Degranulation of individual mast cells in response to Ca2+ and guanine nucleotides: an all-or-none event.

Widespread experience indicates that application of suboptimal concentrations of stimulating ligands (secretagogues) to secretory cells elicits submaximal extents of secretion. Similarly, for permeabilized secretory cells, the extent of secretion is related to the concentration of applied intracellular effectors. We investigated the relationship between the extent of secretion from mast cells (assessed as the release of hexosaminidase) and the degranulation (exocytosis) responses of individual cells. For permeabilized mast cells stimulated by the effector combination Ca2+ plus GTP-gamma-S and for intact cells stimulated by the Ca2+ ionophore ionomycin, we found that exocytosis has the characteristics of an all-or-none process at the level of the individual cells. With a suboptimal stimulus, the population comprised only totally degranulated cells and fully replete cells. In contrast, a suboptimal concentration of compound 48/80 applied to intact cells induced a partial degree of degranulation. This was determined by observing the morphological changes accompanying degranulation by light and electron microscopy and also as a reduction in the intensity of light scattered at 90 degrees, indicative of a change in the cell-refractive index. These results may be explained by the existence of a threshold sensitivity to the combined effectors that is set at the level of individual cells and not at the granule level. We used flow cytometry to establish the relationship between the extent of degranulation in individual rat peritoneal mast cells and the extent of secretion in the population (measured as the percentage release of total hexosaminidase). For comparison, secretion was also elicited by applying the Ca2+ ionophore ionomycin or compound 48/80 to intact cells. For permeabilized cells and also for intact cells stimulated with the ionophore, levels of stimulation that generate partial secretion gave rise to bimodal frequency distributions of 90 degrees light scatter. In contrast, a partial stimulus to secretion by compound 48/80 resulted in a single population of partially degranulated cells, the degree of degranulation varying across the cell population. The difference between the all-or-none responses of the permeabilized or ionophore-treated cells and the graded responses of cells activated by compound 48/80 is likely to stem from differences in the effective calcium stimulus. Whereas cell stimulated with receptor-directed agonists can undergo transient and localized Ca2+ changes, a homogeneous and persistent stimulus is sensed at every potential exocytotic site in the permeabilized cells.

Nitric oxide synergistically potentiates interleukin-1 beta-induced increase of cyclooxygenase-2 mRNA levels, resulting in the facilitation of substance P release from primary afferent neurons: involvement of cGMP-independent mechanisms.

We previously demonstrated that cultured rat dorsal root ganglion (DRG) cells respond to stimulation with interleukin-1 beta (IL-1 beta) by releasing substance P (SP), and this response is regulated via the cyclooxygenase (COX)-2 pathway. In this study, to ascertain the interaction between nitric oxide (NO) and prostaglandins in primary afferent neurons, we investigated the effect of NO on the IL-1 beta-induced release of SP in cultured DRG cells. An NO donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP), did not in itself evoke SP release. However, it potentiated the IL-1 beta-induced release of SP. Similarly, while SNAP did not elicit the expression of COX-2 mRNA, it potentiated the expression induced by IL-1 beta in cultured DRG cells, and this potentiation was significantly suppressed by the NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (carboxy-PTIO). Moreover, SNAP also potentiated the expression of COX-2 protein induced by IL-1 beta in cultured DRG cells. The stimulatory effect of SNAP on the IL-1 beta-induced release of SP was completely inhibited on co-incubation with a selective COX-2 inhibitor, NS-398. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ), a potent inhibitor of soluble guanylate cyclase, did not suppress, and a membrane-permeable cGMP analogue, 8-Br-cGMP, did not mimic the stimulatory effects of SNAP in DRG cells. These results suggest that in cultured DRG cells, NO potentiates the IL-1 beta-induced increase in COX-2 expression via a soluble guanylate cyclase-cGMP-independent pathway, resulting in facilitation of SP release. The interaction between NO and COX in primary afferent neurons might contribute to the change in nociceptive perception in inflammatory hyperalgesia.

Imidazodiazepinediones: a new class of adenosine receptor antagonists.

