Biological Function Of Novel HGHRH Dimers

Objective:Human growth hormone-releasing hormone(h GHRH) stimulates the synthesis and release of human growth hormone(h GH) in the pituitary. The maturation of prepro-h GHRH(1-107/8) precursor is involved in a series of pro-protein processing. The final mature forms of h GHRH include h GHRH(1–44)NH2, h GHRH(1–37)OH, and h GHRH(1–40)OH, but the short half-life and sub-optimal potency is a major limitation. The previous work reported that the N-terminal 1Tyr→Pro-1Pro-/1Pro- replacement and C-terminal GGC extension of h GHRH(1–44)OH effectively regulated its potency in releasing pituitary hormones. In this paper, the relationship of the structure and biological evaluation of the four novel h GHRH dimer agonists were reported to lay the foundation for further studies in animal experiment.Methods: 1 Chemical synthesis and identification of the monomeric GHRH analogues. The structure-related GHRH monomers with both a N-terminal replacement and a C-terminal extension produced in solid-phase polypeptide synthesizer. The molecular weights and purities were determined in ESI mass spectrum and C18 RP-HPLC respectively. 2 Formation and identification of GHRH dimers GHRH dimmers were formed when the monomeric GHRH peptides were incubated in ammonium hydroxide water solution(p H 11.5) for 60 h at 37℃. The final products were obtained through lyophilizing the peptide-ammonium hydroxide solutions. The purities of the dimeric peptides were determined in a peptide-PAGE. 3 In vitro activity assay of the GHRH dimers in stimulating the secretion of pituitary hormones. Sprague-Dawley female rats were acclimated to a light-dark cycle of 12:12 h in the temperature of 26±1℃.The rats were sacrificed by cervical vertebradislocation and their pituitaries were harvested within 30 min. After rinsing with sterilized lactated Ringer’s buffer, immediately the pituitaries were incubated in 1 ml of the lactated Ringer’s buffer at 37℃ in 1×10 cm glass tube. These tubes were incubated for a total of 5 h. Buffer was removed every hour to generate 5 samples(P1, P2, I3, I4, I5) and then 1 ml of fresh buffer was added. The subsequent 3 h incubation(I3,I4,I5) included a continuous 1.927 μM h GHRH peptide treatment. The collected samples were analyzed using ELISA kits for rat GH ACTH, LH, and PRL. 4 Rat pituitary GH-releasing inhibition analysis. According to the pituitary GH-releasing activity experiment(P1, P2, I3, I4, I5). The GHRH dimers containing 1.927 μM in I3-I5 were added 0.482 or 1.927 μM of growth hormone inhibition hormone(GHIH) respectively. The P2 without peptide and S peptide were used as blank and a standard. Rat pituitary GH level was measured with rat GH ELISA kit. 5 In vitro receptor binding studies of the GHRH dimers. Thirty pituitaries, harvested from female S-D rats were homogenized and re-suspended. The protein concentration of the solution was determined using Bradford method. In the binding assay, each of the plate wells was added 100 μl of pituitary homogenate, 5 different concentrations of FITC-labeled h GHRH peptide and the corresponding non-labeled h GHRH peptide with total volume of 300 μl,respectively. Each treatment had 2 replicates and read fluorescence values at wavelength of 490/525 nm using fluorescent detector. 6 Fluorescent staining analysis of rat pituitary tissue. Formalin-fixed S-D rat pituitary tissue sections were stained as follows. The slides were soaked in a HEPES buffer 3 times for every 5 minutes. 200 μl of HEPES buffer containing 76±1 fluorescent intensity of FITC-labeled h GHRH peptide was dropped on each tissue section to incubate for 1h at 37℃. After the slides were washed twice in the HEPES buffer, DAPI water solution(0.01 g/ml) was dropped in the tissue section for 15 min at 37℃. After washing with HEPES buffer, the tissue section was mounted onto the slide with a fluorescent mounting medium. The staining procedure was performed in a dark environment.Results: 1 Purity and molecular mass of the synthetic h GHRH analogs. Using RT-HPLC and ESI mass spectroscopy methods, the purities and the molecular masses of the synthetic monomeric h GHRH peptides were analyzed. The purities were higher than 95% and all of them were coincident with their actual molecular masses, respectively. The purities of h GHRH dimers meet the requirement of the experiment with peptide-PAGE analysis. 2 Rat pituitary hormone-releasing activities of the h GHRH dimmers. 2.1 Rat GH-releasing activities: Compared with the net r GH value of S peptide, the net r GH value of 2F or 2Y peptide showed significant difference in statistics(P<0.05). The maximal net GH value of 2F or 2Y dimer reached 30.6 or 36.5% more than that of S peptide, and the activity of 2D, 2E, 2F, or 2Y covered 104±16.7%, 94±32.6%, 114±16.6%, or 122±14.5% of that of S peptide, respectively. The I3 value of 2F peptide was statistically higher than its P2 value or the I3 value of S peptide(P<0.01). The r GH release in the I5 incubation of 2Y peptide was higher than that in the I5 of S peptide(P<0.01). 2.2 Other pituitary hormone release of h GHRH peptide: These GHRH dimers do not regulate pituitary ACTH, PRL, or LH release as standard S peptide. 3 Rat pituitary GH-releasing inhibition. Based on the 5-h pituitary incubation assay, in the presence of 1.927 μM of GHRH dimer, 0.482 or 1.927 μM of GHIH in I3-I5 incubation produced obvious inhibition effects on r GH release. Compared with P2 value without peptide, the lower data in the I3-I5 incubation suggests that GHIH peptide strongly inhibits the rat GH release in a dose- and time-dependent manners(P <0.05 or 0.01). 4 GHRH dimer binding with pituitary GHRH receptor. The binding of S, 2D, 2E, 2F, or 2Y peptide to rat pituitary cells was specific. The ranking of these h GHRH peptides were 2F>2D>2Y>2E>S. suggesting that 2F peptide had the highest binding capacity to pituitary homogenate(P<0.05). 5 Fluorescent staining of rat pituitary tissue. The staining of FITC-labeled GHRH dimer had a cell membrane distribution. The strength ranking of the fluorescent staining was 2F>2D>2E>2Y>S and2F peptide showed the most abundant distribution in the pituitary cells.Conclusions: 1 From the results of the r GH-releasing activity/inhibition, receptor binding, and tissue chemical staining of the h GHRH dimers, 2D, 2E, 2F, and 2Y have good r GH-releasing activities and similar pituitary hormone-releasing specificities. 2 2F peptide has the strongest and long-lasting activity, the strongest binding to pituitary cells. So 2F peptide may be one promise in future animal experiment. 3 The facts indicate that GHRH is required for GH release and the h GHRH dimers with two N-termini showed better GH-releasing effects than h GHRH(1-44)NH2 with single N-terminus.