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.

Receptor for motilin identified in the human gastrointestinal system.

Motilin is a 22-amino acid peptide hormone expressed throughout the gastrointestinal (GI) tract of humans and other species. It affects gastric motility by stimulating interdigestive antrum and duodenal contractions. A heterotrimeric guanosine triphosphate-binding protein (G protein)-coupled receptor for motilin was isolated from human stomach, and its amino acid sequence was found to be 52 percent identical to the human receptor for growth hormone secretagogues. The macrolide antibiotic erythromycin also interacted with the cloned motilin receptor, providing a molecular basis for its effects on the human GI tract. The motilin receptor is expressed in enteric neurons of the human duodenum and colon. Development of motilin receptor agonists and antagonists may be useful in the treatment of multiple disorders of GI motility.

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.

Stimulation of benzodiazepine binding to rat brain membranes and solubilized receptor complex by avermectin B1a and gamma-aminobutyric acid.

The binding of [3H]flunitrazepam to benzodiazepine receptors in synaptic membranes and a digitonin-solubilized receptor fraction of rat brain is increased by avermectin B1a and gamma-aminobutyric acid (GABA). The effects of avermectin B1a and GABA are both sensitive to inhibition by (+)-bicuculline. Avermectin B1a and GABA both decrease the Kd and increase the Bmax of [3H]flunitrazepam binding to membranes. Kinetic analysis of the binding of [3H]flunitrazepam to rat brain membranes indicates that avermectin B1a and GABA reduce the rate constants of both association and dissociation between the ligand and the receptor. These results suggest a similar mechanism of modulation of benzodiazepine binding by avermectin B1a and GABA. This modulation may involve in interaction among the receptors for benzodiazepine, GABA and avermectin B1a.

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.

Structure-function studies on the new growth hormone-releasing peptide, ghrelin: minimal sequence of ghrelin necessary for activation of growth hormone secretagogue receptor 1a.

The recently discovered growth hormone secretagogue, ghrelin, is a potent agonist at the human growth hormone secretagogue receptor 1a (hGHSR1a). To elucidate structural features of this peptide necessary for efficient binding to and activation of the receptor, several analogues of ghrelin with various aliphatic or aromatic groups in the side chain of residue 3, and several short peptides derived from ghrelin, were prepared and tested in a binding assay and in an assay measuring intracellular calcium elevation in HEK-293 cells expressing hGHSR1a. Bulky hydrophobic groups in the side chain of residue 3 turned out to be essential for maximum agonist activity. Also, short peptides encompassing the first 4 or 5 residues of ghrelin were found to functionally activate hGHSR1a about as efficiently as the full-length ghrelin. Thus the entire sequence of ghrelin is not necessary for activity: the Gly-Ser-Ser(n-octanoyl)-Phe segment appears to constitute the "active core" required for agonist potency at hGHSR1a.

Discovery, production and purification of the Na+, K+ activated ATPase inhibitor, L-681,110 from the fermentation broth of Streptomyces sp. MA-5038.

The maximum yield for the production of L-681,110 by Streptomyces sp. MA-5038 (ATCC 31587) was observed after 5 days' incubation at 28 degrees C and pH about 8.3. L-681,110 was isolated from the fermentation broth by acetone extraction of the mycelia, absorption to Amberlite XAD-2 resin and two separations by thin-layer chromatography. The structure of L-681,110 was found to consist of a sixteen-membered lactone with a new type of substitution. The inhibition of ATPase, activity against Caenorhabditis elegans and stimulation of gamma-aminobutyric acid release indicate that L-681,110 possesses some characteristics of both oligomycin and avermectin. L-681,110 was also active against tapeworm and ticks in an in vivo assay.

Prostaglandin production by methylcholanthrene-transformed mouse BALB/3T3: effect of oxidative phosphorylation inhibitors.

Oligomycin, antimycin, and 2,4-dinitrophenol, compounds that are known to inhibit oxidative phosphorylation by different mechanisms, inhibit the production of prostaglandins by serum-stimulated MC5-5 cells. The prostaglandin production that is stimulated by thrombin and bradykinin is inhibited by 2,4-dinitrophenol. Prostaglandin synthesis by MC5-5 cells from exogeneously-supplied arachidonic acid, however, is not affected by 2,4-dinitrophenol. Antimycin and 2,4-dinitrophenol also inhibit the serum-stimulated release of arachidonic acid from the cellular lipids, suggesting that it is the expression of phospholipase activity, a prerequisite for synthesis of prostaglandins by MC5-5 cells, that is dependent on oxidative phosphorylation.

Avermectin B1a modulation of gamma-aminobutyric acid receptors in rat brain membranes.

