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Supplemental Digital Content is available in the text.A major risk factor for the development of Alzheimer’s disease (AD) is increasing age, but the reason behind this association has not been identified. It is thought that the changes in endocytosis seen in AD patients are causal for this condition. Thus, we hypothesized that the increased risk of developing AD associated with ageing may be because of changes in endocytosis. We investigated using Western blotting whether the expression of endocytic proteins involved in clathrin-mediated and clathrin-independent endocytosis are altered by increasing age in a mouse model of amyloid pathology. We used mice transgenic for human amyloid precursor protein containing the V717I London mutation. We compared the London mutation mice with age-matched wild-type (WT) controls at three ages, 3, 9 and 18 months, representing different stages in the development of pathology in this model. Having verified that the London mutation mice overexpressed amyloid precursor protein and β-amyloid, we found that the expression of the smallest isoform of PICALM, a key protein involved in the regulation of clathrin-coated pit formation, was significantly increased in WT mice, but decreased in the London mutation mice with age. PICALM levels in WT 18-month mice and clathrin levels in WT 9-month mice were significantly higher than those in the London mutation mice of the same ages. The expression of caveolin-1, involved in clathrin-independent endocytosis, was significantly increased with age in all mice. Our results suggest that endocytic processes could be altered by the ageing process and such changes could partly explain the association between ageing and AD.

Lipopolysaccharide (LPS) contributes to asthma exacerbations and development of inhaled corticosteroid insensitivity. Complete resistance to systemic corticosteroids is rare, and most patients lie on a continuum of steroid responsiveness. This study aimed to examine the sensitivity of combined ovalbumin- (Ova) and LPS-induced functional and inflammatory responses to inhaled and systemic corticosteroid in conscious guinea pigs to test the hypothesis that the route of administration affects sensitivity. Guinea pigs were sensitized to Ova and challenged with inhaled Ova alone or combined with LPS. Airway function was determined by measuring specific airway conductance via whole-body plethysmography. Airway hyper-responsiveness to histamine was determined before and 24 hours post-Ova challenge. Airway inflammation and underlying mechanisms were determined from bronchoalveolar lavage cell counts and lung tissue cytokines. Vehicle or dexamethasone was administered by once-daily i.p. injection (5, 10, or 20 mg/kg) or twice-daily inhalation (4 or 20 mg/ml) for 6 days before Ova challenge or Ova with LPS. LPS exacerbated Ova-induced responses, elongating early asthmatic responses (EAR), prolonging histamine bronchoconstriction, and further elevating airway inflammation. Intraperitoneal dexamethasone (20 mg/kg) significantly reduced the elongated EAR and airway inflammation but not the increased bronchoconstriction to histamine. In contrast, inhaled dexamethasone (20 mg/ml), which inhibited responses to Ova alone, did not significantly reduce functional and inflammatory responses to combined Ova and LPS. Combined Ova and LPS–induced functional and inflammatory responses are insensitive to inhaled, but they are only partially sensitive to systemic, dexamethasone. This finding suggests that the route of corticosteroid administration may be important in determining corticosteroid sensitivity of asthmatic responses.

Amyloid-beta (Aβ) is produced from amyloid precursor protein (APP) primarily after APP is internalised by endocytosis and clathrin-mediated endocytic processes are altered in Alzheimer’s disease (AD). There is also evidence that cholesterol and flotillin affect APP endocytosis. We hypothesised that endocytic protein expression would be altered in the brains of people with AD compared to non-diseased subjects which could be linked to increased Aβ generation. We compared protein expression in frontal cortex samples from men with AD compared to age-matched, non-diseased controls. Soluble and insoluble Aβ40 and Aβ42, the soluble Aβ42/Aβ40 ratio, βCTF, BACE1, presenilin-1 and the ratio of phosphorylated:total GSK3β were significantly increased while the insoluble Aβ42:Aβ40 ratio was significantly decreased in AD brains. Total and phosphorylated tau were markedly increased in AD brains. Significant increases in clathrin, AP2, PICALM isoform 4, Rab-5 and caveolin-1 and 2 were seen in AD brains but BIN1 was decreased. However, using immunohistochemistry, caveolin-1 and 2 were decreased. The results obtained here suggest an overall increase in endocytosis in the AD brain, explaining, at least in part, the increased production of Aβ during AD.

