Thomas R. Downs

Fluorometric quantification of DNA in cells and tissue

The validation of a simple and rapid DNA solubilization procedure is described. Quantitative extraction of intact, polymerized DNA was achieved by cell lysis or tissue homogenization in an ammonium hydroxide-Triton X-100 solution. The solubilization procedure inactivates endogenous DNAase and increases the fluorescence-enhancement activity of the extracted DNA, thereby eliminating the need for enzyme treatment or exposure to high salt solutions. The extracts can be utilized directly in a sensitive fluorescence-enhancement assay with bisbenzimidazole (Hoechst 33258) reagent. Estimates of DNA cell content were unaffected by the number of cells lysed or the volume of lysate employed in the assay. In all cases, the solubilized DNA estimates were linear and parallel to the bovine DNA standard. The optimum range for estimation of DNA in this assay is 5-150 ng. In addition, estimates of DNA obtained with this method and the standard diphenylamine assay were in excellent agreement. This simple, one-step DNA extraction procedure can be utilized in conjunction with Hoechst reagent to obtain quantitative estimates of DNA levels in cell or tissue extracts.

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

The plasma enzyme responsible for primary proteolytic cleavage of growth hormone-releasing hormone (GRH) at the 2-3 amino acid bond was characterized. Native GRH[GRH(1-44)-NH2 and GRH(1-40)-OH], and COOH-terminally shortened fragments [GRH(1-32)-NH2 and GRH(1-29)-NH2] were rapidly cleaved, while GRH(2-32)-NH2 was not degraded at this site. Moreover, degradation to GRH(3-44)-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(3-44)-NH2 was blocked by diprotin A, a DPP type IV (DPP IV) competitive inhibitor. D-Amino acid substitution at either position 1 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(3-44)-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).

Measurement of growth hormone-releasing hormone and somatostatin in hypothalamic-portal plasma of unanesthetized sheep. Spontaneous secretion and response to insulin-induced hypoglycemia.

To elucidate the role of growth hormone (GH)-releasing hormone (GRH) and somatostatin (SRIH) in the regulation of the growth hormone (GH) secretory pattern, we collected portal blood from five unanesthetized ovariectomized ewes for repeated measurements of GRH and SRIH simultaneous with those of peripheral GH. Hormones were measured at 10-min intervals for 5.5 h and their interrelationships analyzed. Mean portal GRH was 20.4 +/- 6.7 (SD) pg/ml and the estimated overall secretion rate was 13 pg/min. GRH secretion was pulsatile with peaks of 25-40 pg/ml and a mean pulse interval of 71 min. Mean portal SRIH was 72 +/- 33 pg/ml and the estimated overall secretion rate was 32 pg/min. SRIH secretion was also pulsatile with peaks of 65-160 pg/ml and a mean pulse interval of 54 min. The GH pulse interval was 62 min. A significant association was present between GRH and GH secretory peaks though not between GRH and SRIH or SRIH and GH. Insulin hypoglycemia resulted in a rapid and brief stimulation of SRIH secretion followed by a decline in GH levels. No effect was observed on GRH secretion until 90 min, when a slight increase occurred. The results suggest (a) the presence of an independent neural rhythmicity of GRH and SRIH secretion with a primary role of GRH in determining pulsatile GRH secretion, and (b) that the inhibitory effects of insulin hypoglycemia on GH in this species are attributable to a combination of enhanced SRIH secretion and possibly other factors, though without significant inhibition of GRH.

Rapid enzymatic degradation of growth hormone-releasing hormone by plasma in vitro and in vivo to a biologically inactive product cleaved at the NH2 terminus.

