Alpha-adrenergic receptors on rat ventricular myocytes: characteristics and linkage to cAMP metabolism

1986 ◽  
Vol 251 (2) ◽  
pp. H307-H313 ◽  
Author(s):  
I. L. Buxton ◽  
L. L. Brunton
Keyword(s):  
Dissociation Constant ◽  
Ventricular Myocytes ◽  
Specific Binding ◽  
Single Class ◽  
Adult Rat ◽  
Alpha Adrenergic Receptors ◽  
High Affinity ◽  
Adrenergic Antagonist ◽  
Dependent Protein ◽  
Camp Metabolism

When incubated with purified cardiomyocytes from adult rat ventricle, the alpha 1-antagonist [3H]prazosin binds to a single class of sites (8 X 10(4) per cell) with high affinity [dissociation constant (KD) = 82 pM]. Competition for [3H]prazosin binding by the alpha 2-selective antagonist yohimbine [inhibitor dissociation constant (KI) = 714 nM] and the nonselective alpha-antagonist phentolamine (KI = 168 nM) demonstrates that these receptors are of the alpha 1-subtype. In addition, incubation of myocyte membranes with [3H]yohimbine results in no measurable specific binding. Agonist competition for [3H]prazosin binding to membranes prepared from purified myocytes demonstrates the presence of two components of binding: 28% of alpha 1-receptors interact with norepinephrine with high affinity (KD = 36 nM), whereas the majority of receptors (72%) have a low affinity for agonist (KD = 2.2 microM). After addition of 10 microM GTP, norepinephrine competes for [3H]prazosin binding to a single class of sites with lower affinity (KD = 2.2 microM). Incubation of intact myocytes for 2 min with 1 microM norepinephrine leads to significantly less cyclic AMP (cAMP) accumulation (13.6 pmol/mg) than stimulation with either norepinephrine plus prazosin or isoproterenol (18 pmol/mg). Likewise, incubation of intact myocytes with 10(-6) M norepinephrine leads to significantly less activation of cAMP-dependent protein kinase (71 +/- 4%) than when myocytes are stimulated by both norepinephrine and the alpha 1-adrenergic antagonist, prazosin (95 +/- 1%), or the beta-adrenergic agonist, isoproterenol (100%).(ABSTRACT TRUNCATED AT 250 WORDS)

Dopamine1 receptors in rat kidneys identified with 125I-Sch 23982

1988 ◽  
Vol 255 (5) ◽  
pp. F970-F976 ◽  
Author(s):  
R. A. Felder ◽  
P. A. Jose
Keyword(s):  
Room Temperature ◽  
Specific Binding ◽  
Rat Kidney ◽  
Sch 23390 ◽  
Receptor Density ◽  
Single Class ◽  
Apparent Dissociation Constant ◽  
High Affinity ◽  
High Affinity Binding Site ◽  
Inhibition Constants

Dopamine1 receptors were studied in rat kidney using the selective dopamine1 antagonist 125I-labeled Sch 23982. The specific binding of 125I-Sch 23982 (defined by 5 microM Sch 23390) to renal cortical homogenates incubated at room temperature was rapid, saturable with time and ligand concentration, and reversible. Analysis of Rosenthal plots revealed a single class of receptors with an apparent dissociation constant of 12.2 +/- 1.9 nM and maximum receptor density of 1.03 +/- 0.15 pmol/mg protein (n = 6). However, competition experiments with the dopamine1 antagonist Sch 23390 revealed a low- and high-affinity binding site with inhibition constants of 1 x 10(-6) and 1 x 10(-8) M, respectively. The competition experiments were also indicative of dopamine1 receptors with stereoselectivity noted for dopamine1 but not for dopamine2 antagonists. The inhibition constants for dopamine1 antagonists and agonists were two orders of magnitude greater in renal cortical than striatal homogenates. Different buffers affected striatal but not renal cortical binding. Autoradiographic studies revealed 125I-Sch 23982 binding in renal cortical but not medullary tissue. These studies confirm the presence of dopamine1 receptors in the cortex of the rat kidney.

