Salivary proteomics in bisphosphonate-related osteonecrosis of the jaw.

OBJECTIVE: The objective of this study was to identify differentially expressed salivary proteins in bisphosphonate-related osteonecrosis of the jaw (BRONJ) patients that could serve as biomarkers for BRONJ diagnosis. SUBJECTS AND METHODS: Whole saliva obtained from 20 BRONJ patients and 20 controls were pooled within groups. The samples were analyzed using iTRAQ-labeled two-dimensional liquid chromatography-tandem mass spectrometry. RESULTS: Overall, 1340 proteins were identified. Of these, biomarker candidates were selected based on P-value (<0.001), changes in protein expression (≥1.5-fold increase or decrease), and unique peptides identified (≥2). Three comparisons made between BRONJ and control patients identified 200 proteins to be differentially expressed in BRONJ patients. A majority of these proteins were predicted to have a role in drug metabolism and immunological and dermatological diseases. Of all the differentially expressed proteins, we selected metalloproteinase-9 and desmoplakin for further validation. Immunoassays confirmed increased expression of metalloproteinase-9 in individual saliva (P = 0.048) and serum samples (P = 0.05) of BRONJ patients. Desmoplakin was undetectable in saliva. However, desmoplakin levels tended to be lower in BRONJ serum than controls (P = 0.157). CONCLUSIONS: Multiple pathological reactions are involved in BRONJ development. One or more proteins identified by this study may prove to be useful biomarkers for BRONJ diagnosis. The role of metalloproteinase-9 and desmoplakin in BRONJ requires further investigation.

The clinical significance of intrapulmonary vascular dilations in liver transplant candidates.

Intrapulmonary vascular dilations (IPVD) are common in patients with cirrhosis, but the majority do not have hepatopulmonary syndrome (HPS). The clinical significance of IPVD is unknown. Our aim was to determine the clinical impact due to the entire spectrum of IPVD in liver transplant (LT) candidates. A total of 122 evaluees for LT underwent contrast transthoracic echocardiography (cTTE). The degree of shunting was graded 1-3 (severe). HPS was defined as PaO(2) < 70 mmHg in the presence of IPVD and exclusion of other causes of hypoxemia. IPVD were detected in 57/122 (47%), and of these HPS was found in 5. IPVD were associated with higher Alveolar-arterial (A-a) gradients, with the highest occurring in patients with HPS (IPVD vs. no IPVD: p = 0.003; HPS vs. no IPVD: p = 0.004). All patients with HPS had grade 3 shunting, and had significantly widened A-a gradient and lower PaO(2) compared with grade 1 or 2 IPVDs. Presence of IPVD did not affect survival measured from evaluation or after LT. Other clinical outcomes were also similar among patients with and without IPVD. IPVD are common among LT candidates. HPS is unlikely in presence of only mild to moderate shunting. Clinical outcomes are similar among patients with and without IPVD.

Human temporomandibular joint and myofascial pain biochemical profiles: a case-control study.

Neurobiological mechanisms of human musculoskeletal pain are poorly understood. This case-control study tested the hypothesis that biomarkers within temporomandibular muscle and joint disorders (TMJD) subjects' masseter muscles or temporomandibular joint (TMJ) synovial fluid correlate with plasma biomarker concentrations. Fifty subjects were recruited and categorized into TMJD cases (n=23) and pain-free controls (n=27) at the University of Minnesota School of Dentistry. Prior to specimen collection, pain intensity and pressure pain threshold masseter muscles and the TMJs were assessed. We collected venous blood; biopsied masseter muscle; and sampled TMJ synovial fluid on the subjects' side of maximum pain intensity. We assayed these tissues for the presence of nerve growth factor (NGF), bradykinin (BK), leukotreine B(4) (LTB(4) ) and prostaglandin E(2) (PGE(2) ), F(2) -isoprostane (F(2) I) and substance P (SP). The data was analyzed using Spearman Correlation Coefficients. We found that only plasma concentrations of bradykinin statistically correlated with synovial fluid concentrations (ρ=-0·48, P=0·005), but no association was found between pain intensities. The data suggests that biomarkers used to assess TMJD need to be acquired in a site-specific manner. We also discovered that F(2) I concentrations were associated with muscle pain intensity and muscle pressure pain threshold (PTT) (ß=0·4, 95%CI: 0·03-0·8) and joint PPT (ß=0·4, 95%CI: 0·07-0·8) suggesting that muscle oxidative stress is involved in myofascial pain and that F(2) -I may be a biomarker for myofascial pain.