Current therapies for Alzheimer’s disease only treat the symptoms of the disease. We have previously developed a novel monoclonal antibody, 2B3, which binds to the -secretase cleavage site in amyloid precursor protein (APP) and reduces the production of amyloid- (A) in human cell lines. To determine whether the antibody was likely to be effective in mouse models of amyloid pathology in vivo, we investigated whether 2B3 could also bind to APP in mouse primary cortical neurones. Primary cortical neurones were produced from E15.5–17.5 C57Bl6 wild-type and transgenic APPV717I (London mutation) embryos. The percentage of the neuronal population was determined by immunocytochemistry. Cells were treated with 10 gml 2B3 or an irrelevant IgG for 48 h and A40 levels determined by ELISA. The population of cells was found to contain over 75 neurones and 2B3 bound effectively to these cells. No differences in A40 were detected between wild-type and transgenic cells. Importantly, 2B3 significantly inhibited the production of A40 by 75.15±1.37 of the media control, whereas an irrelevant IgG only significantly reduced A40 levels by 23.35±5.55 of the media control. The reduction in A40 produced by 2B3 was significantly greater than that caused by the IgG. These data indicate that 2B3 binds to APP in mouse neurones and can inhibit A40, similar to our previous findings. The antibody is probably therefore acting by steric hindrance of -secretase and these data suggest that it will be effective in mice in vivoand could be an alternative potential therapy for Alzheimer’s disease.

Inhaled bradykinin causes bronchoconstriction in asthmatic subjects but not nonasthmatics. To date, animal studies with inhaled bradykinin have been performed only in anesthetized guinea pigs and rats, where it causes bronchoconstriction through sensory nerve pathways. In the present study, airway function was recorded in conscious guinea pigs by whole-body plethysmography. Inhaled bradykinin (1 mM, 20 s) caused bronchoconstriction and influx of inflammatory cells to the lungs, but only when the enzymatic breakdown of bradykinin by angiotensin-converting enzyme and neutral endopeptidase was inhibited by captopril (1 mg/kg i.p.) and phosphoramidon (10 mM, 20-min inhalation), respectively. The bronchoconstriction and cell influx were antagonized by the B2kinin receptor antagonist 4-(S)-amino-5-(4-{4-[2,4-dichloro-3-(2,4-dimethyl-8-quinolyloxymethyl)phenylsulfonamido]-tetrahydro-2H-4-pyranylcarbonyl}piperazino)-5-oxopentyl](trimethyl)ammonium chloride hydrochloride (MEN16132) when given by inhalation (1 and 10 μM, 20 min) and are therefore mediated via B2kinin receptors. However, neither intraperitioneal MEN16132 nor the peptide B2antagonist icatibant, by inhalation, antagonized these bradykinin responses. Sensitization of guinea pigs with ovalbumin was not sufficient to induce airway hyperreactivity (AHR) to the bronchoconstriction by inhaled bradykinin. However, ovalbumin challenge of sensitized guinea pigs caused AHR to bradykinin and histamine. Infection of guinea pigs by nasal instillation of parainfluenza-3 virus produced AHR to inhaled histamine and lung influx of inflammatory cells. These responses were attenuated by the bradykinin B2receptor antagonist MEN16132 and H-(4-chloro)DPhe-2′(1-naphthylalanine)-(3-aminopropyl)guanidine (VA999024), an inhibitor of tissue kallikrein, the enzyme responsible for lung synthesis of bradykinin. These results suggest that bradykinin is involved in virus-induced inflammatory cell influx and AHR.

Inhaled bradykinin causes bronchoconstriction in asthmatic subjects but not nonasthmatics. To date, animal studies with inhaled bradykinin have been performed only in anesthetized guinea pigs and rats, where it causes bronchoconstriction through sensory nerve pathways. In the present study, airway function was recorded in conscious guinea pigs by whole-body plethysmography. Inhaled bradykinin (1 mM, 20 s) caused bronchoconstriction and influx of inflammatory cells to the lungs, but only when the enzymatic breakdown of bradykinin by angiotensin-converting enzyme and neutral endopeptidase was inhibited by captopril (1 mg/kg i.p.) and phosphoramidon (10 mM, 20-min inhalation), respectively. The bronchoconstriction and cell influx were antagonized by the B(2) kinin receptor antagonist 4-(S)-amino-5-(4-{4-[2,4-dichloro-3-(2,4-dimethyl-8-quinolyloxymethyl)phenylsulfonamido]-tetrahydro-2H-4-pyranylcarbonyl}piperazino)-5-oxopentyl](trimethyl)ammonium chloride hydrochloride (MEN16132) when given by inhalation (1 and 10 μM, 20 min) and are therefore mediated via B(2) kinin receptors. However, neither intraperitioneal MEN16132 nor the peptide B(2) antagonist icatibant, by inhalation, antagonized these bradykinin responses. Sensitization of guinea pigs with ovalbumin was not sufficient to induce airway hyperreactivity (AHR) to the bronchoconstriction by inhaled bradykinin. However, ovalbumin challenge of sensitized guinea pigs caused AHR to bradykinin and histamine. Infection of guinea pigs by nasal instillation of parainfluenza-3 virus produced AHR to inhaled histamine and lung influx of inflammatory cells. These responses were attenuated by the bradykinin B(2) receptor antagonist MEN16132 and H-(4-chloro)DPhe-2'(1-naphthylalanine)-(3-aminopropyl)guanidine (VA999024), an inhibitor of tissue kallikrein, the enzyme responsible for lung synthesis of bradykinin. These results suggest that bradykinin is involved in virus-induced inflammatory cell influx and AHR.