Prostaglandin E2 binding sites in porcine oxyntic mucosa: effects of salicylates

1986 ◽  
Vol 64 (5) ◽  
pp. 515-520 ◽  
Author(s):  
B. L. Tepperman ◽  
B. D. Soper
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Prostaglandin E ◽  
Affinity Binding ◽  
Variable Degree ◽  
Single Class ◽  
Oxyntic Mucosa ◽  
High Affinity Binding ◽  
Mucosal Ulceration ◽  
High Affinity

These studies were designed to examine the changes in the characteristics of prostaglandin E2 (PGE2) binding to porcine oxyntic mucosa in the response to oral ingestion of salicylates. Either acetylsalicylic acid (ASA) or salicylic acid (SA) was administered to conscious pigs (100 mg/kg in 30 mL of an equimolar concentration of NaHCO3) once a day for 1, 3, 10, or 20 days. In control experiments a similar volume of 0.3 M NaHCO3 was administered for similar durations. Mucosal ulceration and the characteristics of the binding of [3H]PGE2 to a 30 000 × g membrane preparation of oxyntic mucosa were examined. Generation of mucosal PGE2 was measured by radioimmunoassay. ASA treatment resulted in an increase in the number and severity of mucosal ulcers and a decrease in PGE2 levels within the first treatment day. By day 20 the degree of ulceration had decreased in spite of a persistent reduction of mucosal PGE2 generation. A variable degree of ulceration was observed in SA-treated animals. In control animals only a single class of binding sites for [3H]PGE2 was evident. After 3 days of ASA treatment a second class of binding sites with a high affinity dissociation constant appeared. There was a decrease in the high affinity binding of [3H]PGE2 after 20 days of ASA ingestion. Low affinity binding was not altered. ASA treatment resulted in a significant increase in specific binding capacities for both families of binding sites. SA treatment did not consistently alter PGE2 binding characteristics from control at any time period studied. These data suggest that SA treatment results in a small degree of mucosal damage in the absence of a significant reduction in tissue generation of PGE2 or changes in PGE2 binding. Damage in response to ASA ingestion was associated with a reduction in both endogenous synthesis of PGE2 and an increase in the concentration of both low and high affinity binding sites for PGE2. The reduction in mucosal ulceration on day 20 in spite of depressed endogenous PGE2 coincides with an increase in PGE2 binding.

The rainbow trout skeletal muscle β-adrenergic system: characterization and signaling

2003 ◽  
Vol 284 (3) ◽  
pp. R689-R697 ◽  
Author(s):  
Michel B. Lortie ◽  
Thomas W. Moon
Keyword(s):  
Rainbow Trout ◽  
Binding Sites ◽  
Specific Binding ◽  
Adrenergic System ◽  
Camp Production ◽  
Binding Assays ◽  
Single Class ◽  
Rainbow Trout Oncorhynchus Mykiss ◽  
Adrenergic Antagonist ◽  
Number Of Binding Sites

The presence and functionality of β-adrenoceptors (β-ARs) were examined in red (RM) and white muscle (WM) membranes isolated from the rainbow trout Oncorhynchus mykiss. Specific binding assays revealed the presence of a single class of binding sites with similar affinities in both muscle types ( K d in nM: 0.14 ± 0.03 and 0.18 ± 0.03 for RM and WM, respectively) but with a significantly higher number of binding sites in RM compared with WM (Bmax in fmol/mg protein: 3.22 ± 0.11 and 2.60 ± 0.13, respectively). Selective and nonselective β-adrenergic agonists (β-AAs) and antagonists indicated an atypical β-AR pharmacology. This result may represent a nonmammalian β-AR classification or, more likely, the presence of more than one β-AR subtype in trout muscles with similar affinities that could not be kinetically resolved. Adenylyl cyclase (ACase) assays showed a dose-dependent increase in cAMP production as concentrations of β2-AAs increased in both muscle membranes with significantly higher basal cAMP production in RM compared with WM (cAMP production in pmol cAMP · mg protein−1 · 10 min−1: 24.67 ± 3.06 and 9.64 ± 3.45, respectively). The agonist-induced increase in cAMP production was blocked by the β-adrenergic antagonist propranolol, while the ACase activator forskolin increased cAMP production by 7- to 14-fold above basal and ∼3-fold above all β-AAs tested. This study demonstrated the presence of atypical β2-ARs on RM and WM membranes of trout, suggesting that β2-AAs may be a tool to enhance protein accretion through this signaling pathway.