Arthrosopic knot pushers. Does one size fit all?

Little is known about how knot-pusher design affects arthroscopic knot tying. In our practice, we observed the knot-pusher riding onto the arthroscopic knot at the point of maximum tightening. This can lead to snagging of the knot, which is undesirable as it may lead to loosening of, or damage to the knot. The aim of this study is to determine the optimum size of a knot-pusher to efficiently push the knot without overriding or snagging it. We used an apparatus to model arthropcopic knot tying. Ten examples each of the Duncan loop were tied under controlled conditions of load using one polydioxanone (PDS) monofilament absorbable suture (Ethicon, Livingston, UK), two Ethibond, two Fibrewire and two Panacryl. The loop of the knot was then secured and a 50 N force applied to tension the knot. The suture diameter was measured. Then the knot diameter was measured in two planes using an analogue micrometer. The internal diameter of a Mitek knot-pusher was measured. The mean maximum diameter for each knot was respectively PDS, 2.061 +/- 0.13 mm; Panacryl, 1.907 +/- 0.14 mm; Ethibond, 1.717 +/- 0.16 mm and Fibrewire, 1.654 +/- 0.14 mm. There were significant differences in size between knots tied with different materials except between Ethibond and Fibrewire where the difference was not significant. For each set of knots the smallest maximum knot diameter observed was identified. This was respectively PDS, 1.92 mm; Ethibond, 1.476 mm; Fibrewire, 1.488 mm and Panacryl, 1.715 mm. The internal diameter of a Mitek knot-pusher was found to be 1.95 mm. The current Mitek knot-pusher appears to be well suited to one PDS and two Panacryl. It appears less ideal for two Ethibond and two Fibrewire. One knot-pusher does not fit all and we suggest that different knot-pushers be used for different suture materials.

Accelerated degradation of FADD and procaspase 8 in cells expressing human papilloma virus 16 E6 impairs TRAIL-mediated apoptosis.

Viruses have developed sophisticated strategies to evade host defenses and facilitate the production and spread of progeny. In this study, we show that transfection of the human papillomavirus (HPV) 16 E6 oncogene into HCT116 cells provides protection from tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-mediated apoptosis. Additionally, we demonstrate that the protection provided by E6 is dose-dependent because higher levels of E6 provide greater protection. The mechanism underlying this protection involves a rapid reduction in the protein levels of both Fas-associated death domain (FADD) and procaspase 8, which results in suppression of the activation of caspases 8, 3 and 2. Interestingly, E6 does not interfere with the mitochondrial apoptotic pathway even though HCT116 cells have been classified as type II cells with regard to TRAIL signaling. These findings demonstrate that E6 has a more generalized effect on signaling by death ligands than was previously thought and support the notion that E6 can utilize p53-independent mechanisms to modulate cell survival.

The human papillomavirus 16 E6 protein can either protect or further sensitize cells to TNF: effect of dose.

High-risk strains of human papillomavirus, including HPV 16, cause human cervical carcinomas, due in part to the activity of their E6 oncogene. E6 interacts with a number of cellular proteins involved in host-initiated apoptotic responses. Paradoxically, literature reports show that E6 can both protect cells from and sensitize cells to tumor necrosis factor (TNF). To examine this apparent contradiction, E6 was transfected into U2OS cells and stable clones were treated with TNF. Intriguingly, clones with a high level of E6 expression displayed an increased sensitivity to TNF by undergoing apoptosis, while those with low expression were resistant. Furthermore, TNF treatment of cells in which the expression of E6 was regulated by the addition of doxycycline demonstrated clearly that while low levels of E6 protect cells from TNF, high levels sensitize cells. Together, these results demonstrate that virus-host interactions can be complex and that both quantitative and qualitative aspects are important in determining outcome.