cDNAs encoding three splice variants of the P2X2receptor were isolated from rat cerebellum. The first variant has a serine/proline-rich segment deleted from the intracellularly located carboxyl-terminal domain of the P2X2subunit. The second and third variants have the splice site in the second half of the predicted first transmembrane domain. Either a 12-amino acid insertion or a six-amino acid deletion occurs at this position. cRNAs for these isoforms of the P2X2subunit were injected intoXenopus laevisoocytes and tested for function. ATP evoked inward currents only with the splice variant [designated P2X2(b)] having the 69-amino acid deletion. The potencies of various agonists at the homomeric P2X2(b)receptor were not significantly different from those at the P2X2(a)homomeric channel. However, the P2X2(b)receptor showed significantly lower antagonist sensitivity. In contrast to the nondesensitizing P2X2(a)receptor, prolonged application of ATP produced a more rapid desensitization of the P2X2(b)receptor. When the P2X2(a)and P2X2(b)receptor responses were recorded in transfected mammalian cells, this difference was again found. The change in desensitization may be determined by proline/serine-rich segments and/or phosphorylation motifs that are removed from the tail region in formation of the P2X2(b)subunit. In situhybridization of the three newly isolated isoforms of the P2X2subunit was performed at the macroscopic and cellular levels; transcripts for two of them [P2X2(b)and p2x2(c)] but not the third [p2x2(d)], which carries the 12-amino acid addition, were present in many structures in the neonatal rat brain and on sensory and sympathetic ganglia. mRNA for the p2x2(d)splice variant was present only in the nodose ganglion, at a low level.

1The mouse somatostatin (SRIF) sst2receptor exists in two splice variants, sst2(a)and sst2(b), which differ in their intracellular carboxy‐termini only. The murine sst2(b)receptor was reported to be less prone to agonist‐induced desensitization as compared with the sst2(a)receptor. To determine whether a sst2(b)splice variant with similar functional characteristics exists in the rat, we have isolated a cDNA fragment from rat gastric mucosa encoding a sst2(b)receptor and expressed the full‐length protein in CHO‐K1 cells for functional characterization.2This study provides the first evidence for the occurrence in the rat of the sst2(b)receptor, which has a 15 amino acid carboxy‐terminus differing in composition to the 38 amino acid C‐terminus of the rat sst2(a)receptor.3In CHO‐K1 cells expressing rat recombinant sst2(a)or sst2(b)receptors, SRIF caused concentration‐dependent increases in extracellular acidification rates (EAR) with pEC50values of 9.0 and 9.9, respectively. Pre‐treatment with pertussis toxin (Ptx) caused a rightward displacement of the SRIF concentration‐effect curves with pEC50values of 8.3 (sst2(a)) and 8.4 (sst2(b)).4SRIF (3 pM–3 nM) also caused concentration‐dependent inhibition of forskolin‐stimulated cyclic AMP formation in CHO‐sst2(a)cells (pIC5010.5) and CHO‐sst2(b)cells (pIC5010.4). The degree of inhibition was less with higher concentrations of SRIF resulting in bell‐shaped concentration‐effect curves. Following pre‐treatment with Ptx, the inhibitory effect of SRIF was abolished and SRIF caused only increases in cyclic AMP formation.5Both the SRIF‐induced increases in EAR and inhibition of cyclic AMP formation were susceptible to agonist‐induced desensitization, but this was less apparent following pre‐treatment with Ptx.6This demonstrates that the operational characteristics of the recombinant rat sst2(a)and sst2(b)receptors are broadly similar. Both isoforms couple to Ptx‐sensitive as well as ‐insensitive G proteins and are equally prone to agonist‐induced desensitization.

Calcilytics reduce airway hyperresponsiveness and inflammation and may represent effective asthma therapeutics.