Thomas F. Murray

The Origin of the Synergistic Effect of Muramyl Dipeptide with Endotoxin and Peptidoglycan

Although the basis for the high mortality rate for patients with mixed bacterial infections is likely to be multifactorial, there is evidence for a synergistic effect of muramyldipeptide (MDP) with lipopolysaccharide (LPS) on the synthesis of proinflammatory cytokines by mononuclear phagocytes. In this study, co-incubation of human Mono Mac 6 cells with MDP and either LPS or peptidoglycan (PGN) resulted in an apparent synergistic effect on tumor necrosis factor-α (TNF-α) secretion. Although incubation of cells with MDP alone produced minimal TNF-α, it caused significant expression of TNF-α mRNA. These findings suggest that the majority of TNF-α mRNA induced by MDP alone is not translated into protein. Furthermore, simultaneous incubation of cells with MDP and either LPS or PGN resulted in TNF-α mRNA expression that approximated the sum of the amounts expressed in response to MDP, LPS, and PGN individually. These findings indicate that the apparent synergistic effect of MDP on TNF-α production induced by either LPS or PGN is due to removal of a block in translation of the mRNA expressed in response to MDP. In subsequent studies, the effects of MDP alone and its effect on the production of TNF-α by LPS and PGN were determined to be independent of CD14, Toll-like receptor 2, and Toll-like receptor 4. These findings indicate that MDP acts through receptor(s) other than those primarily responsible for transducing the effects of LPS and PGN. Successful treatment of patients having mixed bacterial infections is likely to require interventions that address the mechanisms involved in responses induced by a variety of bacterial cell wall components.

Characterization of a functional neuropeptide F receptor from Drosophila melanogaster

Potential receptors for Drosophila neuropeptide F (DmNPF) were identified in the genome database. One receptor (DmNPFR1) sequence resembled the Lymnaea NPY receptor, an invertebrate homolog of the vertebrate Y-receptor family. DmNPFR1 was cloned and tested for functionality in stably transfected mammalian CHO cells. In whole cell binding assays, DmNPF displaced 125I-NPF in a concentration-dependent manner (IC(50) = 65 nM). DmNPF inhibited forskolin-stimulated adenylyl cyclase activity similarly (IC(50) = 51 nM). Whole-mount in situ hybridization revealed that DmNPFR1 RNA is expressed in CNS and midgut of Drosophila larvae. DmNPFR1, a new invertebrate Y-receptor homolog, apparently is a functional receptor for DmNPF.

Behavioral, pharmacological, and molecular characterization of an amphibian cannabinoid receptor

Abstract : Investigation of cannabinoid pharmacology in a vertebrate with a phylogenetic history distinct from that of mammals may allow better understanding of the physiological significance of cannabinoid neurochemistry. Taricha granulosa, the roughskin newt, was used here to characterize an amphibian cannabinoid receptor. Behavioral experiments demonstrated that the cannabinoid agonist levonantradol inhibits both newt spontaneous locomotor activity and courtship clasping behavior. Inhibition of clasping was dose‐dependent and potent (IC50 = 1.2 μg per animal). Radioligand binding studies using [3H]CP‐55940 allowed identification of a specific binding site (KD = 6.5 nM, Bmax = 1,853 fmol/mg of protein) in brain membranes. Rank order of affinity of several ligands was consistent with that reported for mammalian species (KD, nM) : CP‐55940 (3.8) > levonantradol (13.0) > WIN55212‐2 (25.7) ▴ anandamide (1,665) >> anandamide + 100 μM phenylmethylsulfonyl fluoride (2,398). The cDNA encoding the newt CB1 cannabinoid receptor was cloned, and the corresponding mRNA of 5.9 kb was found to be highly expressed in brain. A nonclonal Chinese hamster ovary cell line stably expressing the newt CB1 cannabinoid receptor was prepared that allowed demonstration of cannabinoid‐mediated inhibition of adenylate cyclase (EC 4.6.1.1) activity. This inhibition was dose‐dependent and occurred at concentrations consistent with affinities determined through radioligand binding experiments. The behavioral, pharmacological, and molecular cloning results demonstrate that a CB1 cannabinoid receptor is expressed in the CNS of the roughskin newt. This amphibian CB1 is very similar in density, ligand binding affinity, ligand binding specificity, and amino acid sequence to mammalian CB1. The high degree of evolutionary conservation of cannabinoid signaling systems implies an important physiological role in vertebrate brain function.

Partial purification and biochemical characterization of a membrane glucocorticoid receptor from an amphibian brain.

