An Extremely Sensitive<i>in Vitro</i>Model for Elucidating Structure-Activity Relationships of Growth Hormone-Releasing Factor Analogs*

Heiman, Mark L.; Nekola, Mary V.; Murphy, William A.; Lance, V. A.; Coy, David H.

doi:10.1210/endo-116-1-410

Cited by 58 publications

(15 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Substitution of Tyr' with residues other than His produced a similar effect (36). Thus, the first superactive GRH analogues were designed by stabilizing the NH2-terminal residues, primarily with D-amino acid substitutions (36)(37)(38). The reported increase in the intrinsic activity of some of these analogues in vitro by some investigators (37, 38) remains controversial (36).…”

Section: Resultsmentioning

confidence: 99%

Dipeptidylpeptidase IV and trypsin-like enzymatic degradation of human growth hormone-releasing hormone in plasma.

Frohman¹,

Downs²,

Heimer³

et al. 1989

J. Clin. Invest.

174

View full text Add to dashboard Cite

The plasma enzyme responsible for primary proteolytic cleavage of growth hormone-releasing hormone (GRH) at the 2-3 amino acid bond was characterized. Native GRHIGRH(144)-NH2 and GRH(140)-OHI, and COOH-terminally shortened fragments [GRH(1-32)-NH2 and GRH(1-29)-NH21 were rapidly cleaved, while GRH(2-32)-NH2 was not degraded at this site. Moreover, degradation to GRH(344)-NH2 was unaffected by an aminopeptidase inhibitor, indicating that this metabolite was generated from a single step cleavage by a dipeptidylpeptidase (DPP) rather than sequential aminopeptidase cleavages. Conversion to GRH(344)-NH2 was blocked by diprotin A, a DPP type IV (DPP IV) competitive inhibitor. DAmino acid substitution at either position I or 2 also prevented hydrolysis, characteristic of DPP IV. Analysis of endogenous plasma GRH immunoreactivity from a human GRH transgenic pig revealed that the major peak coeluted with GRH(344)-NH2. Native GRH exhibited trypsin-like degradation at the 11-12 position but cleavage at the 12-13 site occurred only with GRH(1-32)-NH2 and GRH(1-29)-NH2. Formation of these metabolites was independent of prior DPP IV hydrolysis but was greatly reduced by trypsin inhibitors. Evaluation of plasma stability of potential GRH super analogues, designed to resist degradation by these enzymes, confirmed that GRH degradation in plasma occurs primarily by DPP IV, and to a lesser extent by trypsin-like enzyme(s).

show abstract

Section: Resultsmentioning

confidence: 99%

Dipeptidylpeptidase IV and trypsin-like enzymatic degradation of human growth hormone-releasing hormone in plasma.

Frohman¹,

Downs²,

Heimer³

et al. 1989

J. Clin. Invest.

174

View full text Add to dashboard Cite

show abstract

“…GRF potency was increased up to 50 times following ID-Ala'] substitution in in vitro studies (Heiman et al 1985) or following i.v. injection to sodium pentobarbital anesthetized rats (Lance et al 1984 For personal use only.…”

Section: Discussionmentioning

confidence: 99%

“…Synthetic analogs were then designed to resist the aminopeptidase degradation of amino acid 1-3. N-acetyiation of GRF improved potency both in vivo and in vitro (Heiman et al 1985;Lance et al 1984), whereas D-Ala in position 2 lead to a larger increase in potency as measured by GH release in rats (Lance et al 1984;Coy et al 1986), pigs (Lance et al 1984) and cattle (Hodate et al 1986). Kaiser and Kezdy (1984) have proposed an amphiphilic, helicoidal secondary structure of GRF which would result in the formation of distinct hydrophylic and hydrophobic surfaces (Coy et al 1985).…”

mentioning

confidence: 99%

Effect of a Growth Hormone-Releasing Factor Analog on Growth Hormone, Insulin-Like Growth Factor 1 and Milk Production in Dairy Cows

Lapierre¹,

Pelletier²,

Petitclerc³

et al. 1990

Can. J. Anim. Sci.