Avermectin B1a stimulates high-affinity binding of [3H]-gamma-aminobutyric acid (GABA) to receptors in washed rat brain membranes. Scatchard analysis of the data indicates that the drug does not significantly alter the apparent dissociation constant of GABA binding, but increases the detectable number of binding sites from 3.2 to 5.1 pmol/mg protein, (+)-Bicuculline completely blocks control and avermectin B1a-stimulated GABA binding, whereas picrotoxin antagonizes specifically the avermectin B1a-stimulated GABA binding. The avermectin B1a-stimulated GABA binding is also chloride ion-dependent, whereas GABA binding in the control is not. These observations suggest that the mechanism of avermectin B1a stimulation of GABA binding may involve the chloride ion channel.

Actions of avermectin B1a on GABA nerves.

Avermectin B1a (AVM) is a macrolide derived from Streptomyces avermitilis. It possesses potent anthelmintic and insecticidal activities. It immobilizes nematodes by blocking the signal transmission from the central command interneurons to the peripheral motoneurons; this block can be reversed by the chloride ion channel blocker picrotoxin. The arthropods are paralyzed by AVM through drug inhibition of both inhibitory and excitatory postsynaptic potentials at the neuromuscular junctions. The mechanism of action is reduction of the muscle membrane resistance by opening the gamma-aminobutyric acid (GABA)-controlled chloride ion channels in the membrane--an action also inhibitable by picrotoxin. AVM has specific and high-affinity binding sites in the mammalian brain synaptic membrane. The sites are concentrated in the cerebellum and closely associated with the GABA-benzodiazepine receptor-chloride ion channel complex. GABA release from brain synaptosomes is specifically stimulated by AVM. The density of available postsynaptic GABA receptors is increased by AVM--a process which requires chloride ion and is inhibited by picrotoxin. AVM also stimulates benzodiazepine binding and potentiates the in vivo diazepam activity of muscle relaxation. These findings add up to a conclusive demonstration of the mechanism of AVM action as a potentiation of the GABA action. They also point out the research on GABA nerve in invertebrates as an attractive approach to chemotherapy against nematodes and insects and suggest AVM as a very useful tool in isolating GABA receptors and in-depth understanding of GABA function.

Characterization of a leukotriene D4 receptor in guinea pig lung.

[3H]Leukotriene D4 was found to bind, in a saturable manner and with exceedingly high affinity, to a membrane preparation from guinea pig lung. Measurement of saturation at equilibrium yielded Kd values of 5.46 +/- 0.31 X 10(-11) M at 20 degrees C and 2.12 +/- 0.37 X 10(-10) M at 0 degree C while the numbers of binding sites (Bmax) were 384 +/- 34 and 302 +/- 44 fmol/mg of protein at 20 and at 0 degree C, respectively. The time courses of both association and dissociation were slow but the rate of dissociation was accelerated by either NaCl or GTP. Binding was enhanced by Ca2+, Mg2+, and Mn2+ and inhibited by Na+ but not by Li+ or K+, suggesting that the binding of leukotriene D4 may be regulated by ions. Leukotriene E4, but not leukotriene C4, had a high affinity for the putative receptor, consistent with the pharmacological evidence that the actions of leukotrienes D4 and E4 are mediated by a receptor distinct from that for leukotriene C4. Affinities of stereoisomers and related compounds for the leukotriene D4 binding sites closely paralleled their contractile activities in guinea pig lung parenchymal strips. In addition, the antagonist of slow-reacting substance of anaphylaxis, FPL 55712, inhibited the binding of [3H]leukotriene D4 with a Ki value of 1 X 10(-7) M, which is in agreement with reported Kb values obtained in pharmacological studies.

Prostaglandin synthetase systems of rabbit tissues and their inhibition by nonsteroidal anti-inflammatory drugs.

Prostaglandin synthetase activities in the microsomal fractions of seven rabbit tissues have been partially characterized. All of the microsomal preparations required arachidonic acid, hydroquinone and reduced glutathione for enzymatic activity. The synthetic systems of the renal medulla were most active followed by microsomal preparations from the renal cortex, lung, brain, spleen, uterus and heart. Three structurally distinct nonsteroidal anti-inflammatory drugs, indomethacin, flufenamic acid and aspirin, inhibited the production of prostaglandins by these microsomes. The effectiveness of these drugs depended on the concentration of substrate. When assayed under identical experimental conditions, the inhibiting activity of each of these drugs for the synthetase of each of the tissues was the same.

Biosynthesis of prostaglandins in rabbit renal cortex.

Prostaglandin biosynthetic activity has been found in the microsomal fractions prepared from rabbit renal cortex. The cortex preparation requires arachidonic acid as substrate and hydroquinone and reduced glutathione as cofactors for enzymatic activity and is inhibited by indomethacin. The biosynthetic capacity of the washed microsomes of cortex ranges from one third to equal that of the washed microsomes of medulla. A partially purified medullary cytoplasmic factor and crystalline hemoglobin can stimulate greatly the synthetase activity in the microsomes of the medulla, but they have no effect on the activity of cortical microsomes.

