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Am. J. Respir. Cell Mol. Biol., Volume 25, Number 1, July, 2001 98-103

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

Adolfo Borges, Sinaí Sánchez de Villarroel, Nena J. Winand, Itala Lippo de Bécemberg, Marcelo J. Alfonzo, and Ramona González de Alfonzo

Sección de Biomembranas, Instituto de Medicina Experimental, Facultad de Medicina, Universidad Central de Venezuela, Caracas, Venezuela; and Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, New York

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 beta 1 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.

Abbreviations: atrial NP 1-28, ANP; adenosine triphosphate, ATP; brain NP 1-26, BNP; bovine serum albumin, BSA; bovine tracheal smooth muscle, BTSM; complementary DNA, cDNA; cyclic guanosine monophosphate, cGMP; C-type NP 1-22, CNP; C-type NP 1-53, CNP-53; dithiothreitol, DTT; guanylyl cyclase, GC; ANP-sensitive guanylyl cyclase, GC-A; CNP-sensitive guanylyl cyclase, GC-B; Escherichia coli enterotoxin/guanylin-sensitive guanylyl cyclase, GC-C; guanosine triphosphate, GTP; membrane-bound guanylyl cyclase, mGC; natriuretic peptide, NP; 1H- [1,2,4]oxadiazolo[4,3-alpha ]quinoxalin-1-one, ODQ; polymerase chain reaction, PCR; phenylmethylsulfonyl fluoride, PMSF; reverse transcriptase/ polymerase chain reaction, RT-PCR.




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