C-type natriuretic peptide receptor expression in pancreatic alpha cells.

Atrial natriuretic peptide (ANP), brain type natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) comprise a family of natriuretic peptides that mediate their biological effects through three natriuretic peptide receptor subtypes, NPR-A (ANP, BNP), NPR-B (CNP) and NPR-C (ANP, BNP, CNP). Several reports have provided evidence for the expression of ANP and specific binding sites for ANP in the pancreas. The purpose of this study was to identify the ANP receptor subtype and to localize its expression to a specific cell type in the human pancreas. NPR-C immunoreactivity, but neither ANP nor NPR-A, was detected in human islets by immunofluorescent staining. No immunostaining was observed in the exocrine pancreas or ductal structures. Double-staining revealed that NPR-C was expressed mainly in the glucagon-containing alpha cells. NPR-C mRNA and protein were detected in isolated human islets by RT-PCR and Western blot analysis, respectively. NPR-C expression was also detected by immunofluorescent staining in glucagonoma but not in insulinoma. ANP, as well as BNP and CNP, stimulated glucagon secretion from perifused human islets (1,111 +/- 55% vs. basal [7.3 fmol/min]; P < 0.001). This response was mimicked by cANP(4-23), a selective agonist of NPR-C. In conclusion, the NPR-C receptor is expressed in normal and neoplastic human alpha cells. These findings suggest a role for natriuretic peptides in the regulation of glucagon secretion from human alpha cells.

Identification, regulation and anti-proliferative role of the NPR-C receptor in gastric epithelial cells.

Evidence suggests that functional atrial natriuretic peptide (ANP) receptors occur in surface gastric mucosal epithelial cells. To evaluate functional aspects of ANP in a model of these cells we examined the expression of natriuretic peptide receptors (NPR) subtypes A and C in the non-transformed rat gastric mucosal epithelial cell line RGM1. Transcripts for NPR-A and NPR-C were detected in RGM1 cells by RT-PCR. However, only NPR-C protein was detected by Western blot and immunohistochemical analyses. Specific saturable binding of (125)I-ANP to RGM1 cells revealed a single class of high affinity binding sites (K (d) = 208 +/- 71pM, B (max) = 110,000 +/- 14,000 sites/cell, Hill coefficient = 0.97 +/- 0.05). ANP (IC(50) 130 +/- 47pM), BNP (IC(50) 716 +/- 26 pM), CNP (IC(50) 356 +/- 85pM) and C-ANP (IC(50) 134 +/- 13pM), a specific ligand for NPR-C, effectively displaced (125)I-ANP binding. Cross-linking of (125)I-ANP to cells labeled predominantly a protein of 66,000 Da. These data suggest that (125)I-ANP binding was primarily to NPR-C. ANP and C-ANP inhibited forskolin- and prostaglandin E(2) (PGE(2))-stimulated cAMP in a PTx-sensitive fashion. PGE(2), transforming growth factor-+/-1 (TGF-+/-1), forskolin, 8-bromo-cyclic AMP, and phorbol-12-myristate-13-acetate (PMA) caused a dose-dependent decrease in specific (125)I-ANP binding, whereas epidermal growth factor (EGF), 8-bromo-cyclic GMP and 4+/--phorbol didecanoate had no effect. PGE(2), forskolin, TGF-+/-1 and PMA significantly decreased (125)I-ANP B (max) values, NPR-C protein and steady-state NPR-C transcript levels. H89, a protein kinase A inhibitor, blocked the reduction of NPR-C mRNA produced by both forskolin and PGE(2.) GF109203X, a protein kinase C inhibitor, abolished the PMA-induced decrease in NPR-C transcripts but only partially blocked that produced by TGF-+/-1. RGM1 cells exhibited a dose-dependent decrease in both DNA synthesis and cell proliferation when cultured in the presence of ANP or C-ANP. These findings indicate that RGM1 cells express functional NPR-C receptors that can influence RGM1 cell proliferation and are down-regulated by PGE(2) and TGF-+/-1.

Atrial natriuretic hormone prohormone gene expression in cardiac and extra-cardiac tissues of diabetic Goto-Kakizaki rats.

The present investigation was designed to determine if the mechanism for the increased atrial natriuretic peptides within the circulation of diabetic animals involves atrial natriuretic hormone prohormone (proANH) gene expression upregulation. The tissue specificity of this potential upregulation of the proANH gene was investigated in a spontaneous model of type 2 diabetes, i.e. the Goto-Kakizaki (GK) rat with comparison to age-matched non-diabetic Wistar rats from which the GK colony was originally derived. Reverse transcription-polymerase chain reaction revealed that proANH gene expression was increased 3.1-fold in the left heart ventricle, 5-fold in lung, 2-fold in kidney, 3-fold within mucosa and 1.8-fold within muscle of gastric antrum (p < 0.05 for each) of GK rats compared to Wistar rats. There was no significant increase in proANH gene expression in atria and right ventricle of the heart of GK rats compared to Wistars. These results indicate that steady-state ANH prohormone mRNA levels increase within the left ventricle and extracardiac tissues in type 2 diabetic animals. This enhanced gene expression is a functional increase with its expressed proteins (4 peptide hormones; ANPs) increasing 2-6 fold within the circulation of GKs. The greater increase in proANH messenger RNA in the extracardiac tissues compared to the amount of increase within the heart and the greater tissue mass of these combined extra cardiac tissues suggests the majority of the increase in ANPs within the circulation of diabetics is secondary to increased synthesis in extracardiac tissues. This also suggests that there is a systemic regulatory mechanism of proANH gene expression not only within the heart but also within the lung, gastrointestinal tract and kidney. Diabetes is the first disease in which there is more upregulation of ANH prohormone in extracardiac tissues compared to upregulation within the heart itself.