Nephron specificity of dopamine receptor-adenylyl cyclase defect in spontaneous hypertension

1993 ◽  
Vol 264 (2) ◽  
pp. F274-F279 ◽  
Author(s):  
K. Ohbu ◽  
R. A. Felder
Keyword(s):  
Adenylyl Cyclase ◽  
Dopamine Receptor ◽  
Radioligand Binding ◽  
Spontaneously Hypertensive Rat ◽  
Collecting Duct ◽  
Spontaneous Hypertension ◽  
Sodium Reabsorption ◽  
Binding Studies ◽  
Cytoplasmic Loop ◽  
The Third

Since DA1 receptors regulate renal tubular sodium transport, it is possible that the reported defect in the coupling between the DA1 dopamine receptor and adenylyl cyclase (AC) in the proximal tubule (PT) is a mechanism for the increased sodium reabsorption in animal models of spontaneous hypertension. Because the distal nephron may participate in the increased sodium retention in the spontaneously hypertensive rat (SHR), we determined whether the defective DA1 receptor-AC coupling described in PT of SHR is also present in the cortical collecting duct (CCD). Radioligand binding studies with the DA1 antagonist 125I-Sch 23982 revealed similar dissociation constants and maximum receptor densities in the CCD from Wistar-Kyoto rats (WKY) and SHR. Fenoldopam, a DA1-selective agonist, stimulated AC activity to a similar extent in CCD from both rat groups. Therefore the defective DA1 receptor-AC coupling in SHR has nephron segment specificity, since it is present in PT but not in CCD. One of the AC-linked dopamine receptors is an intronless D1A cloned from brain, which is also present in PT. Because the coupling defect in the PT may reside in the third cytoplasmic loop (involved in G protein coupling), we compared the sequence of this segment of the cloned D1A receptor using genomic DNA. Because no differences were noted between WKY and SHR, the coupling defect in the PT is not due to a mutation at the third cytoplasmic loop of the D1A receptor.

Dopamine recruits D1A receptors to Na-K-ATPase-rich caveolar plasma membranes in rat renal proximal tubules

2004 ◽  
Vol 287 (5) ◽  
pp. F921-F931 ◽  
Author(s):  
Meghna Trivedi ◽  
Vihang A. Narkar ◽  
Tahir Hussain ◽  
Mustafa F. Lokhandwala
Keyword(s):  
Plasma Membrane ◽  
Adenylyl Cyclase ◽  
Plasma Membranes ◽  
Radioligand Binding ◽  
Sch 23390 ◽  
Sucrose Density Gradient ◽  
Proximal Tubules ◽  
Sodium Reabsorption ◽  
Sprague Dawley ◽  
Renal Proximal Tubules

Activation of dopamine D1A receptors in renal proximal tubules causes inhibition of sodium transporters (Na-K-ATPase and Na/H exchanger), leading to a decrease in sodium reabsorption. In addition to being localized on the plasma membrane, D1A receptors are mainly present in intracellular compartments under basal conditions. We observed, using [3H]SCH-23390 binding and immunoblotting, that dopamine recruits D1A receptors to the plasma membrane in rat renal proximal tubules. Furthermore, radioligand binding and/or immunoblotting experiments using pharmacological modulators showed that dopamine-induced D1A receptor recruitment requires activation of cell surface D1-like receptors, activation of adenylyl cyclase, and intact endocytic vesicles with internal acidic pH. A key finding of this study was that these recruited D1A receptors were functional because they potentiated dopamine-induced [35S]GTPγS binding, cAMP accumulation, and Na-K-ATPase inhibition. Interestingly, dopamine increased immunoreactivity of D1A receptors specifically in caveolin-rich plasma membranes isolated by a sucrose density gradient. In support of this observation, coimmunoprecipitation studies showed that D1A receptors interacted with caveolin-2 in an agonist-dependent fashion. The caveolin-rich plasma membranes had a high content of the α1-subunit of Na-K-ATPase, which is a downstream target of D1A receptor signaling in proximal tubules. These results show that dopamine, via the D1-like receptor-adenylyl cyclase pathway, recruits D1A receptors to the plasma membrane. These newly recruited receptors couple to G proteins, increase cAMP, and participate in dopamine-mediated inhibition of Na-K-ATPase in proximal tubules. Moreover, dopamine-induced recruitment of D1A receptors to the caveolin-rich plasma membranes brings them in close proximity to targets such as Na-K-ATPase in proximal tubules of Sprague-Dawley rats.