A series of imidazo[4,5-e][1-4]diazepine-5,8-diones were synthesized from hypoxanthines. Certain of these cyclic homologues of caffeine, theophylline, theobromine, 3-isobutyl-1-methylxanthine, and enprofylline were inhibitors of binding of adenosine analogues to rat brain A1 and A2 adenosine receptors and were antagonists of A2 adenosine receptors stimulatory to adenylate cyclase in rat PC12 cell membranes. Activity at adenosine receptors was lower than the corresponding xanthines, perhaps because imidazodiazepinediones contain a boat-shaped seven-membered ring rather than the planar heteroaryl ring system of the xanthines. The imidazodiazepinediones had low affinity for brain benzodiazepine sites.

7-Deaza-2-phenyladenines: structure-activity relationships of potent A1 selective adenosine receptor antagonists.

A series of derivatives of 7-deazapurines with varying substituents in the 2-, 6-, and 9-position was synthesized in an attempt to improve the adenosine receptor affinity and A1 or A2 selectivity. The adenosine receptor affinities were assessed by measuring the inhibition of [3H]-(R)-N6-(phenylisopropyl) adenosine (R-PIA) binding to rat brain A1 and inhibition of [3H]-5'-(N-ethylcarboxamido)adenosine (NECA) binding to rat striatum A2 adenosine receptors. A selected set of compounds representing the main structural variations was further examined in adenosine receptor coupled adenylate cyclase assays. All tested compounds antagonized the inhibition of adenylate cyclase elicited by interaction of R-PIA with A1 receptors in rat fat cell membranes and the activation of adenylate cyclase elicited by interaction of NECA with A2 receptors of pheochromocytoma PC12 cell membranes. The results indicate that 7-deazahypoxanthines have a potential for A2 selectivity, while all 7-deazaadenines are A1 selective. Introduction of a phenyl residue in the 2-position of 7-deazaadenines increases A1 activity tremendously. 2-(p-Chlorophenyl)-7,8-dimethyl-9-phenyl-7-deazaadenine (29) is potent and specific for the A1 receptors of rat brain (Ki = 122 nM), having no affinity for the A2 receptors of rat striatum. The compound has low activity at the A2 receptors of rat PC12 cell membranes where it appears to act as a noncompetitive inhibitor. A 1-phenylethyl substituent at the 9-position was found to be superior to a phenyl residue in terms of A1 affinity. The most potent A1 antagonist in the present series is the highly A1 selective (790-fold) (R)-7,8-dimethyl-2-phenyl-9-(1-phenylethyl)-7-deazaadenine (31, Ki = 4.7 nM), which is 30-35 times more potent at A1 receptors than its S enantiomer. The solubility of six of the potent 7-deaza-2-phenyladenines was determined by means of an A1 binding assay. Chloro substitution of the 2-phenyl ring appeared to improve the solubility as well as the solubility over A1 affinity ratio of 9-phenyl- and 9-(1-phenylethyl)-substituted 7-deazadenines.

Differential effects on D2 dopamine receptor and prolactin gene expression by haloperidol and aripiprazole in the rat pituitary.

[3H]Spiperone-binding assay to D2 receptors and quantitative ribonuclease protection assay for both isoforms (D2L and D2S receptor) of the D2 receptor mRNA and the prolactin mRNA were performed on pituitaries from the control rat and from the rat injected orally daily with either haloperidol (2 mg/kg) or aripiprazole (24 mg/kg) for 21 days. Haloperidol treatment increased the [3H]spiperone-binding by 28%, the levels of D2L and D2S receptor mRNA by 41% and 38%, respectively, and the level of prolactin mRNA by 26%. In contrast, the treatment with aripiprazole, a newly developed atypical antipsychotic with reduced side effects, decreased the [3H]spiperone-binding by 24% and the levels of D2L and D2S receptor mRNA by 23% and 23%, respectively, and did not have any effect on the level of prolactin mRNA. The same treatment with sulpiride (100 mg/kg) increased the levels of D2L and D2S receptor mRNA by 59% and 62%, respectively, but treatment with clozapine (25 mg/kg) did not cause any effect. Neither treatment changed the ratio of the level of D2S receptor mRNA to the level of D2L receptor mRNA in the pituitary. These findings indicate that D2 receptor densities in the pituitary are influenced differentially by the treatment with these antipsychotics, which could be induced at least partly by the changes in the levels of mRNA without any effects on the splicing mechanisms and thus affect the plasticity of the prolactin mRNA expression. The inhibitory effects of chronic aripiprazole treatment on D2 receptors in the pituitary might underlie this drug's clinical property of reduced hyperprolactinemia side effect.

