Joseph P. Arena

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 Drosophila melanogaster Glutamate-gated Chloride Channel Sensitive to the Antiparasitic Agent Avermectin

Glutamate-gated chloride channels, members of the ligand-gated ion channel superfamily, have been shown in nematodes and in insects to be a target of the antiparasitic agent avermectin. Two subunits of the Caenorhabditis elegans glutamate-gated chloride channel have been cloned: GluCl-α and GluCl-β. We report the cloning of a Drosophila melanogaster glutamate-gated chloride channel, DrosGluCl-α, which shares 48% amino acid and 60% nucleotide identity with the C. elegans GluCl channels. Expression of DrosGluCl-α in Xenopus oocytes produces a homomeric chloride channel that is gated by both glutamate and avermectin. The DrosGluCl-α channel has several unique characteristics not observed in C. elegans GluCl: dual gating by avermectin and glutamate, a rapidly desensitizing glutamate response, and a lack of potentiation of the glutamate response by avermectin. The pharmacological data support the hypothesis that the DrosGluCl-α channel represents the arthropod H-receptor and an important target for the avermectin class of insecticides.

The mechanism of action of avermectins in Caenorhabditis elegans : correlation between activation of glutamate-sensitive chloride current, membrane binding, and biological activity

Xenopus laevis oocytes were injected with mRNA isolated from the free-living nematode Caenorhabditis elegans and the activation and potentiation of a glutamate-sensitive chloride current by a series of avermectin analogs and milbemycin D were determined. There was a strong correlation between the EC50 value determined for current activation in oocytes, the LD95 value for nematocidal activity, and also for the Ki value determined in a [3H]ivermectin competition binding assay. Four of the analogs were tested for potentiation of glutamate-sensitive current and the rank order for potentiation correlated with the EC50 for direct activation of current. We conclude that avermectins and milbemycins mediate their nematocidal effects on C. elegans via an interaction with a common receptor molecule, glutamate-gated chloride channels.

Expression of a glutamate-activated chloride current in Xenopus oocytes injected with Caenorhabditis elegans RNA: evidence for modulation by avermectin

Membrane currents were recorded from Xenopus laevis oocytes injected with C. elegans poly(A)+ RNA. In such oocytes glutamate activated an inward membrane current that desensitized in the continued presence of glutamate. Glutamate-receptor agonists quisqualate, kainate, and N-methyl-D-aspartate were inactive. The reversal potential of the glutamate-sensitive current was -22 mV, and exhibited a strong dependence on external chloride with a 48 mV change for a 10-fold change in chloride. The chloride channel blockers flufenamate and picrotoxin inhibited the glutamate-sensitive current. Ibotenate, a structural analog of glutamate, also activated a picrotoxin-sensitive chloride current. Ibotenate was inactive when current was partially desensitized with glutamate, and the responses to low concentrations of glutamate and ibotenate were additive. The anthelmintic/insecticide compound avermectin directly activated the glutamate-sensitive current. In addition, avermectin increased the response to submaximal concentrations of glutamate, shifted the glutamate concentration-response curve to lower concentrations, and slowed the desensitization of glutamate-sensitive current. We propose that the glutamate-sensitive chloride current and the avermectin-sensitive chloride current are mediated via the same channel.

Evolutionary Relationship of the Ligand-Gated Ion Channels and the Avermectin-Sensitive, Glutamate-Gated Chloride Channels

Abstract. Two cDNAs, GluClα and GluClβ, encoding glutamate-gated chloride channel subunits that represent targets of the avermectin class of antiparasitic compounds, have recently been cloned from Caenorhabditis elegans (Cully et al., Nature, 371, 707–711, 1994). Expression studies in Xenopus oocytes showed that GluClα and GluClβ have pharmacological profiles distinct from the glutamate-gated cation channels as well as the γ-aminobutyric acid (GABA)- and glycine-gated chloride channels. Establishing the evolutionary relationship of related proteins can clarify properties and lead to predictions about their structure and function. We have cloned and determined the nucleotide sequence of the GluClα and GluClβ genes. In an attempt to understand the evolutionary relationship of these channels with the members of the ligand-gated ion channel superfamily, we have performed gene structure comparisons and phylogenetic analyses of their nucleotide and predicted amino acid sequences. Gene structure comparisons reveal the presence of several intron positions that are not found in the ligand-gated ion channel superfamily, outlining their distinct evolutionary position. Phylogenetic analyses indicate that GluClα and GluClβ form a monophyletic subbranch in the ligand-gated ion channel superfamily and are related to vertebrate glycine channels/receptors. Glutamate-gated chloride channels, with electrophysiological properties similar to GluClα and GluClβ, have been described in insects and crustaceans, suggesting that the glutamate-gated chloride channel family may be conserved in other invertebrate species. The gene structure and phylogenetic analyses in combination with the distinct pharmacological properties demonstrate that GluClα and GluClβ belong to a discrete ligand-gated ion channel family that may represent genes orthologous to the vertebrate glycine channels.

