scholarly journals Identification of the Ca2+-release activity and ryanodine receptor in sarcoplasmic-reticulum membranes during cardiac myogenesis

1988 ◽  
Vol 253 (3) ◽  
pp. 631-636 ◽  
Author(s):  
M Michalak
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Binding Sites ◽  
Binding Capacity ◽  
Ryanodine Receptors ◽  
Ruthenium Red ◽  
Scatchard Analysis ◽  
Fetal Sheep ◽  
Cardiac Myogenesis ◽  
High Affinity Binding Site ◽  
Well Differentiated

Ca2+-induced Ca2+ release and pH-induced Ca2+ release activities were identified in sarcoplasmic-reticulum (SR) vesicles isolated from adult- and fetal-sheep hearts. Ca2+-induced Ca2+ release and pH-induced Ca2+ release appear to proceed via the same channels, since both phenomena are similarly inhibited by Ruthenium Red. Ca2+ release from fetal SR vesicles is inhibited by higher concentrations of Ruthenium Red than is that from adult membranes. Both fetal and adult SR vesicles bind ryanodine. Fetal SR shows higher ryanodine-binding capacity than adult SR vesicles. Scatchard analysis of ryanodine binding revealed only one high-affinity binding site (Kd 6.7 nM) in fetal SR vesicles compared with two distinct binding sites (Kd 6.6 and 81.5 nM) in the adult SR vesicles. SR vesicles isolated from fetal and adult hearts were separated on discontinuous sucrose gradients into light (free) and heavy (junctional) SR vesicles. Heavy SR vesicles isolated from adult hearts exhibited most of the Ca2+ release activities. In contrast, Ca2+-induced Ca2+ release, pH-induced Ca2+ release and ryanodine receptors were detected in both light and heavy fetal SR. These results suggest that fetal SR may not be morphologically and functionally as well differentiated as that of adult cardiac muscle and that it may contain a greater number of Ca2+-release channels than that present in adult SR membranes.

Mineralcorticoid receptors along the nephron: [3H]aldosterone binding in rabbit tubules

1981 ◽  
Vol 241 (6) ◽  
pp. F605-F611 ◽  
Author(s):  
A. Doucet ◽  
A. I. Katz
Keyword(s):  
Binding Sites ◽  
Binding Capacity ◽  
Specific Binding ◽  
Rabbit Kidney ◽  
Scatchard Analysis ◽  
Thick Ascending Limb ◽  
Collecting Tubule ◽  
The Difference ◽  
Collecting Tubules ◽  
Cortical Collecting Tubule

To identify the site of mineralocorticoid action along the nephron, we measured the specific binding of [3H]aldosterone to nephron segments microdissected from aldosterone-deficient rabbits. Specific binding was defined as the difference between binding measured in the absence or in the presence of 2,000-fold excess of unlabeled hormone (in 10(-18) mol X cm tubule length-1 +/- SE). High specific binding capacity was found in the branched collecting tubule (108 +/- 4), the cortical collecting tubule (119 +/- 9), and the outer medullary collecting tubule (115 +/- 16), whereas specific binding was negligible in the proximal convoluted tubule (8 +/- 9), pars recta (2 +/- 6), medullary thick ascending limb (4 +/- 6), cortical thick ascending limb (6 +/- 2), and distal convoluted tubule (6 +/- 6). In cortical collecting tubules, Scatchard analysis of the specific [3H]aldosterone binding indicated a dissociation constant (KD) of 2.2 X 10(-9) M and a maximum number of binding sites of 157 X 10(-18) mol X cm tubule length-1. The steroid specificity was assessed from the competition of various steroids for [3H]aldosterone binding sites. Receptors from the cortical collecting tubule revealed the following sequence of affinities: aldosterone greater than DOCA greater than spironolactone greater than dexamethasone greater than 5 alpha-dihydrotestosterone = progesterone = 17 beta-estradiol, indicating that the binding sites in the collecting tubule are mineralocorticoid receptors. These results demonstrate significant [3H]aldosterone binding to receptors of high affinity and mineralocorticoid specificity only in the collecting tubule and suggest that this nephron segment is the target site of mineralocorticoid action in the rabbit kidney.

