Biomarkers associated with disease severity in allergic and nonallergic asthma

Asthma is a complex, chronic respiratory disease with a wide clinical spectrum. Use of high-throughput technologies has generated a great deal of data that require validation. In this work the objective was to validate molecular biomarkers related to asthmatic disease types in peripheral blood samples and define their relationship with disease severity. With this purpose, ninety-four previously described genes were analyzed by qRT-PCR in 30 healthy control (HC) subjects, 30 patients with nonallergic asthma (NA), 30 with allergic asthma (AA), and 14 patients with allergy (rhinitis) but without asthma (AR). RNA was extracted from peripheral blood mononuclear cells (PBMCs) using the TRIzol method. After data normalization, principal component analysis (PCA) was performed, and multiple approaches were used to test for differential gene expression. Relevance was defined by RQ (relative quantification) and corrected P value (<0.05). Protein levels of IL-8 and MSR1 were determined by ELISA and Western blot, respectively.PCA showed 4 gene expression clusters that correlated with the 4 clinical phenotypes. Analysis of differential gene expression between clinical groups and HCs revealed 26 statistically relevant genes in NA and 69 in AA. Protein interaction analysis revealed IL-8 to be a central protein. Average levels of IL-8 were higher in the asthma patients’ sera (NA: 452.28 ± 357.72, AA: 327.46 ± 377 pg/ml) than in HCs (286.09 ± 179.10), but without reaching statistical significance. Nine genes, especially MSR1, were strongly associated with severe NA.In conclusion, several molecular biomarkers of asthma have been defined, some of which could be useful for the diagnosis or prognosis of disease severity.     

Increased matrix metalloproteinase-9 with elastolysis in nocturnal asthma.

BACKGROUND: Matrix metalloproteinase-9 (MMP-9) is capable of degrading elastin, whereas tissue inhibitor of metalloproteinase-1 (TIMP-1) can inhibit MMP-9 activity. We observed reduced airway tissue elastin volume density in six subjects with nocturnal asthma (NA) as compared with seven subjects with nonnocturnal asthma (NNA) and seven normal controls (NL) when endobronchial biopsies were evaluated morphometrically at 4:00 PM and 4:00 AM. OBJECTIVE: We hypothesized that increased metalloproteinases and decreased tissue inhibitors of metalloproteinases in the airways of subjects with NA may be responsible for reduced elastin volume density. METHODS: Ten additional subjects with NA, 10 subjects with NNA, and 7 normal control subjects underwent bronchoscopy with bronchoalveolar lavage at 4:00 PM and 4:00 AM. Levels of MMP-2, MMP-9, TIMP-1, and TIMP-2 were determined in bronchoalveolar lavage by enzyme-linked immunosorbent assay. RESULTS: There was a fourfold circadian increase in bronchoalveolar lavage levels of MMP-9, and there was a twofold increase in MMP-9:TIMP-1 ratio in NA subjects from 4:00 PM to 4:00 AM. There were no circadian changes in the NNA and NL subjects. At 4:00 AM, MMP-9 levels and the MMP-9:TIMP-1 ratio were highest in NA subjects. At 4:00 PM, no significant group differences were observed. The MMP-9 levels positively correlated with the overnight fall in forced expiratory volume in 1 second and the MMP-9/TIMP-1 ratio negatively correlated with the 4:00 AM % predicted forced expiratory volume in 1 second. CONCLUSIONS: Our results from these two pilot studies suggest that increased MMP-9 and decreased TIMP-1 at night in NA may lead to reduced elastin density.     

Orienting in a defensive world: Mammalian modifications of our evolutionary heritage. A Polyvagal Theory

The vagus, the 10th cranial nerve, contains pathways that contribute to the regulation of the internal viscera, including the heart. Vagal efferent fibers do not originate in a common brainstem structure. The Polyvagal Theory is introduced to explain the different functions of the two primary medullary source nuclei of the vagus: the nucleus ambiguus (NA) and the dorsal motor nucleus (DMNX). Although vagal pathways from both nuclei terminate on the sinoatrial node, it is argued that the fibers originating in NA are uniquely responsible for respiratory sinus arrhythmia (RSA). Divergent shifts in RSA and heart rate are explained by independent actions of DMNX and NA. The theory emphasizes a phylogenetic perspective and speculates that mammalian, but not reptilian, brainstem organization is characterized by a ventral vagal complex (including NA) related to processes associated with attention, motion, emotion, and communication. Various clinical disorders, such as sudden infant death syndrome and asthma, may be related to the competition between DMNX and NA.     