A membrane receptor for corticosterone (mGR) in the brain of the roughskin newt (Taricha granulosa) has been previously identified. This manuscript reports the evaluation of several chromatographic resins for enrichment of the newt mGR solubilized from neuronal membranes. A protein with an apparent molecular weight of 63 kDa was purified to near homogeneity following sequential purification using ammonium sulfate fractionation, wheat germ agglutinin (WGA)-agarose chromatography, hydroxylapatite chromatography, and an immobilized ligand affinity resin (Corticosterone-Sepharose). Other studies employed a novel protein differential display strategy and a photoaffinity labeling strategy to visualize candidate receptor proteins following SDS-PAGE. Both of these techniques also identified a 63 kDa protein, agreeing with the estimation of molecular weight from the purification data. Furthermore, the use of 2D SDS-PAGE following the photolabeling procedure showed the candidate 63 kDa protein to have a pI of approximately 5.0. Taken together these data suggest that the newt mGR is an acidic glycoprotein with an apparent molecular weight of 63 kDa. Because these characteristics of newt mGR are inconsistent with the characteristics of intracellular glucocorticoid receptors, these two receptor proteins are apparently distinct.

Domoic acid neurotoxicity in cultured cerebellar granule neurons is controlled preferentially by the NMDA receptor Ca2+ influx pathway

We have monitored real-time alterations in [Ca(2+)](i) in fluo-3-loaded cerebellar granule neurons exposed to domoate, and ascertained the influence of pharmacological blockers of various Ca(2+) entry pathways on intracellular Ca(2+) accumulation, excitatory amino acid (EAA) release and neuronal death. Domoate produced a rapid and concentration-dependent increase in [Ca(2+)](i), the magnitude of which correlated closely with the severity of neuron loss. The increase in [Ca(2+)](i) was derived from activation of NMDA receptors, L-type voltage-sensitive calcium channels (VSCC) and the reversed mode of operation of the Na(+)/Ca(2+) exchanger. When the level of neuroprotection conferred by pharmacological manipulation of these calcium entry pathways was regressed with the corresponding reductions in [Ca(2+)](i) load, it was observed that neuronal vulnerability is controlled preferentially by NMDA receptors. This observation is consistent with our previous study of brevetoxin-induced autocrine excitotoxicity and with the source specificity hypothesis of others [J. Neurochem. 71 (1998) 2349], which suggests that elevation of [Ca(2+)](i) in the vicinity of the NMDA receptor ion channel activates processes leading to neuronal death.

Cannabinoid agonist, CP 55,940, facilitates intake of palatable foods when injected into the hindbrain.

Cannabinoids have been shown to influence food intake, and until recently, the neural pathways mediating these effects have remained obscure. It has been previously shown that intracerebroventricular injection of delta-9-tetrahydrocannabinol (Delta(9)-THC) causes increased consumption of palatable foods in rats, and we postulated the involvement of the hindbrain in this cannabinoid-induced food intake. Cannulated rats (both female and male groups) trained to consume sweetened condensed milk received either lateral or fourth ventricle injections of CP 55,940 and were presented with sweetened condensed milk 15 min after injection. Rats were injected over a range of doses between 100 pg and 10 microg per rat. Milk intake was recorded for a total of 3 h. Lateral ventricle injection of CP 55,940 increased milk intake at doses in the microgram range. However, CP 55,940 was effective in increasing food intake at nanogram doses when injected into the fourth ventricle. Finally, male rats appeared to be more sensitive to CP 55,940 than female rats inasmuch as milk consumption was increased at the 1 ng dose in male rats, whereas only the 10 ng dose was effective in females. These results indicate that CP 55,940 may act in the hindbrain to influence feeding behavior in rats.

Antillatoxin and kalkitoxin, ichthyotoxins from the tropical cyanobacterium Lyngbya majuscula, induce distinct temporal patterns of NMDA receptor-mediated neurotoxicity

Curacin-A, antillatoxin and kalkitoxin, natural products from the marine cyanobacterium Lyngbya majuscula, were tested for neurotoxicity in primary cultures of rat cerebellar granule neurons. Curacin-A was non-toxic, whereas antillatoxin and kalkitoxin produced concentration-dependent cytotoxicity with LC50 values of 20.1+/-6.4 and 3.86+/-1.91 nM, respectively. Antillatoxin neurotoxicity was produced acutely, whereas kalkitoxin caused a delayed neurotoxic response. The cytotoxicity produced by both antillatoxin and kalkitoxin was prevented by the non-competitive NMDA receptor antagonists dextrorphan and MK-801.