The effect of plasma on degradation of human growth hormone-releasing hormone (GRH) was examined in vitro and in vivo using high performance liquid chromatography (HPLC), radioimmunoassay (RIA), and bioassay. When GRH(1-44)-NH2 was incubated with human plasma, the t1/2 of total GRH immunoreactivity was 63 min (RIA). However, HPLC revealed a more rapid disappearance (t1/2, 17 min) of GRH(1-44)-NH2 that was associated with the appearance of a less hydrophobic but relatively stable peptide that was fully immunoreactive. Sequence analysis indicated its structure to be GRH(3-44)-NH2. Identity was also confirmed by co-elution of purified and synthetic peptides on HPLC. Biologic activity of GRH(3-44)-NH2 was less than 10(-3) that of GRH(1-44)-NH2. After intravenous injection of GRH(1-44)-NH2 in normal subjects, a plasma immunoreactive peak with HPLC retention comparable to GRH(3-44)-NH2 was detected within 1 min and the t1/2 of GRH(1-44)-NH2 (HPLC) was 6.8 min. The results provide evidence for GRH inactivation by a plasma dipeptidylaminopeptidase that could limit its effect on the pituitary.

Decreased hypothalamic growth hormone-releasing hormone content and pituitary responsiveness in hypothyroidism.

The effects of thyroidectomy (Tx) and thyroxine replacement (T4Rx) on pituitary growth hormone (GH) secretion and hypothalamic GH-releasing hormone (GRH) concentration were compared to define the mechanism of hypothyroid-associated GH deficiency. Thyroidectomized rats exhibited a complete loss of pulsatile GH secretion with extensive reduction in GRH responsiveness and pituitary GH content. Cultured pituitary cells from Tx rats exhibited reduced GRH sensitivity, maximal GH responsiveness, and intracellular cyclic AMP accumulation to GRH, while somatostatin (SRIF) suppressive effects on GH secretion were increased. Hypothalamic GRH content was also markedly reduced. T4Rx completely restored hypothalamic GRH content and spontaneous GH secretion despite only partial recovery of pituitary GH content, GRH and SRIF sensitivity, and intracellular cyclic AMP response to GRH. The results indicate multiple effects of hypothyroidism on GH secretion and suggest that a critical role of T4 in maintaining normal GH secretion, in addition to restoring GH synthesis, is related to its effect on hypothalamic GRH.

Obesity-Associated Decrease in Growth Hormone-Releasing Hormone Gene Expression: A Mechanism for Reduced Growth Hormone mRNA Levels in Genetically Obese Zucker Rats

The secretion of growth hormone (GH) is impaired in the genetically obese Zucker rat where GH gene expression and plasma GH levels are depressed; however, the underlying mechanism of this abnormality remains unclear. We have evaluated the potential causative role of hypothalamic GH-releasing hormone (GHRH) and somatostatin (SRIH) gene expression in the onset of the decreased GH mRNA levels by studying both GHRH and SRIH mRNA and peptide levels in obese and lean rats at 5 weeks of age when the decrease in GH mRNA is first detected. At that age both GHRH content and GHRH mRNA were significantly reduced in obese rats as compared to lean controls; hypothalamic SRIH content was also decreased in obese rats, but SRIH mRNA levels did not differ. Since GHRH is capable of stimulating GH gene expression, the decreased GHRH mRNA level could be a critical factor in causing the attenuation in GH gene expression and consequent diminution of circulating plasma GH.

Ectopic GRH Syndromes

Although the occasion for this symposium is the commemoration of the centennial of the first description of acromegaly, it has been only 27 years since a patient with an extrapituitary cause for acromegaly was described. In 1959, Altmann and Schutz described a patient with acromegaly of 12 years duration who did not respond to a course of pituitary irradiation, but who experienced a marked regression of the features of GH hypersecretion following excision of a bronchial carcinoid tumor (1). This report in the German literature was overlooked by the numerous authors who, during the next two decades, described patients with coexisting acromegaly and carcinoid tumors of various types (see references 2 and 3 for reviews). Fifteen years later, Dabek reported a similar case history (4) and two years after that, Sonksen added an additional case report of regression of GH hypersecretion after removal of a carcinoid tumor (5). In 1979, we had an opportunity to study a tumor removed from a patient whose history was remarkably similar to that of the original patient described (1), and reported that it stimulated GH release by rat pituitary cell cultures (6). In another report, Shalet et al. (7) described similar findings using a perifusion system. Within the next few years, several other patients with the syndrome were recognized (8) and finally, identification of two patients prior to surgical removal of the extrapituitary tumor resulted in the preservation of two pancreatic islet tumors from which GH-releasing hormone (GRH) was eventually isolated and sequenced (9,10).