Novel inositol containing phospholipids and phosphates: their synthesis and possible new roles in cellular signalling.

Details of the widely employed PtdIns(4,5)P2 hydrolysis receptor-stimulated signalling pathway continue to be elucidated rapidly. However, it has recently become apparent that numerous other inositol lipids and phosphates are widespread and are likely to have important cellular functions. In this review, we focus particularly on three rapidly progressing areas: the synthesis and possible functions of 3-phosphorylated inositol lipids, particularly phosphatidylinositol 3,4,5-trisphosphate; the roles of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate in coordinating intracellular Ca2+ mobilization and Ca2+ influx in stimulated cells; and the metabolism and possible functions of other inositol polyphosphates and of inositol polyphosphate pyrophosphates.

Flavonol and imidazole derivatives block HPV16 E6 activities and reactivate apoptotic pathways in HPV⁺ cells.

High-risk human papillomaviruses (HR-HPVs) cause nearly all cases of cervical cancer, as well as approximately 30% of head and neck cancers. HPV 16 E6, one of two major viral oncogenes, protects cells from apoptosis by binding to and accelerating the degradation of several proteins important in apoptotic signaling, including caspase 8 and p53. We proposed that blocking the interactions between HPV E6 and its partners using small molecules had the potential to re-sensitize HPV(+) cells to apoptosis. To test this idea, we screened libraries of small molecules for candidates that could block E6/caspase 8 binding and identified several candidates from different chemical classes. We tested hits for dose-dependency and specificity in vitro and for toxicity in a cell-based assay and then used this information to select the two best candidates for further testing: myricetin, a flavonol, and spinacine, an imidazole amino-acid derivative of histidine. Both compounds clearly inhibited the ability of E6 to bind in vitro to both caspase 8 and E6AP, the protein that mediates p53 degradation. In addition, both compounds were able to increase the level of caspase 8 and p53 in SiHa cervical cancer cells, resulting in an increase of caspase 3/7 activity. Finally, both myricetin and spinacine sensitized HPV(+) cervical and oral cancer cells, but not HPV(-) cervical and oral cancer cells, to apoptosis induced by the cancer-specific ligand TRAIL, as well as the chemotherapeutic agents doxorubicin and cisplatin. New therapies based on this work may improve treatment for HPV(+) cancer patients.

Inhibition of porcine brain inositol 1,3,4-trisphosphate kinase by inositol polyphosphates, other polyol phosphates, polyanions and polycations.

We have partially purified an enzyme activity that phosphorylates inositol 1,3,4-trisphosphate from porcine brain, rat liver and bovine testis by FPLC chromatography on Q-Sepharose anion-exchange resin and Heparin-agarose. The products of this reaction were inositol 1,3,4,6-tetrakisphosphate and inositol 1,3,4,5-tetrakisphosphate. The same enzyme appears to be responsible for both 6-kinase and 5-kinase activities against inositol 1,3,4-trisphosphate (the 6-kinase: 5-kinase activity ratio is approximately 4 to 1), has a pH optimum of approximately 6.8 and requires Mg2+ for activity. The Km values of the enzyme for inositol 1,3,4-trisphosphate and ATP were approximately 0.5 microM and approximately 100 microM, respectively. Inositol 3,4,5,6-tetrakisphosphate, inositol 1,3,4,6-tetrakisphosphate and inositol 1,3,4,5-tetrakisphosphate are all competitive inhibitors with K(i) values of 0.4 microM, 3 microM and 5 microM, respectively, well within their likely intracellular concentration ranges: they inhibited 6-kinase and 5-kinase activities equally. 2,3-Bisphosphoglycerate and spermine were also competitive inhibitors, with K(i) values of 0.8 mM an 12 mM, respectively. Dextran sulphate was a non-competitive inhibitor with a Ki of approximately 15 microM, and poly-L-lysine (IC50 approximately 200 microM), polyvinylsulphate (IC50 approximately 250 microM) and heparin (IC50 approximately 2 mg/ml) also inhibited. Inhibition by these compounds suggests that inositol 3,4,5,6-tetrakisphosphate (and to a lesser extent inositol 1,3,4,5-tetrakisphosphate and other naturally occurring intracellular ions) may restrict the synthesis of inositol 1,3,4,6-tetrakisphosphate and hence regulate the rate of inositol penta- and hexakisphosphate synthesis from receptor-generated inositol phosphates.