Repeated haloperidol treatment decreases sigma(1) receptor binding but does not affect its mRNA levels in the guinea pig or rat brain.

The effects of chronic treatment with haloperidol on sigma (sigma) receptors were investigated across brain regions and species. The regional distribution of [3H](+)-pentazocine binding to sigma(1) receptor was similar between the guinea pig and rat brains. The highest level of binding was detected in the brain stem and lowest in the striatum and hippocampus. The regional distribution of [3H]1, 3-di (2-tolyl) guanidine ([3H]DTG) binding in the presence of 100 nM (+)-pentazocine to sigma(2) receptor was similar to that of the [3H](+)-pentazocine binding in the guinea pig brain, while in the rat brain high levels of [3H]DTG binding were detected in the cortex, frontal cortex and cerebellum. The intraperitoneal administration of 2 mg/kg of haloperidol to guinea pig and rats once a day for 21 days produced inhibition of [3H](+)-pentazocine binding but did not affect [3H]DTG binding to sigma(2) receptors in any brain region examined. The effects of haloperidol on [3H](+)-penazocine binding in the rat were much weaker than those in the guinea pig. The regional distribution of the level of sigma(1) receptor mRNA determined by the ribonuclease protection assay was similar to that of the [3H](+)-pentazocine binding activity, except in the cortex and cerebellum where the levels of sigma(1) receptor mRNA were low in guinea pig and rat. Treatment with haloperidol did not affect the levels of sigma(1) receptor mRNA in any brain region in either species. These findings suggested that the sigma receptors differentially distributed in brain regions are down regulated by treatment with haloperidol across sigma receptor subtypes and animal species without changing the transcriptional activity of the sigma(1) receptor. The mechanisms by which sigma receptors could be differently regulated in vivo by chronic treatment with haloperidol in different species may contribute to the therapeutic efficacy of haloperidol.

Inactivation of presynaptic 5-HT autoreceptors by lithium in rat hippocampus.

The effect of lithium ion on the electrically stimulated 5-[3H]hydroxytryptamine (5-HT) release from the rat hippocampal slices preloaded with [3H]5-HT was studied. Electrically stimulated [3H]5-HT release decreased when the slices were exposed to 5-HT in a concentration-dependent manner. Lithium (2.5 mM) did not affect [3H]5-HT release when added alone to the superfusion medium. However, the inhibitory effect of 5-HT (1 microM) on [3H]5-HT release was abolished by lithium. The results suggest that lithium may inhibit the regulation of 5-HT release via presynaptic 5-HT autoreceptors in rat hippocampus.

The antinociceptive effect of intrathecally administered adenosine analogs in mice correlates with the affinity for the A1-adenosine receptor.

In the present study, the antinociceptive effects after intrathecal injection of each of 6 N6-substituted adenosine analogs and of 2-phenylaminoadenosine were compared with the affinity for the A1- and A2-adenosine receptors. Adenosine analogs, substituted in the N6-position, had stereoselective structure-dependent antinociceptive effects in the tail flick and hot plate assays after intrathecal injection in mice. The antinociceptive activity for N6-R- and S-phenylisopropyladenosine (R- and S-PIA), N6-R- and S-1-phenylethyladenosine, N6-1,1-dimethyl-2-phenylethyladenosine (methylPIA), and N6-cyclooctyladenosine correlated with the affinity for central A1-adenosine receptors. An adenosine analog, 2-phenylaminoadenosine, selective for A2-adenosine receptors was inactive in the two tests. These results strongly suggest that spinal A1-adenosine receptors are responsible for the antinociceptive effects of adenosine and its analogs after intrathecal injection.

Women with normal mammography describing symptoms at screening.

A retrospective study from May 1989 to June 1995 identified 4327 women with symptoms and normal mammography, from a total population of 139,852. The women with symptoms were divided into two groups: those who had been recalled for assessment, because of potentially significant symptoms, and those who had not. Out of 448 women recalled for assessment with significant symptoms, 8 malignancies were detected and 8 benign biopsies were performed. Review of the remaining 3879 women with symptoms showed that 4 had developed interval cancers in the same breast as the symptoms described at the previous screening attendance. No cancer was detected at subsequent screening examinations. This review shows that breast cancer is so uncommon in women attending for screening with symptoms that a policy of only recalling women with significant symptoms is justifiable.

