Molecular and Biochemical Characterization of a CNP-Sensitive Guanylyl Cyclase in Bovine Tracheal Smooth Muscle

• Published in: American journal of respiratory cell and molecular biology Vol. 25; no. 1; pp. 98 - 103
• Main Authors: Borges, Adolfo, Sanchez de Villarroel, Sinai, Winand, Nena J, Lippo de Becemberg, Itala, Alfonzo, Marcelo J, Gonzalez de Alfonzo, Ramona
• Format: Journal Article
• Language: English
• Published: United States Am Thoracic Soc 01.07.2001; American Thoracic Society
• Subjects: Adenosine Triphosphate - pharmacology; Amino Acid Sequence; Animals; Base Sequence; Cattle; Cloning, Molecular; Cyclic GMP - physiology; DNA Primers; Enzyme Activation; Gastrointestinal Hormones; Guanylate Cyclase - chemistry; Guanylate Cyclase - genetics; Guanylate Cyclase - metabolism; Molecular Sequence Data; Muscle, Smooth - drug effects; Muscle, Smooth - enzymology; Natriuretic Peptide, C-Type - pharmacology; Natriuretic Peptides; Peptides - pharmacology; Reverse Transcriptase Polymerase Chain Reaction; Sequence Homology, Amino Acid; Sodium Chloride - pharmacology; Subcellular Fractions - enzymology; Trachea - drug effects; Trachea - enzymology
• ISSN: 1044-1549; 1535-4989
• DOI: 10.1165/ajrcmb.25.1.4395

Muscarinic activation of bovine tracheal smooth muscle (BTSM) is involved in cyclic guanosine monophosphate (cGMP) production mediated through soluble (sGC) and membrane-bound (mGC) guanylyl cyclases. A muscarinic- and NaCl-sensitive mGC exists in BTSM regulated by muscarinic receptors coupled to G proteins. To identify the mGCs expressed in BTSM, reverse transcriptase/polymerase chain reaction (RT-PCR) from total RNA was performed using degenerate oligonucleotides for amplification of a region conserved among GC catalytic domains. Cloning of amplification products revealed that 76% of all BTSM GC transcripts corresponded to the sGC beta1 subunit and 24% to the B-type (C-type NP 1-22 [CNP]-sensitive) GC receptor. cGMP production by BTSM membrane and soluble fractions confirmed that sGC activity is 3-fold with respect to mGC activity. RT-PCR using specific oligonucleotides revealed that A (atrial NP-sensitive) and C (guanylin-sensitive) mGC subtypes are also expressed in BTSM. Stimulation of basal plasma membrane GC activity by CNP was higher than that by ANP, whereas guanylin showed no effect, indicating that CNP-sensitive guanylyl cyclase (GC-B) is the predominant functional BTSM mGC subtype. Strong adenosine triphosphate inhibition of CNP-stimulated mGC activity supports the finding that the tracheal mGC isoform belongs to the natriuretic peptide-sensitive mGCs. Additionally, CNP was able to reverse the chloride inhibition of BTSM mGC activity, suggesting that this is a novel G protein-coupled GC-B receptor.