Glucocorticoid receptor activation in isolated perfused rat hearts

1989 ◽  
Vol 256 (2) ◽  
pp. C219-C225 ◽  
Author(s):  
S. M. Czerwinski ◽  
E. E. McKee ◽  
R. C. Hickson

The formation of unactivated and activated glucocorticoid receptor complexes was studied in intact, isolated, perfused rat hearts in the presence of [3H]triamcinolone acetonide. Receptor activation, as quantified by the DNA-cellulose-binding assay, began to increase within 30 s of perfusion and reached a final steady-state level (t 1/2 = 4.6 min) with 46% of the steroid-receptor complexes bound to DNA-cellulose. With the use of a linear potassium phosphate (KP) gradient (5-400 mM), unactivated receptors eluted from DEAE-cellulose anion exchange columns at approximately 250 mM KP. Two activated receptor forms appeared, which eluted either in the wash fraction (binder IB) or between 50 and 100 mM KP (binder II) and occurred with half times of 1.3 and 2.7 min, respectively. Postperfusion cytosol preparation did not markedly influence the results as receptor binding was reduced by 10% or less when a 100-fold excess of unlabeled triamcinolone acetonide was included in the homogenizing buffer. We conclude from these results that glucocorticoids are able to exert a direct effect on the heart through binding to their own receptor in the absence of endogenous hormones. The time dependency of receptor activation supports a physiological role for this process. However, activation rates, determined from conformational changes associated with altered DEAE-cellulose elution profiles and appearance of activated receptor forms, occur earlier and may not be coordinated with the rate of activation as quantified by DNA-cellulose binding.

Author(s):  
Joseph P. Zbilut ◽  
Gottfried Mayer-Kress ◽  
Paul A. Sobotka ◽  
Michael O’Toole ◽  
John X. Thomas

1999 ◽  
Vol 276 (2) ◽  
pp. H341-H349 ◽  
Author(s):  
Gavin R. Norton ◽  
Angela J. Woodiwiss ◽  
Robert J. McGinn ◽  
Mojca Lorbar ◽  
Eugene S. Chung ◽  
...  

Presently, the physiological significance of myocardial adenosine A2a receptor stimulation is unclear. In this study, the influence of adenosine A2a receptor activation on A1 receptor-mediated antiadrenergic actions was studied using constant-flow perfused rat hearts and isolated rat ventricular myocytes. In isolated perfused hearts, the selective A2a receptor antagonists 8-(3-chlorostyryl)caffeine (CSC) and 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM-241385) potentiated adenosine-mediated decreases in isoproterenol (Iso; 10−8 M)-elicited contractile responses (+dP/d t max) in a dose-dependent manner. The effect of ZM-241385 on adenosine-induced antiadrenergic actions was abolished by the selective A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (10−7 M), but not the selective A3 receptor antagonist 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (MRS-1191, 10−7 M). The A2a receptor agonist carboxyethylphenethyl-aminoethyl-carboxyamido-adenosine (CGS-21680) at 10−5 M attenuated the antiadrenergic effect of the selective A1 receptor agonist 2-chloro- N 6-cyclopentyladenosine (CCPA), whereas CSC did not influence the antiadrenergic action of this agonist. In isolated ventricular myocytes, CSC potentiated the inhibitory action of adenosine on Iso (2 × 10−7 M)-elicited increases in intracellular Ca2+concentration ([Ca2+]i) transients but did not influence Iso-induced changes in [Ca2+]itransients in the absence of exogenous adenosine. These results indicate that adenosine A2areceptor antagonists enhance A1-receptor-induced antiadrenergic responses and that A2a receptor agonists attenuate (albeit to a modest degree) the antiadrenergic actions of A1 receptor activation. In conclusion, the data in this study support the notion that an important physiological role of A2a receptors in the normal mammalian myocardium is to reduce A1 receptor-mediated antiadrenergic actions.


1984 ◽  
Vol 247 (4) ◽  
pp. H508-H516
Author(s):  
R. A. Kauppinen ◽  
I. E. Hassinen

Optical methods were tested for measuring the membrane potential changes of mitochondria in isolated perfused rat hearts. Safranin was found to be rapidly taken up by the Langendorff-perfused heart, and after loading with the dye there was practically no washout of the stain during perfusion with Krebs-Ringer bicarbonate solution. Staining with safranin induced the appearance of an intense absorption band in the reflectance spectrum of the heart, but the absorbance spectrum changes were not useful for monitoring the mitochondrial membrane potential changes because of interference by endogenous hemoproteins. The fluorescence intensity, however, responded in a manner which indicated that its changes originated from dye attached to the mitochondria. A decrease of the fluorescence was found on energizing the mitochondria by decreasing the cellular energy consumption by arrest induced by 18 mM K+ or by decreasing the beating rate of an electrically paced heart from 5 Hz to the endogenous ventricular frequency of 1.5 Hz. In hearts arrested by Ca2+ depletion, 18 mM K+ did not affect the safranin fluorescence. This was taken to indicate that under these conditions the safranin fluorescence was not sensitive to the plasma membrane potential. The uncoupler carbonyl cyanide m-chlorophenylhydrazone induced an intense enhancement of safranin fluorescence in the perfused heart, demonstrating that the probe is sensitive to mitochondrial membrane potential.(ABSTRACT TRUNCATED AT 250 WORDS)


2016 ◽  
Vol 46 ◽  
pp. 166-173 ◽  
Author(s):  
Erkan KILINÇ ◽  
Ziya KAYGISIZ ◽  
Bedri Selim BENEK ◽  
Kenan GÜMÜŞTEKİN

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