Hormonal regulation of hepatic glycogenolysis in the carp, Cyprinus carpio

1987 ◽  
Vol 252 (4) ◽  
pp. R653-R660 ◽  
Author(s):  
P. A. Janssens ◽  
P. Lowrey
Keyword(s):  
Cyprinus Carpio ◽  
Hormonal Regulation ◽  
Adrenergic Receptors ◽  
Specific Binding ◽  
Arginine Vasotocin ◽  
Dependent Manner ◽  
Beta Adrenergic ◽  
Alpha Adrenergic Receptors ◽  
Adrenergic Antagonist ◽  
Carp Cyprinus Carpio

Carp (Cyprinus carpio) liver maintained normal glycogen content and enzyme complement for several days in organ culture. Epinephrine-stimulated glycogenolysis, phosphorylase activation, and cyclic AMP (cAMP) accumulation in a concentration-dependent manner with EC50s of 100, 100, and 500 nM, respectively. These actions were blocked by the beta-adrenergic antagonist, propranolol, but not by the alpha-adrenergic antagonist phentolamine. Glycogenolysis and tissue cAMP were uninfluenced by 10(-6) M arginine vasotocin, arginine vasopressin, lysine vasotocin, lysine vasopressin, mesotocin, or oxytocin, but were slightly increased by 10(-5) M isotocin and slightly decreased by 10(-6) M angiotensin II. [125I]-iodocyanopindolol (ICP), a beta-adrenergic ligand, bound to isolated carp liver membranes with a KD of 83 pM. Maximum binding of 45 fmol/mg protein was at 600 pM. Propranolol, isoprenaline, epinephrine, phenylephrine, norepinephrine, and phenoxybenzamine displaced ICP with KDs of 100 nM, 2, 20, 20, 60, and 200 microM, respectively. The alpha-adrenergic antagonists, yohimbine and prazosin, showed no specific binding. These data provide evidence that catecholamines act via beta-adrenergic receptors in carp liver and that alpha-adrenergic receptors are not present. Vasoactive peptides play no significant role in regulation of carp liver glycogenolysis.

scholarly journals Phosphatase inhibitor cantharidin blocks adenosine A1 receptor anti-adrenergic effect in rat cardiac myocytes

2000 ◽  
Vol 278 (1) ◽  
pp. H1-H7 ◽  
Author(s):  
Prakash Narayan ◽  
Robert M. Mentzer ◽  
Robert D. Lasley
Keyword(s):  
Ventricular Myocytes ◽  
Ventricular Myocardium ◽  
Receptor Activation ◽  
Camp Content ◽  
Adult Rat ◽  
Phosphatase Inhibitor ◽  
Contractile Parameters ◽  
Dependent Protein ◽  
A1 Receptor ◽  
Rat Cardiac Myocytes

Experiments were performed to examine whether the protein phosphatase inhibitor cantharidin blocks the anti-adrenergic effect of adenosine A1 receptor stimulation. In electrically stimulated adult rat ventricular myocytes loaded with the intracellular calcium concentration ([Ca2+]i) indicator fluo-3, isoproterenol (10 nM) increased systolic [Ca2+]i by 46%, increased twitch amplitude by 56%, and increased total cellular cAMP content by 140%. The adenosine A1 receptor agonist 2-chloro- N 6-cyclopentlyadenosine (CCPA) reduced isoproterenol-stimulated [Ca2+]iand contractility by 87 and 80%, respectively, but reduced cAMP content by only 18%. Cantharidin had no effects on myocyte [Ca2+]i, contractility, or cAMP in the absence or presence of isoproterenol but blocked the effects of CCPA on [Ca2+]i and contractility by ∼44%. Cantharidin had no effect on CCPA attenuation of isoproterenol-induced increases in cAMP. Pretreatment with CCPA also reduced the increase in contractile parameters produced by the direct cAMP-dependent protein kinase A (PKA) activator 8-bromocAMP. These results suggest that activation of protein phosphatases mediate, in part, the anti-adrenergic effect of adenosine A1 receptor activation in ventricular myocardium.

scholarly journals A high-affinity oestrogen-binding protein in cockerel liver cytosol

1979 ◽  
Vol 180 (2) ◽  
pp. 347-353 ◽  
Author(s):  
C B Lazier ◽  
A J Haggarty
Keyword(s):  
Binding Sites ◽  
Proteinase Inhibitors ◽  
Specific Binding ◽  
Binding Protein ◽  
Gel Filtration ◽  
Binding Activity ◽  
Single Class ◽  
High Affinity ◽  
Significant Concentration

In contrast with several earlier reports, cytosol from cockerel liver contains a significant concentration of a protein that binds oestradiol with high affinity. To demonstrate the activity, certain alterations in the conventional method of preparation of cytosol must be made. Homogenization in sucrose-containing buffer at pH 8.4 in the presence of proteinase inhibitors and rapid fractionation of the cytosol with (NH4)2SO4 enables demonstration of a single class of oestradiol-binding sites with a Kd of about 1 nM and specificity only for oestrogens. The concentration is about 300 sites per cell in liver from 2-week-old cockerels. Oestradiol treatment in vivo decreases the number of exchangeable cytosol oestradiol-binding sites by about 80% for 1–4h, after which time it is gradually restored. Gel filtration of the cytosol preparation in the presence of high salt concentrations reveals that most of the oestradiol-binding activity is in high-molecular-weight aggregates, but a mild trypsin treatment generates a specific binding protein with an approximate mol.wt. of 40 000. This protein may be an oestrogen receptor.