View full text Add to dashboard Cite

In a first experiment, 21 cows (98 d in lactation, 609 kg BW) were randomly used to determine the dose-response release of growth hormone (GH) to [desamino-Tyr1, D-Ala2, Ala15] growth hormone-releasing factor (1–29)NH2 analog (GRF-A; 0, 0.12, 0.37, 1.11 and 3.33 μg kg−1 BW) or to human growth hormone-releasing factor (1–29)NH2 (GRF; 3.33 and 10.00 μg kg−1 BW) after s.c. administration. The interaction (P < 0.01) between the two releasing factors and the doses on GH concentrations indicated that the analog was more potent than the original molecule; at the 3.33 μg kg−1 BW dose, the analog elicited a 3.5 times higher GH response than GRF. In a second experiment, the effects of daily s.c. injections for 10 d of either saline, GRF (10 μg kg−1 BW) or GRF-A (0.6 or 1.8 μg kg−1 BW) on GH concentrations and lactational performance were determined using 32 cows (n = 8 per treatment) averaging 91 d in lactation and subjected to a random block design. The three releasing factor treatments similarly increased GH concentrations measured during the 8 h following the injection on d 1 and 10. GRF at the dose of 10 μg kg−1 BW and the analog at the doses of 0.6 and 1.8 μg kg−1 BW increased milk yield by 9.6, 11.7 and 17.6%, respectively, when compared to the control; both releasing factors had no effect on milk composition. Insulin-like growth factor I concentrations were increased 8 h after the last injection, from 73.4 ng mL−1 in control cows to an average of 107.6 ng mL−1 in treated cows. Results show that the [desamino-Tyr1, D-Ala2, Ala15] human growth hormone-releasing factor (1–29)NH2 analog is more potent than human growth hormone-releasing factor (1–29)NH2 in stimulating GH release and milk production in dairy cows. Key words: Growth hormone-releasing factor, analog, growth hormone, milk production, insulin-like growth factor 1, cows (dairy)

show abstract

“…These analogs are essential for the study of the mechanism of action of GHRH and may also be important in clinical application for endocrine disorders and insulin-like growth factor I-dependent tumors (11)(12)(13) (16,17). To eliminate the drawbacks of the static method, we used a modified dynamic in vitro system, the principles of which were described earlier (18-21).…”

Section: Introductionmentioning

confidence: 99%

A method for evaluation of activity of antagonistic analogs of growth hormone-releasing hormone in a superfusion system.

Rékási

1993

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Antagonistic analogs of growth hormonereleasing hormone (GHRH) are being synthesized in our laboratory for various clinical applications, including treatment of certain endocrine disorders and insulin-like growth factor I-dependent tumors. To evaluate the endocrine effect of these GHRH antagonists, a sensitive dynamic in vitro system has been developed. The concentration causing 50% inhibition (IC50) of the standard GHRH antagonist human [N-Ac-Tyr',DArg2]GHRH-(1-29)-NH2 is 4.5 x 10-8 M in our dispersed pituitary cell superfusion system. This value is 11 times less than that measured in earlier static pituitary cell cultures. This reliable dynamic system is simple, fast, and inexpensive and not only makes it possible to obtain quantitative data on the Inhibitory capacity of the antagonists but also provides information about the intrinsic GHRH activity of the analog. The dynamic interactions of the GHRH antagonist, the GHRH receptors, and GH release can also be evaluated by this superfusion system. The pulsatile GH release induced by 10-9 M human GHRH-(1-29)-NH2 was inhibited by two modes of application, preincubation and simultaneous administration of the GHRH antagonist (10-9 to 10-6 M). The reduction in GHRH-stimulated GH response was more pronounced when the cells were preincubated with the antagonist prior to GHRH infusion than for simultaneous application. The inhibitory effect of the antagonist was dose-dependent, temporary, and of the competitive type. GH release induced by nonspecific stimulus (100 mM potassium chloride) was not influenced by the GHRH antagonist. This sensitive dynamic in vitro system appears to be a suitable method for screening the biological activity of various GHRH antagonists and eliminates the drawbacks of static pituitary cell culture.Since the isolation and characterization of growth hormonereleasing hormone (GHRH) (1, 2), various GHRH antagonistic analogs have been synthesized (3-10). These analogs are essential for the study of the mechanism of action of GHRH and may also be important in clinical application for endocrine disorders and insulin-like growth factor I-dependent tumors (11)(12)(13) (16,17). To eliminate the drawbacks of the static method, we used a modified dynamic in vitro system, the principles of which were described earlier (18-21). In our dispersed pituitary cell superfusion system, the tissue culture medium is perfused continuously. The test material can be applied in a more physiological pulsatile way. The dynamics of hormone response and the changes in the responsiveness can also be analyzed.(1-29)-NH2 (3) was reported to inhibit the GHRH-stimulated adenylate cyclase activity in rat pituitary cells with low potency in the micromolar range (4) and suppress the GHRHinduced GH release with a high IC50 value (-5 x 1O-7 M) in static pituitary cell culture (6, 7). This analog was also reported to block endogenous GHRH activity after injection in the rat (22, 23). We used this GHRH analog as the standard antagonist to compare the sensitivity of our system ...

show abstract

An Extremely Sensitivein VitroModel for Elucidating Structure-Activity Relationships of Growth Hormone-Releasing Factor Analogs*

Cited by 58 publications

References 28 publications

Dipeptidylpeptidase IV and trypsin-like enzymatic degradation of human growth hormone-releasing hormone in plasma.

Dipeptidylpeptidase IV and trypsin-like enzymatic degradation of human growth hormone-releasing hormone in plasma.

Effect of a Growth Hormone-Releasing Factor Analog on Growth Hormone, Insulin-Like Growth Factor 1 and Milk Production in Dairy Cows

A method for evaluation of activity of antagonistic analogs of growth hormone-releasing hormone in a superfusion system.

Contact Info

Product

Resources

About