Molecular and biochemical characterization of a CNP-sensitive guanylyl cyclase in bovine tracheal smooth muscle.
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.
Borges
A
A
Sección de Biomembranas, Instituto de Medicina Experimental, Facultad de Medicina, Universidad Central de Venezuela, Sabana Grande, Caracas 1051, Venezuela.
de Villarroel
S S
SS
Winand
N J
NJ
de Bécemberg
I L
IL
Alfonzo
M J
MJ
de Alfonzo
R G
RG
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D006152:Cyclic GMP / Q000502:physiology; D017931:DNA Primers; D004789:Enzyme Activation; D005768:Gastrointestinal Hormones; D006162:Guanylate Cyclase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000187:drug effects* / Q000201:enzymology; D020098:Natriuretic Peptide, C-Type / Q000494:pharmacology*; D045265:Natriuretic Peptides; D010455:Peptides / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D012965:Sodium Chloride / Q000494:pharmacology; D013347:Subcellular Fractions / Q000201:enzymology; D014132:Trachea / Q000187:drug effects* / Q000201:enzymology
D017931:DNA Primers; D005768:Gastrointestinal Hormones; D045265:Natriuretic Peptides; D010455:Peptides; D020098:Natriuretic Peptide, C-Type; C072685:guanylin; D012965:Sodium Chloride; D000255:Adenosine Triphosphate; D006162:Guanylate Cyclase; D006152:Cyclic GMP