Substituted tetraazaacenaphthylenes as potent CRF1 receptor antagonists for the treatment of depression and anxiety.

Two isomers of the hexahydro-tetraazaacenaphthylene templates (1 and 2) are presented as novel, potent, and selective corticotropin releasing factor-1 (CRF1) receptor antagonists. In this paper, we report the affinity and SAR of a series of compounds, as well as pharmacokinetic characterization of a chosen set. The anxiolitic activity of a selected example (2ba) in the rat pup vocalization model is also presented.

Copper efflux from murine microvascular cells requires expression of the menkes disease Cu-ATPase.

Previously, we showed that the transport of Cu by PC12 pheochromocytoma cells and C6 glioma cells correlated with the expression of a Cu-transporting ATPase (Atp7a) that has been linked to Menkes disease. Here, we show that cerebrovascular endothelial (CVE) cells that comprise the blood-brain barrier (BBB) also express the gene for the Cu-ATPase. By using reverse transcription-polymerase chain reaction (RT-PCR) and primers designed from mouse Atp7a cDNA, we amplified a 925-bp and a 760-bp cDNA fragment from two extreme regions of Atp7a mRNA from murine CVE cells; 777 bp of the 925-bp fragment and 677 bp of the 760-bp fragment had a 99.7 and 100% sequence homology, respectively, with mouse Atp7a cDNA. The 777-bp sequences covered the heavy metal binding (Hmb) domain and the 677-bp fragment coded for residues at the -COOH terminus of Atp7a. A functional analysis showed that Cu efflux was blocked by the sulfhydryl reagent p-chloromercuribenzoate (p-CMB), a potential inhibitor of Atp7a function. This study provides strong evidence that a Cu-ATPase in the BBB controls the penetration of Cu into the brain and that lesions to the Cu-ATPase in CVE cells are a primary cause of low brain Cu levels in Menkes disease.

Sequence of a Menkes-type Cu-transporting ATPase from rat C6 glioma cells: comparison of the rat protein with other mammalian Cu-transporting ATPases.

Rat Atp7a occupied a single open reading frame (274502) which coded for a protein of 1492 residues. Rat Atp7a was 98% and 95% identical to published sequences for the mouse and Chinese hamster, respectively, and 94% homologous to human ATP7A. Compared to ATP7A, the rat transcript coded for an additional alanine (A446) in the heavy metal binding (Hmb) domain and showed a 34 bp gap in the 3' UTR. Based on published sequence data, hydropathic profiles for rat, mouse, Chinese hamster, and human Cu-ATPases were practically identical with the exception of 8 additional amino acid residues between the 4th and 5th Hmb sites in the human. As deduced from amino acid sequence data, Hmb was predicted to have regions with helical and beta structures. All four species had five of the six metal binding sites centered within hydrophobic regions. The comparative analyses suggested that the Hmb region of the molecule could experience numerous amino acid substitutions with no apparent disruption to theATPase transport function whereas variations to theATPase domain would be more critical.

A Menkes P-type ATPase involved in copper homeostasis in the central nervous system of the rat.

We previously reported that copper efflux from C6 rat glioma cells was blocked by a brief exposure to sulfhydryl reagents p-chloromercuribenzoate (PCMB) and iodoacetamide as well as dicyclohexylcarbodiimide, suggesting the possible involvement of a Cu-transporting ATPase in the efflux mechanism. In this report, we show that copper efflux from PC12 cells, a neuron-like cell line established from rat adrenal pheochromocytoma, is also inhibited by PCMB exposure. Furthermore, we show that both C6 and PC12 cells express a homolog of the Menkes gene (MNK) as detected by RT-PCR with primers designed from a mouse cDNA and confirmed by sequence analysis of the amplified product. An expected 760-bp fragment representing the transduction and phosphorylation domains and a 925-bp fragment encoding the heavy metal-binding domain of Atp7a were amplified from a RNA extract of C6 and PC12 cells. Sequence data revealed that 690 bp of the 760-bp fragment from C6 cells were an identical match to a similar fragment from PC12 cells. Both fragments encoded a 229 amino-acid polypeptide that had a 98.7% sequence homology to mouse Atp7a. In addition, 880 bp from the 925-bp fragment of the two cell lines were identical and encoded a 293 amino-acid polypeptide with 94.5% sequence homology to mouse Atp7a. These data establish that a Menkes-type Cu-transporting ATPase is expressed in rat C6 and PC12 cells and strongly support the hypothesis that both neurons and glia are involved in maintaining Cu homeostasis in the central nervous system.

Effects of low-level lead exposure on hypothalamic hormones and serum progesterone levels in pregnant guinea pigs.

Pregnant guinea pigs were given a daily oral dose of 0, 5.5, or 11 mg lead (as lead acetate) per kg body weight during days 22-52 or 22-62 of gestation. Maternal serum progesterone levels were measured at the end of treatment, as well as hypothalamic levels of gonadotropin-releasing hormone (GnRH) and somatostatin (SRIF) in both the mothers and fetuses. Lead-treated dams had lower serum concentrations of progesterone at the end of treatment than did vehicle-treated animals. This effect was statistically significant for the higher Pb dose only. Hypothalamic levels of GnRH and SRIF were reduced in a dose-dependent manner by lead treatment in both dams and fetuses. The reduction of SRIF levels in 52-day-old fetuses was particularly severe (92%) in the 11 mg group. However, neither litter size nor body and organ weights, including placental weight, of the dams and fetuses was significantly affected. The relevance of these hormonal decreases is unknown, but could include decreased reproductive capacity in both the dams and fetuses that does not become apparent until later in the life-cycle.

Evaluation of methods for determining 6-hydroxydopamine cytotoxicity.

The toxic effects of 6-hydroxydopamine on the human neuroblastoma cell line SK-N-SH-SY5Y(SY5Y) and the Chinese hamster ovary (CHO) cell line were measured with five viability assays. Four of the assays (attachment efficiency, plating efficiency, amino acid incorporation into acid-precipitable proteins, and Trypan Blue dye exclusion) showed higher drug susceptibility in SY5Y cells than CHO cells. Only growth inhibition (proliferation index) gave results indicating greater sensitivity in CHO cells. Over a time span of 48 hr, injured cell populations lost vital functions in the following order: attachment ability, amino acid incorporation, proliferative capacity, and dye exclusion. Recovery of each of the functions occurred in sublethally injured populations. Monitoring the extinction and recovery of vital functions permitted the accurate determination of a drug concentration (30 micrograms/ml) selectively toxic for SY5Y cells. A strong correlation was noted between relative values for amino acid incorporation 3 hr after drug treatment, attachment efficiency at 24 hr, and dye exclusion at 24 and 48 hr. We concluded that Trypan Blue dye exclusion and amino acid incorporation were suitable methods for comparing the effects of cytotoxins on different cell lines, provided they were performed at the appropriate time after treatment with the toxin. The combined techniques yield both population and individual cell data, are simple to do, and are applicable to nondividing cell populations.