Botulinum toxin injection in the treatment of tennis elbow. A double-blind, randomized, controlled, pilot study.

BACKGROUND: A recent report has suggested that local injection of botulinum toxin type A is an effective method of treatment for chronic tennis elbow. The toxin is thought to provide temporary paralysis of the painful common extensor origin, thereby allowing a healing response to occur. To test this theory, we performed a double-blind, randomized, controlled, pilot trial comparing injections of botulinum toxin type A with those of a placebo (normal saline solution) in the treatment of chronic tennis elbow. METHODS: Forty patients with a history of chronic tennis elbow for which all conservative treatment measures, including steroid injection, had failed were randomized into two groups. Half the patients received 50 units of botulinum toxin type A, and the remainder received normal saline solution. The intramuscular injections were performed 5 cm distal to the maximum point of tenderness at the lateral epicondyle, in line with the middle of the wrist. The two solutions used for the injections were identical in appearance and temperature. The results of a quality-of-life assessment with the Short Form-12 (SF-12), the pain score on a visual analogue scale, and the grip strength measured with a validated Jamar dynamometer were recorded before and three months after the injection. RESULTS: Three months following the injections, there was no significant difference between the two groups with regard to grip strength, pain, or quality of life. CONCLUSIONS: With the numbers studied, we failed to find a significant difference between the two groups; thus, we have no evidence of a benefit from botulinum toxin injection in the treatment of chronic tennis elbow.

Identification and characterization of inositol 1,4,5-trisphosphate receptors in rat testis.

PCR analysis and immunoblotting with isoform specific antibodies was used to identify the presence of type I, II and III inositol 1,4,5-trisphosphate receptors (InsP3Rs) in rat testis. PCR analysis also revealed that rat testis express both forms of the S1 splice variant (S1+ and S1-), but only the S2- from of the S2 splice variant of the type I InsP3 receptor. PCR analysis was also used to identify InsP3R isoform expression at a cellular level using myoid, Sertoli and germ cells derived from the testis of Wistar rats. The extent of [3H]-InsP3 binding was found to be 9 times lower for testicular microsomes than for cerebellar microsomes, with a Bmax of 1.4 pmoles/mg protein compared to 12.5 pmoles/mg protein for cerebellar microsomes. The Kd for InsP3 binding to its receptor in testicular microsomes was 60 +/- 10 nM which was similar to that found for cerebellar microsomes (80 +/- 20 nM). InsP3-induced Ca2+ release (IICR) in testicular microsomes was found to have an EC50 (concentration which causes a half-maximal response) of 0.5 +/- 0.03 microM, also similar to that seen for cerebellar microsomes (0.3 microM). Maximal IICR occurred at about 20 microM InsP3, with up to 4% of total intracellular Ca2+ stores being mobilized as compared to between 10-30% for cerebellar microsomes. Time resolved IICR using stopped-flow spectrofluorimetry, showed the kinetics of IICR for this testis preparation to be monophasic with a maximum rate constant of 0.15 s-1 at 30 microM InsP3. The rate constants are 7 times slower than values for cerebellar microsomes under similar conditions (approximately 1 s-1) and taken together with the binding data support the proposal that the receptor density/Ca2+ store is approximately 8 times lower than seen in cerebellar microsomal vesicles. The pharmacological properties as assessed using heparin and InsP3 analogues also confirmed similar behaviour for testicular InsP3Rs and cerebellar InsP3Rs.