Calcium signaling and protein kinase C for TNF-alpha secretion in a rat mast cell line.

In mast cells, like other nonexcitable cells, receptor activation produces Ca2+-mobilizing second messengers such as inositol 1,4,5-triphosphate or sphingosine-1-phosphate, which induce Ca2+ release from internal stores. The resulting depletion of Ca2+ stores activates Ca2+ channels in plasma membranes designated as Ca2+ release-activated Ca2+ (CRAC) channels. Ionomycin appears to cause activation of CRAC channels by depleting intracellular Ca2+ stores rather than by acting as an ionophore. We compared the effects of azelastine, an anti-allergic drug, on TNF-alpha secretion, on Ca2+ signal, and on degranulation in an antigen- or ionomycin-stimulated rat mast RBL-2H3 cell line. Azelastine inhibited TNF-alpha release at concentrations lower than those needed for the inhibition of degranulation. In antigen-stimulated cells, azelastine also inhibited equipotently TNF-alpha mRNA expression/protein synthesis, TNF-alpha release and Ca2+ influx. In ionomycin-stimulated cells, however, azelastine inhibited TNF-alpha release to a greater extent than TNF-alpha mRNA expression/protein synthesis and Ca2+ influx, indicating that azelastine inhibits the release process more potently than transcription or production of TNF-alpha by interfering with a signal other than Ca2+. Pretreatment with 1 microM azelastine inhibited ionomycin-induced, but not antigen-induced, protein kinase C translocation to the membranes. These results suggest that TNF-alpha transcription/production is mainly regulated by Ca2+ influx, but the release process of TNF-alpha is regulated by additional mechanism(s) possibly involving activation of protein kinase C.

Osteoma of the thyroid cartilage--an unusual cause of difficult intubation.

We describe a case of histologically proven osteoma of the thyroid cartilage that presented because of difficulty in intubation prior to coronary bypass surgery. To our knowledge, this is the first documented case in the English literature.

Development of a preclinical orthotopic xenograft model of ewing sarcoma and other human malignant bone disease using advanced in vivo imaging.

Ewing sarcoma and osteosarcoma represent the two most common primary bone tumours in childhood and adolescence, with bone metastases being the most adverse prognostic factor. In prostate cancer, osseous metastasis poses a major clinical challenge. We developed a preclinical orthotopic model of Ewing sarcoma, reflecting the biology of the tumour-bone interactions in human disease and allowing in vivo monitoring of disease progression, and compared this with models of osteosarcoma and prostate carcinoma. Human tumour cell lines were transplanted into non-obese diabetic/severe combined immunodeficient (NSG) and Rag2(-/-/)γc(-/-) mice by intrafemoral injection. For Ewing sarcoma, minimal cell numbers (1000-5000) injected in small volumes were able to induce orthotopic tumour growth. Tumour progression was studied using positron emission tomography, computed tomography, magnetic resonance imaging and bioluminescent imaging. Tumours and their interactions with bones were examined by histology. Each tumour induced bone destruction and outgrowth of extramedullary tumour masses, together with characteristic changes in bone that were well visualised by computed tomography, which correlated with post-mortem histology. Ewing sarcoma and, to a lesser extent, osteosarcoma cells induced prominent reactive new bone formation. Osteosarcoma cells produced osteoid and mineralised "malignant" bone within the tumour mass itself. Injection of prostate carcinoma cells led to osteoclast-driven osteolytic lesions. Bioluminescent imaging of Ewing sarcoma xenografts allowed easy and rapid monitoring of tumour growth and detection of tumour dissemination to lungs, liver and bone. Magnetic resonance imaging proved useful for monitoring soft tissue tumour growth and volume. Positron emission tomography proved to be of limited use in this model. Overall, we have developed an orthotopic in vivo model for Ewing sarcoma and other primary and secondary human bone malignancies, which resemble the human disease. We have shown the utility of small animal bioimaging for tracking disease progression, making this model a useful assay for preclinical drug testing.

Fluid levels in medical imaging.

