A nonpeptidyl growth hormone secretagogue.

A nonpeptidyl secretagogue for growth hormone of the structure 3-amino-3-methyl-N-(2,3,4,5-tetrahydro-2-oxo-1-([2'-(1H-tetrazol-5 -yl) (1,1'-biphenyl)-4-yl]methyl)-1H-1-benzazepin-3(R)-yl)-butanamid e (L-692,429) has been identified. L-692,429 synergizes with the natural growth hormone secretagogue growth hormone-releasing hormone and acts through an alternative signal transduction pathway. The mechanism of action of L-692,429 and studies with peptidyl and nonpeptidyl antagonists suggest that this molecule is a mimic of the growth hormone-releasing hexapeptide His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (GHRP-6). L-692,429 is an example of a nonpeptidyl specific secretagogue for growth hormone.

A receptor in pituitary and hypothalamus that functions in growth hormone release.

Small synthetic molecules termed growth hormone secretagogues (GHSs) act on the pituitary gland and the hypothalamus to stimulate and amplify pulsatile growth hormone (GH) release. A heterotrimeric GTP-binding protein (G protein)-coupled receptor (GPC-R) of the pituitary and arcuate ventro-medial and infundibular hypothalamus of swine and humans was cloned and was shown to be the target of the GHSs. On the basis of its pharmacological and molecular characterization, this GPC-R defines a neuroendocrine pathway for the control of pulsatile GH release and supports the notion that the GHSs mimic an undiscovered hormone.

Identification of a new G-protein-linked receptor for growth hormone secretagogues.

The potential application of small molecules in GH therapy has recently become a topic of increasing interest. The spiroindoline MK-0677, the benzolactam L-692,429, and the peptides, GHRP-6 and hexarelin, have been shown to possess potent and selective GH-secretory activity in several species including human. Moreover, these synthetic GH secretagogues act on a signal transduction pathway distinct from that of GHRH. A specific high affinity binding site in porcine and rat anterior pituitary membranes that mediates the activity of these secretagogues has now been identified. The binding affinity of these structurally diverse secretagogues is tightly correlated with GH-secretory activity. The binding is Mg(2+)-dependent, is inhibited by GTP-gamma-S, and is not displaced by GHRH and somatostatin. The receptor is distinct from that for GHRH and has the properties of a new G-protein-coupled receptor. It is speculated that these GH secretagogues mimic an unidentified natural hormone that regulates GH secretion in concert with GHRH and somatostatin.

Heparin inhibits bovine testicular hyaluronidase activity in myocardium of dogs with coronary artery occlusion.

Bovine testicular hyaluronidase (BTH) reduces experimental myocardial infarct size and ameliorates electrocardiographic signs of ischemia. This study was done to determine if heparin, an in vitro inhibitor of hyaluronidase activity, blocks the action of BTH in the myocardium of dogs after coronary artery occlusion. BTH was administered intravenously as 5,000 NF units/kg at 0.5 and 2.5 hours after coronary occlusion. Heparin was administered intravenously as a 150-unit/kg loading dose, followed by 10 units/kg per hour i.v., beginning 15 minutes before coronary occlusion. The area of myocardial ischemia at risk was assessed by a radiolabeled microsphere technique; the area that developed necrosis was assessed by a histochemical technique. In vivo activity of BTH was assessed by a colorimetric analysis of the BTH substrate, i.e., hyaluronic acid (HA), extracted from myocardial tissue. For biochemical analysis of HA, the heart was divided into anterior myocardium, which included ischemic tissue and posterior nonischemic myocardium. The myocardial HA content of dogs treated with BTH plus heparin (anterior, 3.44 +/- 0.40 micrograms HA/mg protein; posterior, 3.69 +/- 0.33 micrograms HA/mg protein) was not significantly different from control (anterior, 3.61 +/- 0.29 micrograms HA/mg protein; posterior, 3.55 +/- 0.23 micrograms HA/mg protein). In contrast, BTH lowered myocardial HA content (anterior, 2.16 +/- 0.21 micrograms HA/mg protein; posterior, 2.08 +/- 0.14 micrograms HA/mg protein) compared with either BTH plus heparin or control groups in both anterior myocardium (p = 0.006) and posterior myocardium (p = 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of L-692,429-stimulated rat growth hormone release by a weak substance P antagonist: L-756,867.

H2N,D-Arg,Pro,Lys,Pro,D-Phe,Gln,D-Trp,Phe,D-Trp,Leu, Leu,NH2 (L-756,867), a weak substance P antagonist, inhibited L-692,429-stimulated GH release from rat primary pituitary cells in a dose-dependent manner. At a concentration of 50 nM, L-756,867 shifted the dose-response curve of L-692,429-induced GH release to the right by about tenfold. It also impaired the ability of L-692,429 to potentiate the effect of growth hormone-releasing factor (GRF) on GH release. Substance P (1 microM) had no effect on basal or L-692,429-stimulated GH release. When tested in anesthetized rats, L-756,867 inhibited L-692,429- and growth hormone-releasing hexapeptide- (GHRP-6)-stimulated GH secretion in a dose-dependent manner. Complete inhibition was observed at an i.v. dose of 100 micrograms/kg of L-756,867. However, at the same concentration, it had no effect on GRF-induced GH secretion D-Lys3-GHRP-6, a GHRP-6 antagonist, had no effect on GHRP-6 or L-692,429-induced GH secretion even at an i.v. dose of 2 mg/kg. These results indicate that L-692,429 and GHRP-6 stimulate GH release both in vitro and in vivo via a common receptor and signaling pathway which is different from that of substance P in spite of the fact that their effects are inhibited by a weak substance P antagonist.