Rat testicular myoid cells express vasopressin receptors: receptor structure, signal transduction, and developmental regulation.

Detection of the neurohypophysial hormones vasopressin (AVP) and oxytocin (OT) in the testis of several species has led to the proposal that these peptides may have a physiological role in the regulation of testicular function. Therefore, we investigated whether the contractile myoid cells of rat seminiferous tubules express functional receptors for AVP or OT and, thus, constitute a target for these hormones. This study used primary cultures of purified peritubular myoid cells derived from rats both before and after puberty. By several criteria, myoid cells prepared from adult rats expressed vasopressin receptors (VPRs). We detected specific and saturable [3H]AVP binding to a single population of sites with a Kd of 7.5 nM and a binding capacity of 145 fmol/mg protein. AVP stimulated the accumulation of inositol phosphates in a dose-dependent manner with an EC50 of 1.7 nM. Cloning and sequencing of the myoid cell VPR confirmed it to be the V1a subtype of VPR. VPR expression by myoid cells is under developmental control, as the receptors are present in the adult rat, but absent before puberty. In contrast, OT receptors were not expressed at any stage of development. Peritubular myoid cells are also responsive to endothelin-1 (ET-1), which potently stimulated phosphoinositidase-C. However, unlike AVP, the ET-1 responses were observed both before and after sexual maturity, suggesting different roles for AVP and ET-1 in the control of myoid cell function. Our data establish that the myoid cells of the adult rat seminiferous tubule are a target for AVP. This indicates an additional role for AVP in the regulation of testicular function and male fertility in the adult rat.

Phytoestrogens are potent inhibitors of estrogen sulfation: implications for breast cancer risk and treatment.

We investigated the ability of 37 flavonoids and flavonoid sulfoconjugates, including some abundant dietary constituents, to act as substrates and/or inhibitors of the sulfotransferase and sulfatase enzymes that interconvert active estrogens and inactive estrogen sulfates in human tissues. The enzymes studied include estrogen sulfotransferase, the thermostable phenolsulfotransferase that acts on a range of substrates including estrogens; steroid sulfatase; and two related enzymes, monoamine phenolsulfotransferase and arylsulfatase A. Several dietary flavonoids, including the soy isoflavones genistein and daidzein, were sulfated by these human sulfotransferases. Many flavonoids were potent inhibitors of thermostable phenolsulfotransferase. Genistein and equol were potent mixed inhibitors of hepatic estrogen sulfotransferase, with inhibitory constant values of 500 nM and 400 nM, respectively. Monoamine phenolsulfotransferase activity was relatively unaffected by flavonoids, but this enzyme was mainly responsible for the sulfation of flavonoids at concentrations greater than 1 micro M. Of the compounds tested, only daidzein 4,7-bisulfate, a trace metabolite in humans, significantly inhibited steroid sulfatase in the micromolar concentration range. Hence, dietary flavonoids may be able to influence the bioavailability of endogenous estrogens, and disrupt endocrine balance, by increasing the ratio of active estrogens to inactive estrogen sulfates in human tissues.

A clinical and genetic study of a manifesting heterozygote with X-linked myotubular myopathy.

X-linked myotubular myopathy (XLMTM) characteristically causes severe or fatal muscle weakness in male infants. Mutations in the gene MTM1, encoding the protein myotubularin, can be identified in most families. Prior to this report, XLMTM was thought not to cause symptomatic manifestations in female carriers. We describe an adult female from a large family with typical XLMTM. The patient had progressive disabling muscle weakness of later onset and lesser severity than that observed in affected males. The distribution of weakness resembled typical XLMTM with facial weakness, marked limb-girdle weakness, respiratory muscle involvement and dysphagia. Analysis of the MTM1 gene identified a heterozygous missense mutation (G378R) within the highly conserved tyrosine phosphatase site of myotubularin. We did not identify significantly skewed X-inactivation. We conclude that XLMTM is capable of causing significant disability in heterozygotes.