Why are some children with early onset of asthma getting better over the years? - Diagnostic failure or salutogenetic factors

Among children earlier having been identified with a hospital or primary care diagnosis of asthma at least once between 0-7 years of age, almost 40 % of their parents reported in the ISAAC-questionnaire as never having had asthma (NA). These are further analysed and compared with the persisting asthma cases (A) in this study. All these children''s medical records were scrutinized concerning their asthma diagnose retrospectively.The aim of this study was to analyse possible factors related to the outcome in an Asthma diagnosis reassessment by parental questionnaire at the age of ten of the children earlier having been identified with a hospital or primary health care diagnosis of asthma at least once between 0-7 years of age in a total birth-year cohort in a defined Swedish geographical area.A multiple logistic analysis revealed four significant and independent factors associated to the improvement/non-report of asthma at the age of ten. These factors were; not having any past experiences of allergic symptoms (p<0.0001), only having one or two visits at the hospital for asthma diagnosis in the 0-7 interval (p=0.001), not living in a flat but a villa at the age of ten (p=0.029) and no previous perception of mist or mould damage in the house (p=0.052).In the early postnatal stage, obstructive and bronchospastic symptoms typical of asthma may be unspecific, and those cases not continuing to persisting disease tend to have identifiable salutogenetic factors of constitutional rather than environmental nature, namely, an overall reduced allergic predisposition.     

Increased cytotoxicity of CD4+ invariant NKT cells against CD4+CD25hiCD127lo/- regulatory T cells in allergic asthma

CD4+CD25(hi)CD127(lo/-) regulatory T cells (Treg) have been implicated in the resolution of asthma-associated inflammation while the opposite role of CD4+ invariant NKT (iNKT) cells has been the subject of recent investigations. Studies here focused on mechanisms of interaction between CD4+ iNKT cells and Treg to further explore their roles in allergic asthma (AA). Flow cytometry analysis revealed a significant increase in the expression of the natural cytotoxicity receptors NKp30 and NKp46 by CD4+ iNKT cells in AA subjects compared to healthy controls (HC) and non-allergic asthmatics (NA). Subsequent intracellular staining showed that CD4+ iNKT cells also expressed higher levels of granzyme B and perforin in AA than HC. In in vitro killing assays, AA CD4+ iNKT cells selectively killed autologous Treg, but not CD4+CD25- T cells, more potently than HC and NA counterparts. This increased cytotoxicity positively correlated with asthma severity and granzyme B/perforin expression of CD4+ iNKT cells. Furthermore, it could be abrogated by either inhibition of the granzyme B-/perforin-dependent cell death pathway or oral corticosteroid administration. Altogether, these findings suggest that increased cytotoxicity of CD4+ iNKT cells against Treg might contribute to dysfunctional cellular interactions in AA.     

Magnesium depletion with hypo- or hyper- function of the biological clock may be involved in chronopathological forms of asthma.