Characterization of prazosin-sensitive alpha 2 B-adrenoceptors expressed by cultured rat IMCD cells

1991 ◽  
Vol 261 (5) ◽  
pp. F760-F766 ◽  
Author(s):  
G. Yasuda ◽  
L. Sun ◽  
S. Umemura ◽  
W. A. Pettinger ◽  
W. B. Jeffries
Keyword(s):  
Dna Sequences ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Radioligand Binding ◽  
Collecting Duct ◽  
Maximal Response ◽  
Camp Accumulation ◽  
Binding Studies ◽  
Uk 14,304

alpha 2-Adrenoceptor subtype expression was investigated in cultured rat inner medullary collecting duct (IMCD) cells using radioligand binding studies, Northern blot analysis, and adenosine 3',5'-cyclic monophosphate (cAMP) assays. [3H]rauwolscine bound to a single class of alpha 2-adrenoceptors with high affinity [Kd = 1.7 +/- 0.3 nM, maximum binding (Bmax) = 45.2 +/- 10.8 fmol/mg protein]. alpha 2-Adrenoceptor ligands inhibited [3H]rauwolscine binding with a rank order of potency characteristic of interaction with the alpha 2B-adrenoceptor [inhibitory constant (Ki) values (in nM) rauwolscine (1.95) greater than ARC-239 (8.52) greater than prazosin (237) greater than oxymetazoline (30,000)]. Northern blot analysis was performed using poly(A)+ RNA isolated from 90% confluent rat IMCD cells and probes derived from alpha 2-adrenoceptor DNA sequences from the rat nonglycosylated alpha 2B-adrenoceptor and the human alpha 2A-adrenoceptor. The alpha 2B probe hybridized to a 4.2-kb band under high stringency conditions, but the alpha 2A-adrenoceptor probe did not hybridize to this band. In functional studies, the full alpha 2-adrenoceptor agonists epinephrine and UK-14,304 potently inhibited vasopressin-stimulated cAMP accumulation by 50 to 70% [half-maximal response (EC50) (in nM) epinephrine = 11.2, UK-14,304 = 6.4]. Guanabenz and clonidine were partial agonists, inhibiting cAMP accumulation by 30 to 40% and were less potent than the full agonists [EC50 (in nM) 56.0 guanabenz and 94.5 clonidine]. Epinephrine-induced inhibition of cAMP accumulation was blocked by rauwolscine, prazosin, and ARC-239 but not by the alpha 1-adrenoceptor antagonist corynanthine. We conclude that rat IMCD cells in primary culture express functional alpha 2-adrenoceptors of the alpha 2B-subtype.

Absence of DA1/DA2 dopamine receptor interactions in proximal tubules of spontaneously hypertensive rats

1996 ◽  
Vol 270 (1) ◽  
pp. F98-F105 ◽  
Author(s):  
P. Vachvanichsanong ◽  
S. Sela ◽  
A. Sidhu
Keyword(s):  
Dopamine Receptor ◽  
Spontaneously Hypertensive Rats ◽  
Spontaneously Hypertensive Rat ◽  
Proximal Tubules ◽  
Hypertensive Rats ◽  
Binding Studies ◽  
Spontaneously Hypertensive ◽  
Receptor Interactions ◽  
Wistar Kyoto ◽  
The Impact

The impact of defective DA1 dopamine receptors in proximal tubules (PT) of the spontaneously hypertensive rat (SHR) on DA1/DA2 receptor interactions was assessed with the DA1-selective photoaffinity ligand, (+/-)-7-[125I]iodo-8-hydroxy-3-methyl-1-(4-azidophenyl)- 2,3,4,5-tetrahydro-1H-3-benzazepine ([125I]MAB). In PT membranes from both normotensive (Wistar-Kyoto, WKY) and spontaneously hypertensive rats (SHR), [125I]MAB was specifically incorporated into a polypeptide with an M(r) of 74,000 Da, corresponding to the DA1 receptor. The labeling of this band by [125I]MAB in both SHR and WKY was not prevented by SKF-82526, a potent DA1-selective agonist. However, in the presence of the DA2 antagonist, (-)-sulpiride, but not DA2 agonist, LY-171555, SKF-82526 abolished photoincorporation of [125I]MAB into the 74,000-Da band in WKY. In SHR, (-)-sulpiride failed to enhance the ability of SKF-82526 to compete with [125I]MAB for binding to the 74,000-Da subunit. In competition binding studies with SKF-82526, (-)-sulpiride induced the formation of agonist high-affinity binding sites in WKY but not in SHR. These data suggest that in membranes of SHR, but not WKY, DA1/DA2 dopamine receptor interactions are lacking.

scholarly journals A novel short isoform of the D3 dopamine receptor generated by alternative splicing in the third cytoplasmic loop.