Peripheral blood lymphocyte sub-populations and multiple sclerosis.

Serial studies of lymphocyte sub-populations in patients with multiple sclerosis (MS) show periodic reductions in OKT8 cells (%). This alteration is neither immunologically nor disease-specific and also occurs in some apparently healthy normal individuals. In twice-monthly serial studies of lymphocyte sub-populations carried out over a 6-month period, we have found significant reductions in OKT8 cells on at least 2 occasions in 9/9 patients, 7/7 spouses and 3/11 siblings, but rarely in unrelated controls living in the same city. Abnormalities in these unaffected people occurred at the same time as those seen in their affected relative. Cyclical reduction in OKT8 cells is probably one factor associated with susceptibility to MS, perhaps only abnormal in terms of the frequency and duration with which it occurs, and may be related to environmental conditions affecting patients with MS and their close contacts but not most other unaffected individuals.

Adenosine potentiates immunological histamine release from rat mast cells by a novel cyclic AMP-independent cell-surface action.

Adenosine enhanced histamine release and prolonged the adenosine 3':5'-cyclic monophosphate (cyclic AMP) response in purified rat peritoneal mast cells following immunological challenge. The effect on the cyclic AMP response, which was blocked by 8-phenyltheophylline, probably results from an interaction with A2-purinoceptors. Enhancement of histamine release showed different characteristics. It was not inhibited by dipyridamole or hexobendine, thereby indicating an action at the cell surface. However, the relative potencies of adenosine analogues and nucleotides, together with the observation that this effect was not antagonized by 8-phenyltheophylline or theophylline, suggest that it is not mediated by a previously recognised purinoceptor. Thus, enhancement of histamine release may represent a novel cell surface action of adenosine which is independent of its effects on adenylate cyclase.

Studies on the receptor mediating cyclic AMP-independent enhancement by adenosine of IgE-dependent mediator release from rat mast cells.

Adenosine produced a concentration-related enhancement of antigen-induced 5-hydroxytryptamine (5-HT) release from rat serosal mast cells. This potentiation was maximal following the simultaneous addition of adenosine with antigen. Enhancement of 5-HT release was accompanied by potentiation of the adenosine 3':5'-cyclic monophosphate (cyclic AMP) response to challenge. The cyclic AMP response, which was antagonized by 8-phenyltheophylline, was characterized as an A2-purinoceptor-mediated effect by the use of 5'-N-ethylcarboxamideadenosine (NECA) and L-N6-phenylisopropyladenosine (L-PIA). Enhancement of 5-HT release, conversely, was not blocked by 8-phenyltheophylline suggesting it to be mediated by a cyclic AMP-independent mechanism. The effect of adenosine on 5-HT release was not reduced by the inhibition of the facilitated uptake of adenosine with dipyridamole, hexobendine or p-nitrobenzylthioguanosine, therefore, suggesting it to be mediated by a cell surface receptor. The receptor mediating enhancement of 5-HT does not appear to belong to the P2-purinoceptor subtype as adenosine was more potent than both adenosine monophosphate (AMP) and adenosine diphosphate (ADP) and alpha, beta-methylene ATP was inactive. Furthermore, the effects of AMP were blocked by alpha, beta-methylene ADP, which inhibits the conversion of AMP to adenosine. Adenosine, NECA, L- and D-PIA were all of equal potency in enhancing 5-HT release. Inosine and 3-deazaadenosine were also active. The rank order of potency of these adenosine analogues is not consistent with an effect at A1- or A2-purinoceptors. There appear to be two adenosine receptors on rat mast cells, an A2-purinoceptor which stimulates adenylate cyclase and a separate purinoceptor, stimulation of which produces enhancement of mediator release by an unknown mechanism. The effects mediated by these receptors appear to be independent of each other.