An Amino Acid Substitution in the Pore Region of a Glutamate-gated Chloride Channel Enables the Coupling of Ligand Binding to Channel Gating

Many of the subunits of ligand-gated ion channels respond poorly, if at all, when expressed as homomeric channels in Xenopus oocytes. This lack of a ligand response has been thought to result from poor surface expression, poor assembly, or lack of an agonist binding domain. The Caenorhabditis elegans glutamate-gated chloride channel subunit GluClβ responds to glutamate as a homomeric channel while the GluClα subunit is insensitive. A chimera between GluClα and GluClβ was used to suggest that major determinants for glutamate binding are present on the GluClα N terminus. Amino acid substitutions in the presumed pore of GluClα conferred direct glutamate gating indicating that GluClα is deficient in coupling of ligand binding to channel gating. Heteromeric channels of GluClα+β may differ from the prototypic muscle nicotinic acetylcholine receptor in that they have the potential to bind ligand to all of the subunits forming the channel.

Identification of neuron-specific ivermectin binding sites in Drosophila melanogaster and Schistocerca americana.

High affinity avermectin binding sites have been identified and partially characterized in membranes from two insect species, Drosophila melanogaster and the locus Schistocerca americana. There is a 10-fold increase in the density of ivermectin binding sites associated with membranes isolated from Drosophila heads (a neuronally enriched tissue source) compared to the bodies (Bmax values were 3.5 and 0.22 pmol/mg, respectively) with only a small difference in the apparent dissociation constant (Kd values of 0.20 and 0.34 nM for heads and bodies, respectively). Membranes prepared from metathoracic ganglia of the locust, Schistocerca americana, were highly enriched in high affinity avermectin binding sites (Kd = 0.2 nM and Bmax = 42 pmol/mg). Using an [125I]arylazido-avermectin analog as a photoaffinity probe, a 45 kDa protein was identified in both the Drosophila head and body tissue preparations. A 45 kDa protein was also specifically labeled with [125I]azido-avermectin in the locust neuronal membranes.

Mechanism of Action of GHRP-6 and Nonpeptidyl Growth Hormone Secretagogues

Growth hormone (GH) secretion from the pituitary gland is regulated by the hypothalamic peptide hormones growth hormone releasing hormone (GHRH) and somatotropin release inhibiting factor (SRIF) (Scheme 11.1). The factor controlling the episodic nature of GH release is unknown but its effects are probably mediated by feedback loops involving the positive effector GHRH and the negative regulator SRIF (1). In 1984, Bowers and Momany and coworkers (2, 3) described the synthesis and properties of a series of small peptide GH secretagogues that were based on the structure of Leu and Met enkephalins. Growth hormone releasing peptide (GHRP)- 6 was the most potent of these peptides and was subsequently shown to be active in man (4, 5). Because of the limited oral bioavailability of peptides we sought a class of GH secretagogues more amenable to chemical modification so that oral bioavailability and pharmacokinetic properties could be optimized. Implicit in establishing assays to identify new small molecules was an understanding of the mechanisms regulating GH release from the anterior pituitary gland. Based on its size, GHRP-6 was considered a potential template for a small molecule peptide mimetic. Our approach was based on screening selected structures in functional and mechanism based assays. Following identification of a benzolactam lead structure, L-692,429 was synthesized and used as a prototype to investigate specificity and efficacy in clinically relevant target populations (6–11).

Detection of intracellular calcium elevations in Xenopus laevis oocytes: aequorin luminescence versus electrophysiology

Detection of receptor expression in Xenopus oocytes often relies upon functional coupling to second messengers such as Ca2+ or cyclic adenosine monophosphate. To detect intracellular Ca2+, electrophysiological measurement of the endogenous Ca(2+)-activated chloride current (ICl(Ca)) is often used (Dascal, 1987). An alternative utilizes the Ca2+ sensing, bioluminescent protein aequorin (Parker and Miledi(1986) Proc. R. Soc. Lond. B, 228: 307-315; Giladi and Spindel (1991) BioTechniques, 10: 744-747). In the present study the sensitivities of aequorin and electrophysiology for detecting receptor-mediated Ca2+ transients were compared. Assays were performed on the same batches of oocytes using either animal serum or ligands of exogenous receptors to generate inositol 1,4,5-trisphosphate (InsP3) and ultimately elevate intracellular Ca2+. Signal amplitudes were controlled by titrating the concentration of animal serum, or titrating the amount of receptor mRNA injected. Both assays detected signals with high concentrations of animal serum, or with high receptor density. However, aequorin signals were not detected in experiments with average ICl(Ca) current amplitudes below 200 nA. To further evaluate the differences between these two techniques, membrane current and bioluminescence were measured simultaneously. Results of these studies suggest that the signals differ due to the spatial distribution of aequorin, the chloride channels, and the calcium release sites.

Avermectins: Idiosyncratic Toxicity in a Subpopulation of Collie Dogs

Avermectins are a family of macrocyclic lactones isolated from Streptomyces avermitilis 1,2 which have potent anthelmintic3 and insecticidal activity.4,5 The name ‘avermectin’ reflects their efficacy against worms or ‘vermes’ as well as their activity against ectoparasites. The avermectins are composed of a 16-membered ring and are distinguished from other macrocyclic antibiotics by their characteristic spiroketal, hexahydrobenzofuran unit and the disaccharide component at the 13-position (Fig. 1). The naturally occurring avermectins are separated into four major components (Ala, A2a, Bla and B2a), of which the B series are generally more biologically active. Abamectin is the non-proprietary name for a marketed miticide and insecticide composed of avermectin Bla (>80%) and avermectin Blb ( 80% 22,23-dihydroavermectin Bla and <20% 22,23-dihydroavermectin Blb) widely used as an endectocide for farm animals. Other avermectin analogs have been reported to have good efficacy against various insect species.6 Milbemycins are natural products closely related to the avermectins, differing only in their lack of a C-13 disaccharide substituent.