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Pharmacological characterization of adenylyl cyclase isoforms in rabbit kidney membranes

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Abstract

Polycystic kidney disease (PKD) is the most common life-threatening genetic disorder with bilateral cysts caused by increased level of cyclic adenosine 3′,5′-monophosphate (cAMP). Since adenylyl cyclases (ACs) catalyze cAMP formation, pharmacological characterization of renal AC isoforms is essential. Therefore, we analyzed differences in activation, inhibition, and regulation of AC isoforms in rabbit cortex and medulla membranes. Glucagon, [8-arginine]vasopressin (AVP) and catecholamines significantly activated cortical AC. However, in medulla only glucagon and AVP activated AC. Under Mg2+ conditions the profile of cortical membrane AC enzyme kinetics and the inhibitory profile of 2′(3′)-O-(N-methylanthraniloyl) (MANT) nucleotides resembled recombinant AC5. In contrast, the K i values of MANT nucleotides for medullary membrane AC and its kinetic properties were similar to those of recombinant AC1. Reverse-transcriptase PCR confirmed the presence of AC1 and AC5 in medulla and cortex, respectively. Cortical AC was sensitive to inhibition by Ca2+, corroborating the importance of AC5. However, Ca2+/CaM dependency specific for AC1 was not found in medulla. In conclusion, according to expression, kinetics and inhibition by MANT nucleotides both parts of the kidney differ in their AC isoforms. Whereas Ca2+-inhibitable AC5 was confirmed in renal cortex, the initially assumed AC1 activation in medulla could not be confirmed, pointing to the involvement of another AC isoform with some similarity to AC1. Since PKD is characterized by predominant involvement of the collecting duct and the distal nephrons located in renal cortex, AC5 may be the major AC isoform in this part of the kidney where cAMP increases cyst growth. Thus, potent and selective AC5 inhibitors could constitute a novel approach to treat PKD.

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Abbreviations

AC:

Adenylyl cyclase

cAMP:

Cyclic adenosine 3′,5′-monophosphate

GPCR:

G-protein-coupled receptor

IBMX:

3-isobutyl-1-methylxanthine

MANT-NTP:

2′(3′)-O-(N-methylanthraniloyl)-nucleoside 5′-triphosphate

MANT-NTPγS:

2′(3′)-O-(N-methylanthraniloyl)-nucleoside 5′-[γ-thio]triphosphate

CaM:

Calmodulin

FS:

Forskolin

AVP:

[8-arginine]vasopressin

GTPγS:

Guanosine 5′-[γ-thio]triphosphate

PKD:

Polycystic kidney disease

RT-PCR:

Reverse-transcription polymerase chain reaction

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Acknowledgments

The authors thank PD. Dr. K. Höcherl (Institute of Physiology, University of Regensburg, Regensburg, Germany) for his help with the AC primer selection. Thanks are also due to Mrs. Susanne Brüggemann and Mrs. Astrid Seefeld for expert technical assistance. Thanks are also due to the reviewers of this paper for their helpful critique. This work was supported by Deutsche Forschungsgemeinschaft [Grant Se 529/5-2] to R. S.

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  1. Department of Pharmacology and Toxicology, University of Regensburg, Regensburg, Germany

    Miriam Erdorf

  2. Institute of Pharmacology, Medical School of Hannover, Carl-Neuberg-Str. 1, 30625, Hannover, Germany

    Roland Seifert

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  1. Miriam Erdorf
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  2. Roland Seifert
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Correspondence to Roland Seifert.

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Erdorf, M., Seifert, R. Pharmacological characterization of adenylyl cyclase isoforms in rabbit kidney membranes. Naunyn-Schmied Arch Pharmacol 383, 357–372 (2011). https://doi.org/10.1007/s00210-011-0600-7

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