Adenosine inhibits and potentiates IgE-dependent histamine release from human basophils by an A2-receptor mediated mechanism.

Adenosine added to human basophils before anti-IgE challenge inhibited histamine release, whereas addition after challenge potentiated release. Peak responses for the two effects occurred 15 min before and after challenge respectively. The effects of adenosine on histamine secretion were dose-related over concentration ranges of 1-100 microM for inhibition and 0.01-1 microM for potentiation. The capacity of adenosine to inhibit and potentiate histamine secretion was inversely related to the strength of immunological challenge. The ability of theophylline (50 microM) to inhibit and dipyridamole (1 microM) to enhance slightly adenosine-induced responses, and the differing pharmacological effect of 2',5'-dideoxyadenosine suggested that adenosine's effects on basophil histamine secretion were mediated by stimulation of cell surface adenosine receptors. The order of potency of adenosine and its analogues L- and D- N6-phenylisopropyladenosine (PIA) and 5'-N-ethylcarboxamideadenosine (NECA) in inhibiting and potentiating IgE-dependent histamine release from basophils indicated that both responses were mediated by stimulation of the adenosine A2-receptor subtype. The capacity of adenosine to cause a transient increase of cyclic AMP levels in 40-70% basophil-enriched leucocytes confirmed the association between stimulation of A2-receptors and activation of adenylate cyclase.

Inhibition of immunological and nonimmunological histamine release from human basophils by adenosine analogues that act at P-sites.

2',5'-Dideoxyadenoside (DDA) inhibited both anti-IgE and ionophore A23187 induced histamine secretion from human basophils. Whereas DDA inhibited IgE-dependent histamine secretion when added at all times prior to challenge, release induced by A23187 was inhibited only with simultaneous addition of DDA and secretagogue. Dipyridamole, but not theophylline, abrogated DDA mediated inhibition of histamine release suggesting an intracellular mechanism of action of DDA. The observations that 2'-deoxyadenosine and 9-beta-D-arabinofuranosyladenine also inhibited release suggest that the its inhibitory effect was enhanced by manganese and reversed by islet activating protein from Bordetella pertussis suggest that DDA inhibits basophil histamine release by interacting with a guanine nucleotide binding protein which may be linked to either adenylate cyclase or other second messenger system(s).

Adenosine inhibits and potentiates IgE-dependent histamine release from human lung mast cells by an A2-purinoceptor mediated mechanism.

Adenosine, at physiological concentrations, may modulate histamine release from mechanically dispersed human lung mast cells. Addition of adenosine to the dispersed mast cells at times up to 5 min before immunological challenge with anti-human IgE inhibited histamine release. When added after this time adenosine caused a small potentiation of immunological histamine release, maximum potentiation occurring with addition of adenosine 5 min after challenge, coincidental with the end of the rapid phase of histamine release. Both inhibition and potentiation of histamine release were more pronounced with low levels of immunological challenge. Theophylline, 8-phenyltheophylline, dipyridamole and analogues of adenosine were used to determine the site of action of adenosine on mast cell mediator release. Theophylline and 8-phenyltheophylline displaced the concentration-response lines for both inhibition and potentiation of mediator release by adenosine to the right whilst dipyridamole, 1 microM, was without significant effect. This suggests that both effects result from interaction of adenosine with cell surface receptors. This was confirmed by demonstrating that the P-site agonist 2',5'-dideoxyadenosine produced only inhibition of histamine release, an effect which was inhibited by dipyridamole but not by theophylline. The rank potency order of adenosine analogues, NECA much greater than adenosine greater than or equal to L-PIA greater than or equal to D-PIA in both inhibiting and potentiating immunological histamine release suggests that both effects are mediated through activation of cell surface A2-purinoceptors. Since adenosine is released into the circulation of asthmatic subjects following bronchial provocation with antigen, causes bronchoconstriction and has the ability to modulate mast cell histamine release, this nucleoside should be considered as an additional inflammatory mediator of allergic reactions.