Prostaglandin production by methylcholanthrene-transformed mouse BALB/3T3: inhibition by cytochalasin B.

Cytochalasin B inhibits the production of prostaglandins by serum-, thrombin-, and bradykinin-stimulated MC5-5 cells. The serum-stimulated release of arachidonic acid from cellular phospholipids also is inhibited. Cytochalasin B does not affect the cells' prostaglandin synthetase activity when exogenous arachidonic acid is present. Deacylation of phospholipids may be the step affected by cytochalasin B possibly as a result of disruption of microfilament organization. Colchicine and vinblastine, two drugs that can disrupt microtubule organization, do not inhibit prostaglandin production by cells.

Ionic and GTP regulation of binding of platelet-activating factor to receptors and platelet-activating factor-induced activation of GTPase in rabbit platelet membranes.

Specific binding of 3H-labeled platelet-activating factor (PAF) to rabbit platelet membranes was found to be regulated by monovalent and divalent cations and GTP. At 0 degrees C, inhibition of [3H]PAF binding by sodium is specific, with an ED50 of 6 mM, while Li+ is 25-fold less effective. On the contrary, K+, Cs+, and Rb+ enhance the binding. The divalent cations, Mg2+, Ca2+, and Mn2+ enhance the specific binding 8-10-fold. From both Scatchard and Klotz analyses, the inhibitory effect of Na+ is apparently due to an increase in the equilibrium dissociation constant (KD) of PAF binding to its receptors. However, the Mg2+-induced enhancement of the PAF specific binding may be attributed to an increased affinity of the receptor and an increased availability of the receptor sites. In the presence of Na+, PAF receptor affinity decreased with increasing temperature with a 100-fold sharp discontinuous decrease in receptor affinity at 24 degrees C. In contrast, the Mg2+-induced increase is independent of temperature suggesting that the Mg2+ regulatory site is different from Na+ regulatory site. [3H]PAF binding is also specifically inhibited by GTP; other nucleotides have little effect. PAF also stimulates hydrolysis of [gamma-32P]GTP with an ED50 of 0.7 nM, whereas 3-O-hexadecyl-2-O-acetyl-sn-glyceryl-1-phosphorylcholine showed no activity even at 10 microM. Moreover, such stimulatory effect of PAF is dependent on Na+ and can be abolished by the PAF-specific receptor antagonist, kadsurenone, but not by an inactive analog, kadsurin B. These results suggest that the PAF receptor may be coupled with the adenylate cyclase system via an inhibitory guanine nucleotide regulatory protein.

Solubilization and characterization of a growth hormone secretagogue receptor from porcine anterior pituitary membranes.

The discovery of a potential new GH therapy by small molecules that induce GH secretion (GHRP-6, L-692,429, MK-0677), has increased the interest in these GH secretagogues and their receptor and mechanism of action, which is different from the one of GHRH. We report the solubilization of the GH-secretagogue-receptor-ligand-G-protein complex (apparent molecular mass of approximately 255 kDa) from porcine anterior pituitary membranes using digitonin, after labelling the receptor with [35S]MK-0677. The solubilized receptor showed high affinity (KD = 122.2 +/- 14.4 pM) and low capacity (Bmax = 3.8 +/- 0.9 fmol/mg protein). These values and the inhibition constants (Ki) for a series of GH secretagogues were similar to the values determined in membranes isolated from porcine anterior pituitary gland. The solubilization of the GH secretagogue receptor opens up the possibility for further molecular characterization and sequencing of the receptor protein, necessary step prior to the identification of the natural ligand that would act as a GHRH amplifying hormone, and that the GH secretagogues would mimic.

Distribution of prostaglandin E 9-ketoreductase and NAD+-dependent and NADP+-dependent 15-hydroxyprostaglandin dehydrogenase in the renal cortex and medulla of various species.

Regional distributions of PGE 9-ketoreductase and 15-hydroxy-prostaglandin dehydrogenase were examined in the cytoplasmic fractions from the kidneys of seven species. All species contained an NADPH-dependent reductase, as well as NAD+- and NADP+-dependent dehydrogenases in both cortex and medulla. A previously unrecognized cytoplasmic NADH-dependent PGE 9-ketoreductase was also detected in the cortex and medulla of rat and bovine kidney. Total NAD+- and NADP+-dependent dehydrogenase activity was about equally distributed between the two renal regions of monkey, dog, rat, and swine. Bovine, rabbit, and cat had greater cortical than medullary dehydrogenase activity with ratios of 3, 5, and 10 respectively. The activities of NAD+- and NADP+-dependent dehydrogenase varied among the renal tissues.