Inhibition of TNF-alpha expression by adenosine: role of A3 adenosine receptors.

Adenosine agonists inhibit TNF-alpha production in macrophage and monocytes, but the mechanism is unknown. Therefore, we studied the human macrophage cell line U937 to determine the adenosine receptor subtypes responsible and the intracellular signaling mechanisms involved. The A1/A3 agonist N6-(4-amino-3-iodobenzyl)adenosine (I-ABA) decreased LPS-stimulated TNF-alpha protein production by 79 +/- 5% (p = 0.003). The mechanism was pretranslational, as adenosine receptor stimulation caused a marked decrease in TNF-alpha mRNA. IL-1 beta, IL-6, and IL-8 mRNA were not changed by adenosine agonists. The rank order of agonists as TNF-alpha inhibitors suggested that the A3 receptor might be involved (N6-(3-iodobenzyl)-9-[5-(methylcarbamoyl)-beta-D-ribofuranosyl] adenosine > 2-chloroadenosine > or = I-ABA > N6 benzyl 5'-N-ethylcarboxamidoadenosine (NECA) > NECA > CGS21680 > N6-cyclohexyladenosine), and this was supported by the fact that a mixed A1/A3 antagonist (xanthine amine congener) reversed the effect, whereas A1-specific (1,3-dipropyl-8-cyclopentylxanthine) and A2-specific (3,7-dimethyl-1-propargylxanthine) antagonists did not. Receptor signaling did not involve cAMP or protein kinase A, nor did it alter the activation and binding characteristics of the transcription factor NF-kappa B. However, the composition of the AP-1 transcription complex was altered by I-ABA. These data suggest that stimulation of the A3 adenosine receptor can alter the cytokine milieu by decreasing TNF-alpha. Adenosine agonists or adenosine regulating agents have potential therapeutic uses in acute and chronic inflammatory diseases.

cDNA cloning and characterization of A3i, an alternatively spliced rat A3 adenosine receptor variant.

A cDNA encoding variant form of the A3 adenosine (Ado) receptor was isolated from rat by reverse transcription of brain mRNA followed by PCR. The full-length receptor (A3i) cDNA encodes 337 amino acids and shares complete sequence identity with the rat A3 Ado receptor, except for the presence of a seventeen amino acid insert located in the second intracellular domain. In contrast to the rat A3 receptor, stable expression of A3i in CHO cells resulted in poor coupling to Gi proteins. Analysis of receptor transcripts by RT-PCR suggests that the A3 Ado receptor mRNAs are products of alternative splicing. Sequence analysis of A3 genomic DNA identified a 1.7 kb intron that is likely alternatively spliced to produce the A3 and A3i receptors.

The skeletal muscle transverse tubular Mg-ATPase: identity with Mg-ATPases of smooth muscle and brain.

Purified chicken skeletal muscle transverse tubule (T-tubule, TT) membrane preparations contain a very active Ca- or Mg-ATPase (EC 3.6.1.3) previously thought to be a T-system-specific marker enzyme. The function of the Mg-ATPase has not yet been determined although its prominent activity and concentration in junctional complexes supports a possible role in the excitation-contraction cycle. An essential component of the Mg-ATPase has been identified as a M(r) 85,000 glycoprotein (85k-GP). Polyclonal antibodies raised against the TT 85k-GP were specific and exhibited no cross-reactivity with other skeletal muscle proteins on immunoblots. Using this anti-85k-glycoprotein IgG, we have explored other chicken tissues to determine the tissue distribution of the 85k-GP. Antibody reactive polypeptides of M(r) 85,000 were found in gizzard smooth muscle, brain, heart, spleen, and lung tissue. The brain and smooth muscle membrane proteins were further purified and characterized for 85k-GP-associated Mg-ATPase activity. The brain and smooth muscle enzymes exhibited properties indistinguishable from the skeletal muscle TT-specific Mg-ATPase with regard to a series of activators and inhibitors, amino terminal amino acid sequences, and the effects of deglycosylation. The enzyme in all three tissues was inhibited by the diacylglycerol kinase inhibitor R 59022. Identification of the TT Mg-ATPase in gizzard smooth muscle has allowed the investigation of the Mg-ATPase membrane topology using isolated whole smooth muscle cells. The data support an ecto-orientation for the smooth muscle cell enzyme. Although the orientations of the brain and skeletal muscle enzymes have not been conclusively determined, the nearly identical properties of all three enzymes argues for an ecto-orientation of the active sites of these enzymes as well. The responsiveness of the three enzymes to regulatory lipids suggests that the ecto-Mg-ATPase may serve as a master switch controlling extracellular ATP concentrations and ligand accessibility to P1- and P2-purinoceptors. It is also proposed that the ecto-MgATPase may regulate ATP accessibility to ectoprotein kinases in a variety of tissues, and, in brain, the ecto-MgATPase may modulate the neurotransmitter role of ATP.

The Rhino-Probe nasal curette for detecting respiratory syncytial virus in children.

During two outbreaks of respiratory syncytial virus (RSV) infection, 68 children with acute respiratory illnesses were cultured for RSV using a Rhino-Probe (RP) nasal curette and either a nasopharyngeal (NP) swab or a nasal wash (NW). In the first outbreak isolations of RSV by the RP nasal curette and NP swab methods were compared. RSV was cultured from 25 of 42 (60%) subjects using the RP nasal curette and from 20 of 42 (48%) subjects using the NP swab. In the second outbreak the RP nasal curette and the NW collection techniques were compared. RSV was isolated from 15 of 26 (58%) children evaluated. RSV was cultured from 14 of 15 (93%) patients by RP and 13 of 15 (87%) when using NW. In the group of culture-positive subjects, the TESTPACK RSV rapid antigen test was positive in 10 of 15 (67%) using the RP and in 6 of 15 (40%) using the NW. Like the NP swab the RP nasal curette was simple, noninvasive and relatively inexpensive, yet it was as sensitive as the NW for detection of RSV.