Sarcoplasmic Reticulum Calcium Release Channels in Ventricles of Older Adult Hamsters

2006 ◽  
Vol 25 (1) ◽  
pp. 107-113 ◽  
Author(s):  
Peter A. Nicholl ◽  
Susan E. Howlett
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Older Adult ◽  
Binding Sites ◽  
Calcium Release ◽  
Ryanodine Receptors ◽  
Calcium Release Channels ◽  
Site Density ◽  
Age Related ◽  
Sarcoplasmic Reticulum Calcium ◽  
Age Related Changes

ABSTRACTWhether the density of sarcoplasmic reticulum (SR) calcium release channels / ryanodine receptors in the heart declines with age is not clear. We investigated age-related changes in the density of «3H»-ryanodine receptors in crude ventricular homogenates, which contained all ligand binding sites in heart and in isolated junctional SR membranes. Experiments utilized young (120 days) and older adult (300 days) hamsters. «3H»-ryanodine binding site density did not change with age in crude homogenate preparations, although total heart protein concentration increased significantly with age. In contrast, the density of «3H»-ryanodine binding sites decreased markedly in heavy SR membranes purified from older hearts. These results show that demonstration of age-related changes in cardiac ryanodine receptor density depends upon the preparation used. Furthermore, the increase in total ventricular protein with age suggests that normalization of data by membrane protein should be used with caution in studies of aging heart.

scholarly journals Influence of divalent cations on the detection of somatogenic and lactogenic binding sites in mouse liver cells

1985 ◽  
Vol 228 (3) ◽  
pp. 761-764 ◽  
Author(s):  
G N Ciccia-Torres ◽  
J M Dellacha
Keyword(s):  
Binding Sites ◽  
Mouse Liver ◽  
Binding Capacity ◽  
Specific Binding ◽  
Divalent Cations ◽  
Isolated Hepatocytes ◽  
Liver Cells ◽  
Scatchard Analysis ◽  
Male Mice ◽  
Ovine Prolactin

Specific binding of 125I-labelled human somatotropin was demonstrated in isolated hepatocytes from male mice. In the presence of divalent cations (Ca2+ and Mg2+) the binding of 125I-labelled human somatotropin was competitive with ovine prolactin. Scatchard analysis of competition data indicated a KD of 1.4 +/- 0.2 nM and a binding capacity of 13 000 +/- 2000 sites/cell. In the absence of divalent cations and in the presence of EDTA, human and bovine somatotropins were found to be equally effective to displace bound 125I-labelled human somatotropin, while ovine prolactin showed a weak competition. In this case, the binding capacity was 8400 +/- 1500 sites/cell and the KD was 1.1 +/- 0.1 nM.

Characteristics and developmental changes of corticotrophin-releasing hormone-binding sites in the fetal sheep anterior pituitary gland

1991 ◽  
Vol 130 (2) ◽  
pp. 223-229 ◽  
Author(s):  
F. Lü ◽  
K. Yang ◽  
J. R. G. Challis
Keyword(s):  
Binding Sites ◽  
Anterior Pituitary ◽  
Time Course ◽  
Scatchard Analysis ◽  
Maximum Output ◽  
Similar Time ◽  
Single Class ◽  
Fetal Sheep ◽  
Releasing Hormone ◽  
Receptor Number

ABSTRACT The responses of the fetal sheep pituitary to corticotrophin-releasing hormone (CRH) change during gestation with maximum output of ACTH around days 120–130, and decreased ACTH output near term. However, there is no information available concerning the extent to which these responses may be modulated by alterations in the number of CRH receptors. Therefore we measured specific CRH-binding sites, and changes in binding characteristics in membrane preparations from fetal sheep anterior pituitaries collected at days 65–70, 85–88, 100–110, 125–130 and at term (approximately 145 days). Binding assays were carried out using 125I-labelled Tyr-ovine CRH (125I-Tyr-oCRH), incubated with crude membrane fractions for 90 min at 22 °C. Binding was time- and temperature-dependent, linear with protein concentration, saturable and specific for oCRH. Scatchard analysis of binding data for individual tissues revealed a single class of CRH-binding sites with high affinity (Kd ≃1 nmol/l) that did not change significantly with gestational age. However, the number of CRH-binding sites increased progressively from days 65–70 to a maximum at days 125–130, then decreased at term. These results demonstrate the presence of specific CRH-binding sites in the fetal sheep anterior pituitary. Furthermore, the change in CRH receptor number with advancing pregnancy follows a similar time-course to the changes reported previously in responsiveness of the fetal sheep anterior pituitary to exogenous CRH stimulation in vivo. These results suggest that alterations in CRH receptor number may contribute to changes in responsiveness of the fetal sheep anterior pituitary to CRH during gestation. Journal of Endocrinology (1991) 130, 223–229