Effects of a beta-adrenergic agonist on protein turnover in muscle cells in culture.

beta-Adrenergic agents powerfully stimulate muscle growth in animals. Whether the mechanism of action involves a direct effect on muscle cell beta-receptors or is secondarily due to a beta-induced alteration in the hormonal environment is not known. To assess whether direct beta-receptor activation results in muscle protein accretion, we examined the effect of the beta-agonist zinterol on several anabolic processes in L8 muscle cells in culture. In vivo feeding of zinterol (26.5 ppm) to rats significantly increased muscle weight by 15%. In vitro, zinterol stimulated lactate release from L8 cells whereas propranolol inhibited this process, demonstrating that these cells have functional beta-receptors both before and after fusion. We measured several anabolic processes, in both serum-stimulated and quiescent cells, over a wide range of zinterol concentrations. Zinterol had no effect on protein or DNA synthesis, protein degradation, or rates of amino acid uptake. These data suggest that the in vivo muscle growth stimulation is either indirect or some in vivo requirements (e.g. tension and nerve interactions) are necessary for expression of the effect.

The farnesyltransferase inhibitor, FPT inhibitor III upregulates Bax and Bcl-xs expression and induces apoptosis in human ovarian cancer cells.

Deregulation in the ras oncogene is a common event in many types of human cancer. Our previous study clearly demonstrated that genetic alterations of ras oncogene are frequently found in human epithelial ovarian cancer. Recent reports have indicated that farnesyltransferase is involved in the regulation of post-translational modification and biological function of Ras proteins. Here, we report that a newly synthesized farnesyltransferase inhibitor, FPT inhibitor III, upregulates Bax and Bcl-xs expression and induces apoptosis in human ovarian cancer cells. This is a critical finding that farnesyltransferase inhibitors may directly activate apoptotic signaling pathways in cancer cells and may help to provide a new strategy in the treatment of human cancer.

Protein turnover and growth of L8 muscle cultures in serum from rats fed clenbuterol.

Beta-adrenergic agonists stimulate muscle growth in chronically treated animals. The response may be primary (receptor binding directly stimulates anabolism) or secondary (circulating levels of hormones or other factors regulate muscle mass). Although B-receptors are functional in L8 muscle cells in culture, a direct effect of B-agonists on protein turnover in culture has not been demonstrated. To determine whether the stimulation of muscle growth is a secondary effect, we assessed the anabolic activity of sera from both clenbuterol-treated rats and normal rats in L8 cultured muscle cells. Rats were fed either normal diet or diet containing ten parts per million clenbuterol for seven days. Blood was collected from the inferior vena cava of rats under metofane anaesthesia and serum prepared. The anabolic activities of serum were measured in cultures of either myoblasts (still dividing) or fused myotubes (non-dividing) to distinguish mitogenic action from alteration in rates of protein turnover. Sera from both normal and clenbuterol-treated rats gave the same stimulation of protein synthesis in myotube cultures. Sera from both groups of rats contained factors which inhibited protein degradation, stimulated amino isobutyric acid (AIB) and thymidine uptake (myoblasts only). These processes were not augmented by sera from rats treated with clenbuterol. Protein accumulation was equivalent after 48 hours of exposure to either test sera. In conclusion, adult rat serum contained anabolic factors but there was no evidence for an increase in activity in serum from clenbuterol-treated rats.

Mononuclear phagocytic cells in peritoneal exudates of guinea pigs: a comparison of inducing agents.

Peritoneal macrophages are used for immunologic assessment of the lymphokine, migration inhibitory factor (MIF). For this purpose, these cells are induced intraperitoneally in animals with inflammatory agents such as mineral oil (MO) and peptone water (PW). Purpose of the present study was two-fold: To assess the changes in biologic properties of guinea pig macrophages induced peritoneally with MO or PW as compared to control cells after administration of normal saline (NS); and to examine the suitability of induced macrophages to respond to MIF in vitro. Peritoneal exudate cells were harvested from guinea pigs 7 days following intraperitoneal injection of MO, PW or NS. They were enumerated in hemocytometers, their differential counts determined by Wright's stain and morphologic characteristics assessed by scanning electron microscopy. Functional activation of peritoneal macrophages was determined by lysosomal enzyme functions, as well as by yeast phagocytosis in vitro. Random cell migration and responses to migration inhibitory factor (MIF) were determined in Mackaness chambers. Mineral oil injection resulted in significantly higher yield of peritoneal macrophages. Greater than 70% of peritoneal exudate cells were macrophages in all three groups. Spread out structures and ruffled borders were seen in electron micrographs of MO induced cells. Such structures were less evident in PW induced cells and were absent in controls. Acid phosphatase (ACP) and beta-N-acetylglucosaminidase (B-NAG) activities as well as yeast phagocytosis significantly increased in MO and PW induced cells. Random migration and responses to MIF in Mackaness chambers remained comparable in the three experimental groups.