1993 ◽  
Vol 268 (8) ◽  
pp. 5872-5878 ◽  
Author(s):  
C.S. Fishburn ◽  
D. Belleli ◽  
C. David ◽  
S. Carmon ◽  
S. Fuchs
Keyword(s):  
Alternative Splicing ◽  
Dopamine Receptor ◽  
Cytoplasmic Loop ◽  
The Third

Bronchospasmolytic and Adenosine Binding Activity of 8- (Proline / Pyrazole)-Substituted Xanthine Derivatives

2020 ◽  
Vol 17 ◽  
Author(s):  
Sneha Singh ◽  
Madhwi Ojha ◽  
Divya Yadav ◽  
Sonja Kachler ◽  
Karl-Norbert Klotz ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Radioligand Binding ◽  
Binding Activity ◽  
Receptor Subtypes ◽  
Significant Protection ◽  
Binding Studies ◽  
Standard Drug ◽  
Xanthine Derivatives ◽  
Radioligand Binding Studies ◽  
Micromolar Range

Background: ABSTRACT: Background: 8-Phenyltheophylline derivatives exhibit prophylactic effects at a specific dose but do not produce the cardiovascular or emetic side effects associated with xanthines, thereby exhibiting unique characteristics of potential therapeutic importance. Methods: Novel series of 8-(proline/pyrazole)-substituted xanthine analogs has been synthesized. The affinity and selectivity of compounds to adenosine receptors have been assessed by radioligand binding studies. The synthesized compounds also showed good bronchospasmolytic properties (increased onset of bronchospasm; decreased duration of jerks) with 100% survival of animals in comparison to the standard drug. Besides, compound 8f & 9f showed good binding affinity in comparison to other synthesized compounds in the micromolar range. Results: The maximum binding affinity of these compounds was observed for A2B receptors, which is ~ 7 or 10 times higher as compared to A1, A2A and A3 receptors. The newly synthesized derivatives 8f, 9a-f, 17g-m, and 18g-m displayed significant protection against histamine aerosol induced bronchospasm in guinea pigs. Conclusion: Newly synthesized proline/pyrazole based xanthines compounds showed a satisfactory binding affinity for adenosine receptor subtypes. Replacement or variation of substituted proline ring with substituted pyrazole scaffold at 8thposition of xanthine moiety resulted in the reduction of adenosine binding affinity and bronchospasmolytic effects.

Effects of forskolin on inotropic performance and phospholamban phosphorylation in exercise-trained hypertensive myocardium

2007 ◽  
Vol 102 (2) ◽  
pp. 628-633 ◽  
Author(s):  
Stephen C. Kolwicz ◽  
Hajime Kubo ◽  
Scott M. MacDonnell ◽  
Steven R. Houser ◽  
Joseph R. Libonati
Keyword(s):  
Heart Rate ◽  
Adenylyl Cyclase ◽  
Spontaneously Hypertensive Rat ◽  
Weight Ratio ◽  
Left Ventricular ◽  
Treadmill Running ◽  
Chronic Hypertension ◽  
Wistar Kyoto ◽  
Developed Pressure ◽  
Phospholamban Phosphorylation

β-Adrenergic receptor (β-AR) responsiveness is downregulated in left ventricular (LV) hypertrophy induced by chronic hypertension. While exercise training in hypertension enhances β-AR responsiveness, the role of adenylyl cyclase remains unclear. The purpose of the present study was to test whether treadmill running in the spontaneously hypertensive rat (SHR) model improves LV responsiveness to forskolin (FOR) or the combination of FOR + isoproterenol (FOR+ISO). Female SHR (16-wk) were randomly placed into sedentary (SHR-SED; n = 7) or treadmill-trained (SHR-TRD; n = 8) groups. Wistar-Kyoto (WKY; n = 7) animals acted as normotensive controls. Langendorff, isovolumic LV performance was established at baseline and during incremental FOR infusion (1 and 5 μmol/l) and FOR+ISO (5 μmol/l + 1×10−8 mol/l). Heart rate, systolic blood pressure, and heart-to-body weight ratio were lower in WKY relative to both SHR groups ( P < 0.05). LV performance and heart rate significantly increased in all groups to a similar extent with incremental FOR infusion. However, in the presence of 5 μmol/l FOR, ISO increased LV developed pressure, positive change in LV pressure, and negative change in LV pressure to a greater extent in SHR-TRD relative to SHR-SED ( P < 0.05). Phospholamban phosphorylation at the Thr17 was greater in SHR-TRD relative to SHR-SED and WKY ( P < 0.05). Absolute LV developed pressure was moderately correlated with phospholamban phosphorylation at both the Ser16 ( r = 0.64; P < 0.05) and Thr17 ( r = 0.52; P < 0.05). Our data suggest that the adenylyl cyclase step in the β-AR cascade is not downregulated in the early course of hypertension and that the enhanced β-AR responsiveness with training is likely mediated at levels other than adenylyl cyclase. Our data also suggest that β-AR inotropic responsiveness in the presence of direct adenylyl cyclase agonism is improved in trained compared with sedentary SHR hearts.

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