Effects of hindlimb suspension on cytosolic Ca2+ and [3H]ryanodine binding in cardiac myocytes

1999 ◽  
Vol 276 (4) ◽  
pp. H1131-H1136
Author(s):  
Guillaume Halet ◽  
Patricia Viard ◽  
Jean-Luc Morel ◽  
Jean Mironneau ◽  
Chantal Mironneau
Keyword(s):  
Ventricular Myocytes ◽  
Binding Capacity ◽  
Ryanodine Receptors ◽  
Hindlimb Suspension ◽  
Scatchard Analysis ◽  
Channel Activity ◽  
Intrinsic Properties ◽  
Release Channel ◽  
Voltage Dependent ◽  
Cell Capacitance

Effects of a 14-day hindlimb suspension were examined on [3H]ryanodine binding to rat ventricular microsomes and on cytosolic Ca2+ concentration ([Ca2+]i) and voltage-dependent Ca2+channels in isolated ventricular myocytes. In suspended rats, the amplitude of the twitch [Ca2+]itransient was increased without significant modifications of the basal [Ca2+]iand sarcoplasmic reticulum content. Because cell capacitance, L-type Ca2+-current density, and Ca2+-channel gating were not significantly modified after suspension, the increase in [Ca2+]iwas expected to reside in a change in ryanodine receptors. Scatchard analysis of [3H]ryanodine binding revealed that suspension enhanced binding by increasing the affinity of the receptors for [3H]ryanodine without affecting the maximal binding capacity. Both Ca2+-release channel activity and [3H]ryanodine binding are modulated by Ca2+. However, the Ca2+ sensitivity of [3H]ryanodine binding remained unchanged after suspension. Taken together, these results suggest that the increase in twitch [Ca2+]itransients after suspension may result from a change in the intrinsic properties of the ryanodine receptors but not from a change in the expression level of these receptors.

Caffeine- and ryanodine-sensitive Ca2+ stores of canine cerebrum and cerebellum neurons

1991 ◽  
Vol 261 (6) ◽  
pp. C1048-C1054 ◽  
Author(s):  
L. G. Meszaros ◽  
P. Volpe
Keyword(s):  
Binding Sites ◽  
Striated Muscle ◽  
Binding Capacity ◽  
Ruthenium Red ◽  
Hill Coefficient ◽  
Stopped Flow ◽  
High Affinity ◽  
Protein Dissociation ◽  
Dissociation Constant Kd ◽  
Slight Inhibition

[3H]ryanodine binding to and Ca2+ release from microsomal fractions derived from canine cerebrum (CBR) and cerebellum (CBL) were investigated. High-affinity ryanodine binding sites were detected in both cerebrum and cerebellum microsomes [CBR: maximal binding capacity (Bmax) = 446 fmol/mg protein, dissociation constant (Kd) = 9 nM, Hill coefficient (n) = 0.95; CBL: Bmax = 650, Kd = 12, n = 1.8]. Ryanodine binding in both fractions was increased by millimolar concentrations of ATP [or its nonhydrolyzable analogue beta, gamma-methyleneadenosine 5'-triphosphate (AMP-PCP)] and micromolar concentrations of Ca2+ but was decreased by micromolar concentrations of ruthenium red, similar to that found in sarcoplasmic reticulum (SR) of striated muscle. The addition of caffeine or the sudden elevation of extravesicular Ca2+ induced a rapid La(3+)-sensitive Ca2+ release from both CBR and CBL microsomal fractions with rate constants of approximately 100 s-1, as determined by stopped-flow photometry of the Ca2+ indicator arsenazo III. The release of Ca2+ was activated by either millimolar ATP or AMP-PCP, blocked by micromolar concentrations of La3+, and significantly inhibited by 50 microM ryanodine. Mg2+ and ruthenium red in millimolar and micromolar concentrations, respectively, caused only a slight inhibition of Ca2+ release. These results indicate that rapid Ca2+ release occurs from caffeine-, Ca2+- and ryanodine-sensitive Ca2+ stores in both CBR and CBL microsomal fractions.

