potency of NKA did not change.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Characterization of High Affinity Binding Sites for Charybdotoxin in Sarcolemmal Membranes from Bovine Aortic Smooth Muscle

1989 ◽  
Vol 264 (35) ◽  
pp. 20902-20909 ◽  
Author(s):  
J Vázquez ◽  
P Feigenbaum ◽  
G Katz ◽  
V F King ◽  
J P Reuben ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Binding Sites ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
Aortic Smooth Muscle ◽  
High Affinity

High Affinity Binding Sites for Activated Protein C and Protein C on Cultured Human Umbilical Vein Endothelial Cells

1994 ◽  
Vol 72 (03) ◽  
pp. 465-474 ◽  
Author(s):  
Neelesh Bangalore ◽  
William N Drohan ◽  
Carolyn L Orthner
Keyword(s):  
Binding Sites ◽  
Protein C ◽  
Umbilical Vein ◽  
Activated Protein C ◽  
Affinity Binding ◽  
Cell Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Gla Domain ◽  
Umbilical Vein Endothelial Cells

SummaryActivated protein C (APC) is an antithrombotic serine proteinase having anticoagulant, profibrinolytic and anti-inflammatory activities. Despite its potential clinical utility, relatively little is known about its clearance mechanisms. In the present study we have characterized the interaction of APC and its active site blocked forms with human umbilical vein endothelial cells (HUVEC). At 4° C 125I-APC bound to HUVEC in a specific, time dependent, saturable and reversible manner. Scatchard analysis of the binding isotherm demonstrated a Kd value of 6.8 nM and total number of binding sites per cell of 359,000. Similar binding isotherms were obtained using radiolabeled protein C (PC) zymogen as well as D-phe-pro-arg-chloromethylketone (PPACK) inhibited APC indicating that a functional active site was not required. Competition studies showed that the binding of APC, PPACK-APC and PC were mutually exclusive suggesting that they bound to the same site(s). Proteolytic removal of the N-terminal γ-carboxyglutamic acid (gla) domain of PC abolished its ability to compete indicating that the gla-domain was essential for cell binding. Surprisingly, APC binding to these cells appeared to be independent of protein S, a cofactor of APC generally thought to be required for its high affinity binding to cell surfaces. The identity of the cell binding site(s), for the most part, appeared to be distinct from other known APC ligands which are associated with cell membranes or extracellular matrix including phospholipid, thrombomodulin, factor V, plasminogen activator inhibitor type 1 (PAI-1) and heparin. Pretreatment of HUVEC with antifactor VIII antibody caused partial inhibition of 125I-APC binding indicating that factor VIII or a homolog accounted for ∼30% of APC binding. Studies of the properties of surface bound 125I-APC or 125I-PC and their fate at 4°C compared to 37 °C were consistent with association of ∼25% of the initially bound radioligand with an endocytic receptor. However, most of the radioligand appeared not to be bound to an endocytic receptor and dissociated rapidly at 37° C in an intact and functional state. These data indicate the presence of specific, high affinity binding sites for APC and PC on the surface of HUVEC. While a minor proportion of binding sites may be involved in endocytosis, the identity and function of the major proportion is presently unknown. It is speculated that this putative receptor may be a further mechanisms of localizing the PC antithrombotic system to the vascular endothelium.

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