scholarly journals Characterization of the high-affinity receptors on Swiss 3T3 cells which mediate the binding, internalization and degradation of the mitogenic peptide bombesin

1988 ◽  
Vol 252 (1) ◽  
pp. 227-235 ◽  
Author(s):  
K D Brown ◽  
M S Laurie ◽  
C J Littlewood ◽  
D M Blakeley ◽  
A N Corps
Keyword(s):  
Enzyme Inhibitors ◽  
Competitive Inhibition ◽  
Specific Binding ◽  
Scatchard Analysis ◽  
3T3 Cells ◽  
Single Class ◽  
High Affinity ◽  
Binding Data ◽  
Molecular Masses ◽  
Swiss 3T3

Bombesin and bombesin-related peptides such as gastrin-releasing peptide (GRP) stimulate DNA synthesis and proliferation of Swiss 3T3 cells in culture. We have used 125I-labelled [Tyr4]bombesin and 125I-labelled GRP to characterize and identify the receptors for these peptides on Swiss 3T3 cells. The binding of 125I-[Tyr4]bombesin, which retained full biological activity, was maximal between 20 and 30 min incubation at 37 degrees C, after which continued incubation led to a decline in cell-associated radioactivity. This decline was markedly slowed by the presence of lysosomal enzyme inhibitors. Specificity of the binding site was indicated by the competitive inhibition of binding by bombesin-related peptides, but not by unrelated peptides and growth factors. Scatchard analysis of binding data indicated a single class of high-affinity receptors. The calculated value for the dissociation constant (Kd) was 2.1 nM and each cell possesses approx. 240,000 receptors. Because [Tyr4]bombesin has no free amino group, 125I-GRP was used in chemical cross-linking studies. When disuccinimidyl suberate was used to covalently couple 125I-GRP to the cells, two major radiolabelled complexes were detected with molecular masses of approx. 80,000-85,000 and 140,000. The binding of 125I-[Tyr4]bombesin to the cells was pH-dependent with maximal binding at pH 6.5-7.5 and effectively no specific binding at pH values below 4.5. At 37 degrees C, cell-associated 125I-[Tyr4]bombesin quickly became resistant to removal by acidic buffers, suggesting its rapid transfer to an intracellular compartment. However, pre-incubation with unlabelled [Tyr4]bombesin did not induce down-regulation of bombesin receptors as measured by the subsequent binding of 125I-[Tyr4]bombesin. In contrast with the Swiss 3T3 cells, specific binding of 125I-[Tyr4]bombesin was not detectable in two cell lines which are biologically unresponsive to bombesin-related peptides.

Thromboxane binding and signal transduction in rat glomerular mesangial cells

1993 ◽  
Vol 264 (2) ◽  
pp. F292-F299 ◽  
Author(s):  
R. F. Spurney ◽  
J. J. Onorato ◽  
F. J. Albers ◽  
T. M. Coffman
Keyword(s):  
Signal Transduction ◽  
Dissociation Constant ◽  
Binding Sites ◽  
Mesangial Cells ◽  
Specific Binding ◽  
Maximal Response ◽  
Scatchard Analysis ◽  
Glomerular Mesangial Cells ◽  
Single Class ◽  
Alpha 7

Thromboxane A2 (TxA2) stimulates contraction of glomerular mesangial cells. However, mesangial cell TxA2 receptors have not been previously characterized. We therefore investigated TxA2 binding and TxA2-associated signal transduction pathways in rat glomerular mesangial cells using the specific thromboxane receptor agonist (1S-[1 alpha,2 beta(5Z),3 alpha-(1E,3S)4 alpha])-7-(3-[3-hydroxy-4-(p- iodophenoxy)-1-butenyl]7-oxabicyclo[2.2.1]hept-2-yl)-5-heptenoic acid (IBOP). In these cells, [125I]BOP binding was saturable, displaceable, and of high affinity. Scatchard analysis revealed a single class of binding sites with a dissociation constant (Kd) of 293 pM and a maximal density of binding sites (Bmax) of 33 fmol/mg protein. Specific binding was inhibited by the thromboxane agonist (15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U-46619) [inhibitor dissociation constant (Ki) = 297 nM] and the TxA2 receptor antagonists SQ 29548 (Ki = 1 nM) and (1R-[1 alpha(Z),2 beta,3 beta,5 alpha])-(+)-7-(5-[(1,1'-biphenyl)- 4-yl-methoxy]-3-hydroxy-2-(1-piperidinyl)cyclopentyl]-4-heptenoic acid (GR 32191) (Ki = 92 nM). Binding was also highly specific for thromboxane because prostaglandin E2 (Ki = 16 microM) and the inactive thromboxane metabolite, TxB2 (Ki = 41 microM), were approximately 1,000-fold less potent at inhibiting binding. IBOP stimulated phosphatidylinositol hydrolysis with an effective concentration of drug that produces 50% of the maximal response of 229 pM, which correlated well with the equilibrium Kd and enhanced phosphorylation of an acidic 80-kDa protein substrate for protein kinase C.(ABSTRACT TRUNCATED AT 250 WORDS)