Asthma is a chronic, inflammatory disorder of the airways leading to airflow limitation. Its worldwide rise, mainly in developed countries, is a matter of concern. Nocturnal asthma (NA) frequently occurs and concerns two thirds of asthmatics. But, it remains controversial whether NA is a distinct entity or is a manifestation of more severe asthma. Generally, it is considered as an exacerbation of the underlying pathology. The pathological mechanisms most likely involve endogenous circadian rhythms with pathological consequences on both respiratory inflammation and hyperresponsiveness. A decrease in blood and tissue magnesium levels is frequently reported in asthma and often testifies to a true magnesium depletion. The link with magnesium status and chronobiology are well established. The quality of magnesium status directly influences the Biological Clock (BC) function, represented by the suprachiasmatic nuclei and the pineal gland. Conversely, BC dysrythmias influence the magnesium status. Two types of magnesium deficits must be clearly distinguished: deficiency corresponding to an insufficient intake which can be corrected through mere nutritional Mg supplementation and depletion due to a dysregulation of the magnesium status which cannot be corrected through nutritional supplementation only, but requires the more or less specific correction of the dysregulation mechanisms. Both in clinical and in animal experiments, the dysregulation mechanisms of magnesium depletion associate a reduced magnesium intake with various types of stress including biological clock dysrhythmias. The differenciation between Mg depletion forms with hyperfunction of BC (HBC) and forms with hypofunction of BC (hBC) is seminal and the main biological marker is melatonin (MT) production alteration. We hypothesize that magnesium depletion with HBC or hBC may be involved in chronopathological forms of asthma. Nocturnal asthma would be linked to HBC, represented by an increase in MT levels. The corresponding clinical forms associate diverse expressions of nervous hypoexcitability such as depression, cluster headaches, dyssomnia, mainly advanced sleep phase syndrome, some clinical forms of chronic fatigue syndrome and of fibromyalgia. The main comorbidities are depression and/or asthenia. They take place during the night or the "bad" seasons (autumn and winter) when sunshine is at a minimum. The corresponding chronopathological therapy relies on bright light phototherapy sometimes with additional psychoanaleptics. Conversely, asthma forms linked to hBC are less frequently studied as a whole and present a decrease in MT levels. They associate various signs of nervous hyperexcitability such as anxiety, diurnal cephalalgia (mainly migraine), dyssomnia, mainly delayed sleep phase syndrome, and some clinical forms of chronic fatigue syndrome and of fibromyalgia. The treatment relies on diverse forms of "darkness therapy", possibly with the help of some psycholeptics. Finally, the treatment of asthma involves the maintenance of a standard dosing schedule of anti-asthma drugs, a balanced magnesium intake and the appropriate treatment of the chronopathological disorders.     

糖皮质激素诱导的亮氨酸拉链蛋白(GILZ)在人气道上皮细胞中的表达及对MAPK信号通路的影响

目的:探讨糖皮质激素诱导的亮氨酸拉链蛋白(GILZ)在人气道上皮细胞9HTE0中的表达以及对MAPK信号通路因子Raf-1、Mek1/2、Erk1/2的影响。方法:采用RT-PCR及Western blot法检测地塞米松(Dex)作用后GILZ mRNA及蛋白的表达,同时细胞免疫荧光法检测GILZ蛋白的定位;合成三条GILZ-SiRNA分别转染人气道上皮细胞9HTE0,用Q-PCR及Western blot法筛选出沉默效果最佳的一条;Western blot法检测MAPK信号通路因子Raf-1、Mek1/2、Erk1/2磷酸化蛋白及其总蛋白的表达。结果:Dex能够明显刺激人气道上皮细胞9HTE0GILZ mRNA及蛋白的表达,GILZ蛋白主要定位在细胞浆中,Dex抑制了Raf-1、Mek1/2、Erk1/2磷酸化蛋白的表达,而GILZ-SiRNA转染后,Raf-1.Mek1/2、Erk1/2磷酸化水平有所回升。结论:糖皮质激素作为支气管哮喘一线用药,虽然能够诱导GILZ表达并发挥一定的抗炎作用,但同时GILZ却抑制了在气道上皮修复中起重要作用的MAPK信号通路的激活,这为临床上研究哮喘防治新措施提供了理论依据。     