Adaptation of the skeletal muscle calcium-release mechanism to weight-bearing condition

1996 ◽  
Vol 270 (6) ◽  
pp. C1588-C1594 ◽  
Author(s):  
S. C. Kandarian ◽  
D. G. Peters ◽  
T. G. Favero ◽  
C. W. Ward ◽  
J. H. Williams
Keyword(s):  
Ryanodine Receptor ◽  
Calcium Release ◽  
Binding Capacity ◽  
Polyclonal Antibodies ◽  
Weight Bearing ◽  
Ryanodine Receptors ◽  
Release Mechanism ◽  
Surgical Removal ◽  
Scatchard Analysis ◽  
Western Blots

In the present study, we examined whether weight-bearing condition can regulate the sarcoplasmic reticulum (SR) Ca(2+)-release mechanism. Measurements of alpha 1-subunit dihydropyridine (alpha 1-DHP) and ryanodine receptor levels were made in hypertrophied fast-twitch plantaris muscles 5 wk after surgical removal of synergist muscles (increased weight bearing) and in atrophied slowtwitch soleus muscles (14 days of non-weight bearing) of the rat. Rates of AgNO3-induced SR Ca2+ release were measured with fura 2 as the Ca2+ indicator and pyrophosphate as the precipitating ion during vesicular Ca2+ loading. Ca(2+)-release rates were 38, 49, and 58% lower in vesicles from hypertrophied vs. control muscles at AgNO3 concentrations of 0.05, 0.5, and 5 microM, respectively (control = 18.2 +/- 1.4 microM.mg-1. min-1). Western blots showed no differences in the relative expression of alpha 1-DHP or ryanodine receptor when IIID5 (monoclonal) or GP3 (polyclonal) antibodies were used. There was also no difference in ryanodine (10 nM) binding in Ca(2+)-incubated SR vesicles from hypertrophied muscles, suggesting no difference in the number of channels. In contrast, expression of alpha 1-DHP and ryanodine receptors was increased by 144 and 157% in non-weight-bearing soleus muscles, respectively. Scatchard analysis of DHP binding showed a 40% increase in maximum binding capacity and no change in the dissociation constant with non-weight-bearing muscles. The direction of modification of the SR Ca(2+)-release mechanism is opposite with increased and decreased weight bearing, but the mechanism by which this is achieved appears to be different.

scholarly journals Effects of ATP on the Interaction of Ca++, Mg++, and K+ with Fragmented Sarcoplasmic Reticulum Isolated from Rabbit Skeletal Muscle

1967 ◽  
Vol 50 (5) ◽  
pp. 1327-1352 ◽  
Author(s):  
Arselio P. Carvalho ◽  
Barbara Leo
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Binding Sites ◽  
Membrane Fraction ◽  
Binding Capacity ◽  
Cation Binding ◽  
The Other ◽  
Surface Binding ◽  
Ph Values ◽  
Number Of Binding Sites

Fragmented sarcoplasmic reticulum isolated from skeletal muscle of the rabbit has a cation-binding capacity of about 350 µeq/g of protein at neutral pH. The same binding sites bind Ca, Mg, K, and H ions and, consequently, the selective binding of Ca induced by ATP releases an amount of the other cations equivalent to the Ca taken up. At pH values below 6.2, an increasing number of binding sites are associated with H+, and ATP induces exchange of Ca mostly for H+. At pH values above 6.2, the binding sites exist in the form of Mg and K, and Ca is bound in exchange for these cations. The total bound Ca + Mg + K, expressed in microequivalents of cations bound per gram of protein, is approximately constant at various pCa values, which indicates a stoichiometric exchange of Ca for the other cations. To accomplish the same degree of exchange of Ca for other cations bound, in the absence of ATP, concentrations of free Ca++ of about 1000-fold higher than those needed in the presence of ATP are required in the medium. We cannot distinguish between a mechanism whereby Ca actively transported into a compartment of the microsomal vesicles containing also the binding sites is bound passively to these sites in exchange for Mg, K, and H and another in which ATP selectively increases the affinity of surface-binding sites for Ca. Irrespective of the mechanism of accumulation, the Ca retained does not contribute to the activity of the cation in the membrane fraction. Caffeine (10 mM) has no effect on the binding of Ca, but releases a more labile fraction of Ca, which presumably accumulates in excess of the bound Ca. Procaine (5 mM) antagonizes the effect of caffeine. Acetylcholine and epinephrine have no effect on the binding of Ca.

scholarly journals Purified ryanodine receptor from rabbit skeletal muscle is the calcium-release channel of sarcoplasmic reticulum.