α1-Adrenergic activation of L-type Ca current in rat ventricular myocytes: perforated patch-clamp recordings

1998 ◽  
Vol 274 (6) ◽  
pp. H2203-H2207 ◽  
Author(s):  
Shi J. Liu ◽  
Richard H. Kennedy
Keyword(s):  
Ventricular Myocytes ◽  
Cell Voltage ◽  
Adrenergic Stimulation ◽  
Whole Cell ◽  
Rat Ventricular Myocytes ◽  
Perforated Patch ◽  
Adult Rat ◽  
Transient Decrease ◽  
Adrenergic Antagonist ◽  
Patch Configuration

α1-Adrenergic stimulation has little effect on L-type Ca2+channel current ( I Ca,L) in adult cardiac myocytes measured using conventional whole cell voltage-clamp techniques. In this study using perforated-patch techniques, we reevaluated the effect of α1-adrenergic stimulation on I Ca,L in adult rat ventricular myocytes. Action potentials and I Ca,L were examined in the presence of 1 μM nadolol, a β-adrenergic antagonist, in myocytes internally dialyzed with Na+- and K+-free solutions (Cs+ and tetraethylammonium as substitutes). Phenylephrine (PE; 30 μM) increased the action potential duration measured at 25 and 70% of repolarization by 104 and 86%, respectively. In the perforated-patch configuration, PE elicited a transient decrease followed by a ∼60% increase in I Ca,L, whereas only the transient decrease in I Ca,L was observed in myocytes when the conventional whole cell configuration was used. The PE-induced increase in I Ca,L was reversibly blocked by 1 μM prazosin, an α1-adrenergic antagonist. These results suggest that α1-adrenergic stimulation enhances cardiac I Ca,L and that obligatory intracellular mediators for this action are lost during whole cell recordings.

scholarly journals Characterization of high-affinity ryanodine-binding sites of rat liver endoplasmic reticulum. Differences between liver and skeletal muscle

1991 ◽  
Vol 276 (1) ◽  
pp. 41-46 ◽  
Author(s):  
V Shoshan-Barmatz ◽  
T A Pressley ◽  
S Higham ◽  
N Kraus-Friedmann
Keyword(s):  
Skeletal Muscle ◽  
Endoplasmic Reticulum ◽  
Binding Sites ◽  
Specific Binding ◽  
Dna Amplification ◽  
Single Class ◽  
High Affinity ◽  
Sodium Dantrolene

In this study, the binding of [3H]ryanodine to liver microsomal subfractions was investigated. The specific binding of [3H]ryanodine, as determined both by vacuum filtration and by ultracentrifugation, is to a single class of high-affinity binding sites with a Kd of 10 +/- 2.5 nM and density of 500 +/- 100 and 1200 +/- 200 fmol/mg of protein by the filtration and centrifugation methods respectively. [3H]Ryanodine binding reached equilibrium in about 1 min and 2 min at 36 degrees C and 24 degrees C respectively, and the half-time of dissociation at 37 degrees C was approx. 15 s. The binding of [3H]ryanodine is Ca(2+)-independent: it is slightly stimulated by NaCl, Mg2+, ATP and InsP3 but strongly inhibited by caffeine, diltiazem and sodium dantrolene. Thus the binding of ryanodine to endoplasmic reticulum membranes shares some of the characteristics of its binding to the sarcoplasmic reticulum but also differs from it in several important properties, such as its Ca(2+)-independence, its rapid association and dissociation, and its inhibition by caffeine. The structural similarities between the skeletal muscle and liver binding sites were further explored by employing in vitro DNA amplification techniques, using the known sequence of the skeletal muscle receptor as reference point. The data obtained with this method indicate that the liver does not process mRNA for the skeletal muscle ryanodine receptor.

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