Somatostatin secretion and action in the regulation of growth hormone secretion.

The inhibition of growth hormone (GH) secretion by the hypothalamic peptide, somatostatin, is mediated by two critical factors: the concentration of the peptide in hypothalamic portal plasma and the number of somatostatin (SRIF) receptors on the somatotroph. The secretory patterns of SRIF and GH-releasing hormone (GRH) in portal blood of unanesthetized sheep is pulsatile and a close relationship of GRH pulses to those of GH secretion was documented, while those of SRIF appear to have more of a modulatory role on the responses to GRH. Peripheral infusion of SRIF at a rate to provide concentrations comparable to those in the portal system leads to a desensitization of SRIF effects on the somatotroph, likely mediated by down-regulation of SRIF receptors. These effects are believed to modulate the GH responses to GRH secretion in the generation of pulsatile GH secretion.

Growth Hormone‐Releasing Hormone: Structure, Gene Expression and Molecular Heterogeneity

Growth hormone-releasing hormone (GHRH) is unique among the hypothalamic hypophyseotrophic hormones for two reasons. First, it was initially isolated, characterized, sequenced, and its gene cloned from human tissue rather than that of laboratory animals. Second, it was first isolated not from the hypothalamus but from pancreatic islet tumours (associated with acromegaly) from which its ectopic secretion had been clinically recognized (1). Once sequenced, the peptide was found to consist of 44 amino acids with an amidated carboxyl terminus. Two other forms of the GHRH peptide were also observed in tumour tissue. These were non-amidated and truncated at the carboxyl terminus (GHRH (1-40) and GHRH (1 -37)); GHRH (1 -40) was the most abundant. Shortly after the discovery of these forms, human GHRH (hGHRH) was isolated from the hypothalamus. It was shown to have an identical structure to the tumour-derived forms, and to exist in both the 40and 44-residue forms (2, 3). The entire hGHRH gene has now been cloned using oligonucleotide probes and cDNA technology (4-6).

Mouse hypothalamic growth hormone-releasing hormone and somatostatin responses to probes of signal transduction systems

Signal transduction mechanisms involved in mouse growth hormone-releasing hormone (GRH) and somatostatin (SRIH) release were investigated using an in vitro perifusion system. Hypothalamic fragments were exposed to depolarizing agents, protein kinase A and C activators, and a calcium ionophore. The depolarizing agents, KCl (60 mM) and veratridine (50 microM), induced similar patterns of GRH and SRIH release. Somatostatin release in response to both agents was twofold greater than that of GRH. Forskolin (10 microM and 100 microM), an adenylate cyclase activator, stimulated both GRH and SRIH release, though with different secretory profiles. The SRIH response was prolonged and persisted beyond removal of the drug from the system, while the GRH response was brief, ending even prior to forskolin removal. Neither GRH nor SRIH were stimulated by 1,9-dideoxy-forskolin (100 microM), a forskolin analog with cAMP-independent actions. A23187 (5 microM), a calcium ionophore, stimulated the release of SRIH to a much greater extent than that of GRH. The GRH and SRIH secretory responses to PMA (1 microM), a protein kinase C activator, were similar, though delayed. The results suggest that 1) GRH and SRIH secretion are regulated by both protein kinase A and C pathways, and 2) depolarizing agents are important for the release of both hormones.