Fluid levels are commonly observed on a range of imaging methods in both normal and abnormal circumstances. Radiologists must be familiar with the appearances and significance of fluid levels, but more fundamentally, require an understanding of the mechanisms by which fluid levels occur and the principles necessary for the demonstration of fluid levels. These are the prerequisites of a cavity and at least two types of immiscible fluid, and most importantly, the requirement of an image that is orientated in the vertical plane.

A rare cause of spinal cord compression: imaging appearances of gout of the cervical spine.

Gout is a metabolic disorder typically affecting the peripheral joints, more commonly in males. Spinal involvement is uncommon and is usually associated with hyperuricemia. We present the imaging findings of a case of spinal gout in a female patient with no previous history of hyperuricaemia, involving multiple spinal segments.

Juvenile spondylolysis: a comparative analysis of CT, SPECT and MRI.

OBJECTIVE: To evaluate whether MRI correlates with CT and SPECT imaging for the diagnosis of juvenile spondylolysis, and to determine whether MRI can be used as an exclusive image modality. DESIGN AND PATIENTS: Juveniles and young adults with a history of extension low back pain were evaluated by MRI, CT and SPECT imaging. All images were reviewed blindly. Correlative analyses included CT vs MRI for morphological grading and SPECT vs MRI for functional grading. Finally, an overall grading system compared MRI vs CT and SPECT combined. Statistical analysis was performed using the kappa statistic. RESULTS: Seventy-two patients (mean age 16 years) were recruited. Forty pars defects were identified in 22 patients (31%), of which 25 were chronic non-union, five acute complete defects and ten acute incomplete fractures. Kappa scores demonstrated a high level of agreement for all comparative analyses. MRI vs SPECT (kappa: 0.794), MRI vs CT (kappa: 0.829) and MRI vs CT/SPECT (kappa: 0.786). The main causes of discrepancy were between MRI and SPECT for the diagnosis of stress reaction in the absence of overt fracture, and distinguishing incomplete fractures from intact pars or complete defects. CONCLUSIONS: MRI can be used as an effective and reliable first-line image modality for diagnosis of juvenile spondylolysis. However, localised CT is recommended as a supplementary examination in selected cases as a baseline for assessment of healing and for evaluation of indeterminate cases.

Percutaneous vertebroplasty: history, technique and current perspectives.

Percutaneous vertebroplasty is a safe and efficacious technique for the treatment of persistent pain from a fractured vertebral body. Injection of cement into the vertebral body is made after insertion of a large-bore needle, frequently by a trans-pedicular approach. Vertebroplasty is most commonly used to treat painful osteoporotic fracture resistant to conservative therapy, but may be helpful in other conditions such as malignant collapse. NICE guidelines are now available for this procedure, which is relatively new in the UK, but has been performed for more than 15 years in continental Europe.

In vivo determination of 5-hydroxytryptamine receptor-stimulated phosphoinositide turnover in rat brain.

Phosphoinositide turnover stimulated by 5-hydroxytryptamine (5-HT) receptors in the intact rat brain was studied using an in vivo method. Phosphoinositides in the rat brain were prelabeled with [3H]inositol injected into the lateral cerebral ventricles. The rats were killed by microwave irradiation after 48 h and the contents in the frontal cortex of 3H-inositol phosphates, [3H]inositol-1-monophosphate [( 3H]IP1), [3H]inositol-1,4-bisphosphate [( 3H]IP2), and a mixture of [3H]inositol-1,4,5-trisphosphate and [3H]inositol-1,3,4-trisphosphate [( 3H]IP3) were assayed by HPLC. Lithium treatment (10 mEq/kg, i.p., 2 h before) increased the content of [3H]IP1 and [3H]IP2. 5-Methoxy-N,N-dimethyltryptamine (5-MeODMT) and quipazine, 5-HT agonists, significantly increased the amount of 3H-inositol phosphates under lithium pretreatment. The response to 5-MeODMT was inhibited by ritanserin, a 5-HT2 antagonist, but not by (-)-propranolol, a 5-HT1 antagonist. These results suggest that phosphoinositide turnover in the rat frontal cortex in vivo is stimulated by 5-HT2 receptor activation. It is considered that this method will be useful for measurement of 5-HT2 receptor-stimulated phosphoinositide turnover in vivo to examine the in vivo effects of various psychotropic drugs such as antidepressants.