Presence of Growth Hormone Secretagogue Receptor Messenger Ribonucleic Acid in Human Pituitary Tumors and Rat GH 3 Cells 1 (original) (raw)

Abstract

A novel G 11-protein-coupled receptor specific for synthetic GHreleasing peptides (GHRPs) has recently been cloned and sequenced. Two forms exist, types 1a and 1b, the latter of which is biologically inactive. Using RT-PCR, we looked for the presence in tumorous pituitary cells of messenger ribonucleic acid (mRNA) for this novel GH secretagogue receptor (GHS-R). Both subtypes of GHS-R mRNA were detected in all six human pituitary somatotropinomas removed from patients with acromegaly. In culture, four of the tumors exhibited strong responses to GHRP-2 in terms of both phosphatidylinositol (PI) hydrolysis and GH secretion, but two were resistant. There was no apparent difference in the type 1a and type 1b expression pattern, as judged by RT-PCR, between responsive and nonresponsive tumors. Similarly, the rat pituitary tumor cell line, GH 3 , was found to express GHS-R mRNA, although these cells also did not respond to GHRPs. RT-PCR failed to detect GHS-R mRNA in eight functionless human pituitary tumors. In contrast, prolactinomas were found to express the receptor and, in culture, significant stimulation of PRL secretion and PI hydrolysis occurred in two of three tumors tested. These results demonstrate that tumorous somatotrophs express the GHS-R gene and that the occasionally observed nonresponsiveness of somatotropinomas to GHRPs is not due to the absence of the biologically active type 1a receptor. Additionally, human pituitary prolactinomas also express GHS-R and are able to respond to GHRPs in terms of PI hydrolysis and PRL secretion. In contrast, GHS-R gene expression does not appear to be associated with human functionless pituitary tumors.

Figures (7)

Fic. 1. RT-PCR for type 1a GHS-R cDNA using RNA derived from six human pituitary somatotropinomas (lanes 1—6). Single bands were observable for all tumors, running just behind the 244-bp marker. M, Markers; bp, number of base pairs in marker bands (arrowed). Table 1 summarizes the clinical and biochemical details of

Fic. 1. RT-PCR for type 1a GHS-R cDNA using RNA derived from six human pituitary somatotropinomas (lanes 1—6). Single bands were observable for all tumors, running just behind the 244-bp marker. M, Markers; bp, number of base pairs in marker bands (arrowed). Table 1 summarizes the clinical and biochemical details of

Fic. 3. RT-PCR for type 1b GHS-R cDNA using RNA derived from the same six human pituitary somatotropinomas (lanes 1—6) as those used in Fig. 1. Single bands were observable for all tumors, running just in front of the 244-bp marker. M, Markers; bp, number of base pairs in marker bands (arrowed). Fic. 2. Partial sequence of RT-PCR DNA bands depicted in Fig. 1. The sequence reads 3'-CCGTTTGTGGTGATGTCGGACGTAAA-5’, is identical to type la GHS-R cDNA (mRNA), and is without the genes’s intervening intronic region situated, as arrowed, between the two underlined G residues (GenBank accession no. U60179) (12).

Fic. 3. RT-PCR for type 1b GHS-R cDNA using RNA derived from the same six human pituitary somatotropinomas (lanes 1—6) as those used in Fig. 1. Single bands were observable for all tumors, running just in front of the 244-bp marker. M, Markers; bp, number of base pairs in marker bands (arrowed). Fic. 2. Partial sequence of RT-PCR DNA bands depicted in Fig. 1. The sequence reads 3'-CCGTTTGTGGTGATGTCGGACGTAAA-5’, is identical to type la GHS-R cDNA (mRNA), and is without the genes’s intervening intronic region situated, as arrowed, between the two underlined G residues (GenBank accession no. U60179) (12).

Fic. 4. Partial sequence of RT-PCR DNA bands depicted in Fig. 3. The sequence reads 3’-GACTCTGGGTGGGTC-5’ and is identical to a specific 3'-terminal region of the type 1b GHS-R cDNA (mRNA; GenBank accession no. U60181) (12).

Fic. 4. Partial sequence of RT-PCR DNA bands depicted in Fig. 3. The sequence reads 3’-GACTCTGGGTGGGTC-5’ and is identical to a specific 3'-terminal region of the type 1b GHS-R cDNA (mRNA; GenBank accession no. U60181) (12).

TABLE 1. Characteristics of six human pituitary somatotrophinomas and in vitro effect of GHRP-2 (100 nmol/L) on PI hydrolysi

TABLE 1. Characteristics of six human pituitary somatotrophinomas and in vitro effect of GHRP-2 (100 nmol/L) on PI hydrolysi

Fic. 5. RT-PCR for the GHS-R cDNA using RNA derived from rat GH, cells (lane 1) and a human pituitary prolactinoma (lane 2). M, Markers; bp, number of base pairs in marker bands (arrowed).

Fic. 5. RT-PCR for the GHS-R cDNA using RNA derived from rat GH, cells (lane 1) and a human pituitary prolactinoma (lane 2). M, Markers; bp, number of base pairs in marker bands (arrowed).

Fic. 6. Stimulatory effect of GHRP-2 (100 nmol/L) on PI hydrolysis (left panels) and PRL secretion (right panels) by cell cultures of two human pituitary prolactinomas (tumors A and B). Cells were incu- bated in the absence (control) or presence of GHRP-2 for 2 h. *, P< 0.05; **, P < 0.01; ***, P < 0.001 (vs. control).

Fic. 6. Stimulatory effect of GHRP-2 (100 nmol/L) on PI hydrolysis (left panels) and PRL secretion (right panels) by cell cultures of two human pituitary prolactinomas (tumors A and B). Cells were incu- bated in the absence (control) or presence of GHRP-2 for 2 h. *, P< 0.05; **, P < 0.01; ***, P < 0.001 (vs. control).

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