Effect of continuous and intermittent clenbuterol feeding on rat growth rate and muscle.

1. The growth response to clenbuterol is a dynamic process. 2. Body weight gain is stimulated within two days of treatment and the effect attenuates by two weeks of treatment. 3. Intermittent feeding prevents the attenuation of the growth response. 4. Muscle weight increased 14-22% by both feeding regimens. 5. Clenbuterol decreased cathepsin B activity in the EDL and gastrocnemius and increased the activity in the soleus after two weeks of continuous clenbuterol treatment.

Prostaglandin production in myotube cultures. Influence on protein turnover.

The production of prostaglandins (PG) E2 and F2 alpha and their possible role in regulation of protein turnover in cultured skeletal-muscle cells were examined. Primary chick myoblasts and myotubes, and L8 myotubes, produced PGE2 and PGF2 alpha from endogenous arachidonic acid. PG production by all three cell types was increased manyfold by the addition of exogenous arachidonic acid. Arachidonate-stimulated PG production was inhibited by the addition of indomethacin (0.1 mM). When L8 and chick myotubes were treated with PGE2, PGF2 alpha, arachidonic acid (0.01 mM) or indomethacin (0.1 mM), no significant alterations in rates of protein synthesis or degradation were observed. Rates of protein synthesis and degradation in these cells were responsive to the addition of 10% fetal-bovine serum under identical experimental conditions. Thus, in contrast with incubated adult skeletal muscle, it appears that the production of prostaglandin metabolites from arachidonic acid is unrelated to regulation of protein turnover in cultured muscle cells.

Clenbuterol-induced muscle growth: investigation of possible mediation by insulin.

The role of insulin as a possible mediator of the beta-adrenergic agonist stimulation of muscle growth was investigated. To exclude possible action of the beta-agonist on the pancreatic release of insulin, diabetes was induced in rats by a streptozotocin injection (100 mg/kg). Insulin levels were almost not detectable in these rats. Feeding either normal diet or diet containing the beta-adrenergic agonist clenbuterol (10 parts/million) did not alter plasma insulin concentrations. The effects of clenbuterol on muscle and weight gain were determined in diabetic rats given daily insulin replacement (D + I) and fed either a normal diet or clenbuterol-treated diet. Clenbuterol, fed for 1 wk, increased the wet weight of the gastrocnemius, soleus, and extensor digitorum longus muscles (15-23%) in both normal and D + I rats. Although clenbuterol increased body weight gain, it did not alter feed consumption and, therefore, feed efficiency (g gain/g food) was improved. Activities of cathepsin B and N-acetyl-beta-glucosaminidase, but not cathepsin D, were elevated in the soleus muscles of clenbuterol-treated rats. The clenbuterol-induced increase in muscle growth in the insulin-replaced diabetic rats indicated that this beta-adrenergic agonist effect was not mediated by an alteration of circulating levels of insulin, secondary to beta-agonist action on pancreatic insulin release.

The serum kinetics of bovine testicular hyaluronidase in dogs, rats and humans.

There is experimental and clinical evidence that i.v. injection of bovine testicular hyaluronidase (BTH) reduces the extent of necrosis during myocardial infarction. The fate of i.v. administered BTH has not been described. In this study, serum kinetics of BTH enzyme activity in dogs, rats and humans were determined. Tissue distribution of BTH was determined with an 125I-labeled preparation of purified BTH. Serum BTH activity initially decreased exponentially with half-life 2.0 +/- 0.1 min in dogs with coronary artery occlusion (n = 8; 500 U of BTH/kg); 3.2 min in humans with acute myocardial infarction (n = 2; 500 U of BTH/kg); and 3.2 +/- 0.3 min in rats (n = 5; 5,000 U of BTH/kg). In dogs BTH disappearance showed two distinct phases. After injection of high dose BTH (5,000 U of BTH/kg), during the first 7 min serum half-life of BTH was 2.1 +/- 0.2 min (n = 8), but increased to 9.4 min in later serum samples. After the injection of 125I-labeled BTH into the rat, protein-bound 125I disappeared from serum with a half-life (3.4 min) that is similar to the serum half-life of BTH enzyme activity (3.2 min). Twenty minutes after injection of 125I-labeled BTH, 30% of the label was recovered in the liver. It is concluded that BTH activity has a short serum half-life of less than 10 min in dogs, rats and humans. In the rat model, the disappearance of serum BTH activity results from physical removal of circulating BTH molecules rather than serum inhibition or inactivation of BTH enzymatic activity.