1988 ◽  
Vol 92 (1) ◽  
pp. 1-26 ◽  
Author(s):  
J S Smith ◽  
T Imagawa ◽  
J Ma ◽  
M Fill ◽  
K P Campbell ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Ryanodine Receptor ◽  
Calcium Release ◽  
Binding Capacity ◽  
Ruthenium Red ◽  
Rabbit Skeletal Muscle ◽  
Permeability Ratio ◽  
Calcium Release Channel ◽  
Release Channel

The ryanodine receptor of rabbit skeletal muscle sarcoplasmic reticulum was purified as a single 450,000-dalton polypeptide from CHAPS-solubilized triads using immunoaffinity chromatography. The purified receptor had a [3H]ryanodine-binding capacity (Bmax) of 490 pmol/mg and a binding affinity (Kd) of 7.0 nM. Using planar bilayer recording techniques, we show that the purified receptor forms cationic channels selective for divalent ions. Ryanodine receptor channels were identical to the Ca-release channels described in native sarcoplasmic reticulum using the same techniques. In the present work, four criteria were used to establish this identity: (a) activation of channels by micromolar Ca and millimolar ATP and inhibition by micromolar ruthenium red, (b) a main channel conductance of 110 +/- 10 pS in 54 mM trans Ca, (c) a long-term open state of lower unitary conductance induced by ryanodine concentrations as low as 20 nM, and (d) a permeability ratio PCa/PTris approximately equal to 14. In addition, we show that the purified ryanodine receptor channel displays a saturable conductance in both monovalent and divalent cation solutions (gamma max for K and Ca = 1 nS and 172 pS, respectively). In the absence of Ca, channels had a broad selectivity for monovalent cations, but in the presence of Ca, they were selectively permeable to Ca against K by a permeability ratio PCa/PK approximately equal to 6. Receptor channels displayed several equivalent conductance levels, which suggest an oligomeric pore structure. We conclude that the 450,000-dalton polypeptide ryanodine receptor is the Ca-release channel of the sarcoplasmic reticulum and is the target site of ruthenium red and ryanodine.

Melatonin receptors are present in adult rat Leydig cells and are coupled through a pertussis toxin-sensitive G-protein

1997 ◽  
Vol 136 (6) ◽  
pp. 633-639 ◽  
Author(s):  
Sandra Valenti ◽  
Massimo Giusti ◽  
Roberta Guido ◽  
Giulio Giordano
Keyword(s):  
G Protein ◽  
Pertussis Toxin ◽  
Binding Sites ◽  
Leydig Cells ◽  
Melatonin Receptors ◽  
Scatchard Analysis ◽  
Adult Rat ◽  
Testosterone Secretion ◽  
High Affinity Binding Site ◽  
17 Hydroxyprogesterone

Abstract Previous studies have suggested that melatonin (MLT) acts directly on rat Leydig cells by modulating androgen production. In the present study, the site of action of MLT was investigated. The binding of 2-[125I]iodomelatonin (125I-MLT; 7–240 pmol/l) to Leydig cell membrane fragments was tested in the presence or absence of guanosine 5′-O-(3-thiotriphosphate) (GTP-γ-S; 50 μmol/l). Saturation studies and Scatchard analysis revealed the existence of a high-affinity binding site with a Bmax of 46·70± 3·50 fmol/mg protein and a Kd of 88·70±6·20 pmol/l; treatment with GTP-γ-S reduced the concentration of 125I-MLT binding sites (Bmax 34·03±4·50), while increasing the Kd to 106·5± 2·61 pmol/l. Pretreatment of the cells with pertussis toxin (PTX; 10 ng/ml for 16 h) resulted in a decreased binding of I-MLT and a lack of effect of GTP-γ-S. Moreover, the effect of MLT on testosterone secretion induced by LH (30 mIU/ml), forskolin (1 μmol/l) and LHRH (100 nmol/l) was studied after 3-h incubation of cells which had been precultured with or without PTX. The inhibition of testosterone secretion due to MLT administration was eliminated by PTX pretreatment during forskolin and LH, but not during LHRH administration. However, 17-hydroxyprogesterone levels were higher in all groups incubated in the presence of MLT, irrespective of PTX pretreatment. Our data suggest that: (a) MLT receptors are present on the membranes of adult rat Leydig cells; (b) they couple through PTX-sensitive G-protein-coupled binding sites; (c) the mechanism by which MLT blocks 17–20 desmolase enzymatic activity (thus leading to increased 17-hydroxyprogesterone levels), and testosterone secretion during LHRH stimulation is likely to depend on one or more different mechanism(s) of action. European Journal of Endocrinology 136 633–639

Sign in / Sign up

Export Citation Format

Share Document