Persistence of rhinovirus RNA after asthma exacerbation in children

BACKGROUND: Rhinoviruses (RVs) are believed to cause most asthma exacerbations but their role in the severity of acute asthma and subsequent recovery of airway function is not defined. The importance of atopy in virus-host interactions is also not clear. OBJECTIVE: We postulated that RV infection and atopic skin prick responses influence the severity of asthma exacerbations as measured by peak expiratory flow (PEF). METHODS: Patients aged 4-12 years admitted with acute severe asthma to a hospital emergency room (ER) were recruited. PEF measurements were obtained and nasal aspirates (NA) were taken. Atopy was diagnosed by skin prick responses to allergen and the presence of RV RNA and respiratory syncytial virus (RSV) RNA in NAs was detected using validated PCR assays. Patients were restudied after 6 weeks and after 6 months. RESULTS: Fifty children with acute asthma (mean age+/-SD, 7.4+/-2.7) were enrolled; atopy was present in 37 (74%). RV RNA was detected in 41 (82%) and RSV RNA in six (12%) subjects. After 6 weeks 41 patients were restudied and RV RNA was again detected in 18 (44%). RV RNA was detected after 6 months in four of 16 patients restudied (25%; P=0.008 vs. ER) and in two of nine children from a control group with stable asthma (22%; P=0.009 vs. ER). Overall PEF measurements were reduced in asthmatics admitted to ER (% predicted, 63.4+/-16.4%) but did not differ between patients with RV RNA, RSV RNA or neither virus present. In subjects with RV RNA detectable in ER and after 6 weeks, measurements of PEF in ER were significantly lower than in patients in whom RV RNA was present in ER but absent after 6 weeks (P=0.009). Regression analysis linked persistence of RV RNA, but not skin prick responses to allergen, to severity of PEF reductions in ER. CONCLUSION: RV RNA was detectable in >40% of asthmatic children 6 weeks after an acute exacerbation. Asthma exacerbations were more severe in patients with persistence of RV RNA suggesting that the severity of acute asthma may be linked to prolonged and possibly more severe RV infections.     

Tyramine-induced endogenous noradrenaline efflux from in situ cardiac sympathetic nerve ending in cats

With the use of dialysis technique, the effects of tyramine on in situ cardiac sympathetic nerve endings were examined in anaesthetized cats. Dialysis probes were implanted in the left ventricular myocardium, and the concentration of dialysate noradrenaline (NA) served as an indicator of NA output at the cardiac sympathetic nerve ending. Locally applied tyramine (600 microM) increased dialysate NA levels from 17 +/- 1 (pg mL-1) to 3466 +/- 209 (pg mL-1). Pretreatment with reserpine (vesicle transport NA blocker 1 microM) did not affect tyramine-induced NA efflux. The tyramine-induced NA efflux was augmented by pretreatment with pargyline (1 mM) but suppressed by pargyline (10 mM). Pretreatment with alpha-methyl-tyrosine suppressed NA efflux evoked by tyramine. These pretreatments did not affect the time course of NA efflux but only altered peak height of NA efflux. The efflux of NA evoked by tyramine was not associated with any reduction of dihydroxyphenylglycol (DHPG). In contrast, in the pretreatment with reserpine, the efflux of NA was associated with a reduction of DHPG. This result suggests that NA graduation between axoplasm and stored vesicle contributes to maintaining the axoplasmic NA level during carrier-mediated outward NA transport. The tyramine-induced NA efflux provides a close reflection of the NA content at the nerve ending. With the use of dialysis, this experimental model is suitable for studying the mechanism of sympathomimetic amine-induced neurotransmitter efflux.     

The response of noradrenergic axons to systemically administered DSP-4 in the rat: an immunohistochemical study using antibodies to noradrenaline and dopamine-beta-hydroxylase

The response of noradrenaline (NA) axons to the effects of systemic injections of N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) was studied in the rat brain. Antibodies to NA and to dopamine-beta-hydroxylase (DBH) were employed to assess by immunohistochemistry the effects of DSP-4 on NA axons between 6 h and 2 weeks after drug administration. The changes in NA and DBH staining after DSP-4 treatment were restricted to brain regions innervated by the locus coeruleus. In these areas, DSP-4 induced profound loss of both NA and DBH from NA axons, but with a distinctly different time-course. While NA disappeared within hours after drug treatment, DBH staining of NA axons remained unchanged during the first 4 days after DSP-4 treatment. Thereafter, there was an abrupt loss of DBH staining which coincided with the appearance of numerous brightly stained, thick and swollen NA axons. The distribution of these fibres suggests that they represent the distal ends of preterminal NA axons. Two weeks after drug treatment, NA axons could no longer be visualized by either NA or DBH immunohistochemistry in regions affected by DSP-4. During this 2-week time-period, the staining of cell bodies in the locus coeruleus and of ascending NA axons in the dorsal bundle was unaffected. The results suggest two phases in the response of NA axons to DSP-4: an acute phase, marked by loss of transmitter, and a neurodegenerative phase, characterized by loss of DBH and structural disintegration of NA axons.     