Brevetoxin‐Induced Autocrine Excitotoxicity Is Associated with Manifold Routes of Ca2+ Influx

Abstract: Real‐time alterations in intracellular Ca2+ ([Ca2+]i) were monitored in fluo‐3‐loaded cerebellar granule neurons (CGNs) exposed to the brevetoxin PbTx‐1. [Ca2+]i was measured using a fluorescent plate reader (FLIPR), which measures simultaneously the mean intracellular Ca2+ change in a population of cultured cells in each well of a 96‐well plate. PbTx‐1 produced rapid and concentration‐dependent increases in neuronal [Ca2+]i with a potency nearly identical to that determined previously for PbTx‐1‐induced neurotoxicity. The NMDA receptor antagonists MK‐801, dextrorphan, and D(‐)‐2‐amino‐5‐phosphonopentanoic acid, and tetanus toxin, an inhibitor of Ca2+‐dependent exocytotic neurotransmitter release, effected significant reductions in both the integrated fluo‐3 fluorescence response and excitatory amino acid release and protected CGNs against PbTx‐1 neurotoxicity. The L‐type Ca2+ channel antagonist nifedipine produced a modest reduction in the fluo‐3 response but reduced substantially the plateau phase of the PbTx‐1 increment in [Ca2+]i when combined with MK‐801. When nifedipine and MK‐801 were combined with the Na+/Ca2+ exchanger (reversed mode) inhibitor KB‐R7943, the PbTx‐1 increment in [Ca2+]i was nearly completely attenuated. These data show that Ca2+ entry into PbTx‐1‐exposed CGNs occurs through three primary routes: NMDA receptor ion channels, L‐type Ca2+ channels, and reversal of the Na+/Ca2+ exchanger. There was a close correlation between reduction of the integrated fluo‐3 fluorescence response and the level of neuroprotection afforded by blockers of each Ca2+ entry pathway; however, simultaneous blockade of L‐type Ca2+ channels and the Na+/Ca2+ exchanger, although reducing the integrated [Ca2+]i response to a level below that provided by NMDA receptor blockade alone, failed to completely attenuate PbTx‐1 neurotoxicity. This finding suggests that in addition to total [Ca2+]i load, neuronal vulnerability is governed principally by the NMDA receptor Ca2+ influx pathway.

Orphanin FQ has an inhibitory effect on the guinea pig ileum and the mouse vas deferens.

The activity of the recently isolated endogenous opioid-like peptide orphanin FQ (OFQ) was measured in two classical bioassays of opioid action. OFQ potently and concentration-dependently suppressed the electrically stimulated contractions of the guinea pig ileum (GPI) and the mouse vas deferens (MVD) with EC50 values of 1.82 +/- 0.16 and 2.97 +/- 0.01 nM, and Emax values of 56 +/- 3% and 96 +/- 4%, respectively. This effect of OFQ, in both the GPI and MVD, was insensitive to the opioid receptor antagonist naloxone. OFQ competed with [3H]diprenorphine binding to mu-, delta- or kappa-opioid receptors stably expressed in Chinese hamster ovary cell lines with IC50 values of 2.1 +/- 0.4, 2.2 +/- 0.3, 0.75 +/- 0.3 microM, respectively. Low affinity for the classical opioid receptors together with the inability of naloxone to antagonize its effect suggest that the inhibitory action of OFQ is mediated via a distinct OFQ receptor in the GPI and MVD. Consequently, the MVD could serve as a valuable bioassay of potential OFQ receptor antagonists.

Cyclopentyladenosine‐Induced Homologous Down‐Regulation of A1 Adenosine Receptors (A1AR) in Intact Neurons Is Accompanied by Receptor Sequestration but Not a Reduction in A1AR mRNA Expression or G Protein α‐Subunit Content

Abstract: We showed previously that exposure of cerebellar granule cells to the A1 adenosine receptor (A1AR)‐selective agonist, cyclopentyladenosine, decreases A1AR density and G protein coupling corresponding to blunted agonist‐induced adenylyl cyclase (EC 4.6.1.1) inhibition. We have now determined that A1AR‐mediated adenylyl cyclase inhibition was desensitized in a homologous manner. Carbachol‐ and baclofen‐induced inhibition of adenylyl cyclase was unaffected by 48‐h exposure to 10 µM cyclopentyladenosine. Expression of G protein α‐subunits was not affected dramatically by agonist exposure. The fraction of sequestered A1AR was increased significantly at 4, 24, and 48 h of cyclopentyladenosine exposure (35, 57, and 81% increase over control, respectively). The time course of agonist‐induced A1AR sequestration was slower than that reported for other G protein‐coupled receptors. Incubation with the adenosine receptor antagonist, 8‐p‐sulfophenyltheophylline or adenosine deaminase did not alter sequestration significantly. Neither steady‐state A1AR mRNA levels nor transcript stability was affected by 48‐h agonist exposure. We determined that A1AR half‐life in cerebellar granule cells is 20.9 h, which is considerably longer than that reported for several other G protein‐coupled receptors. The slow time course of A1AR sequestration and the stability of the corresponding mRNA may be a reflection of the tonic inhibitory tone exerted by adenosine in brain.