Tyramine‐induced endogenous noradrenaline efflux from in situ cardiac sympathetic nerve ending in cats

With the use of dialysis technique, the effects of tyramine on in situ cardiac sympathetic nerve endings were examined in anaesthetized cats. Dialysis probes were implanted in the left ventricular myocardium, and the concentration of dialysate noradrenaline (NA) served as an indicator of NA output at the cardiac sympathetic nerve ending. Locally applied tyramine (600 μM ) increased dialysate NA levels from 17 ± 1 (pg mL^?1) to 3466 ± 209 (pg mL^?1). Pretreatment with reserpine (vesicle transport NA blocker 1 μM ) did not affect tyramine‐induced NA efflux. The tyramine‐induced NA efflux was augmented by pretreatment with pargyline (1 m M ) but suppressed by pargyline (10 m M ). Pretreatment with α‐methyl‐tyrosine suppressed NA efflux evoked by tyramine. These pretreatments did not affect the time course of NA efflux but only altered peak height of NA efflux. The efflux of NA evoked by tyramine was not associated with any reduction of dihydroxyphenylglycol (DHPG). In contrast, in the pretreatment with reserpine, the efflux of NA was associated with a reduction of DHPG. This result suggests that NA graduation between axoplasm and stored vesicle contributes to maintaining the axoplasmic NA level during carrier‐mediated outward NA transport. The tyramine‐induced NA efflux provides a close reflection of the NA content at the nerve ending. With the use of dialysis, this experimental model is suitable for studying the mechanism of sympathomimetic amine‐induced neurotransmitter efflux.     

NA antigen frequencies in the Tunisian population

The biallelic NA antigen system is of special interest as the NA antigens are frequent targets of neutrophil antibodies, causing alloimmune neonatal neutropenia, blood transfusion reactions, and chronic benign autoimmune neutropenia in infancy. Neutrophils isolated from the peripheral blood of 119 unrelated individuals at the National Blood Center were phenotyped for NA1 and NA2 using a granulocyte immunofluorescence assay. A subsequent analysis of the phenotyping study showed that the NA1 and NA2 antigen frequencies were 0.529 and 0.865 respectively, and that the estimated NA1 and NA2 gene frequencies were 0.313 and 0.632 respectively. In conclusion, it was determined that the Tunisian population is of Caucasian origin. However, to validate this finding, further investigations are necessary.     

Expression of influenza neuraminidase in baculovirus-infected cells.

Recombinant influenza neuraminidase (NA, subtype 2, A/NT/60/68) was produced by recombinant baculovirus-infected insect cells. The recombinant NA retained enzyme activity and was located on the cell surface. Enzyme activity was both cell-associated and in the cell free supernatant; maximal NA activity was found in the supernatant. Recombinant NA was recognised by polyclonal antisera and by three monoclonal antibodies specific for NA (subtype 2). Enzyme activity was also neutralised by polyclonal antisera. Recombinant NA thus retains most of the immunological and activity properties of authentic influenza NA. Immunoprecipitation of [35S]Methionine-labelled cells and supernatant and partial purification of NA indicated that a approximately 50-kDa form of NA was present in the supernatant, whilst the expected size (approximately 67-kDa) was cell-associated. Purified recombinant extracellular virus was also enzymatically active, and contained the 67-kDa NA which was located on the membrane capsule of the virus. This suggests that the virus had acquired the cell-associated form of recombinant NA during the budding process from the cell.     

Plasma noradrenaline and dopamine in renin-mediated hypertension

Noradrenaline (NA) and dopamine (DA) have opposite effects on the kidney; NA causes vasoconstriction and increased sodium reabsorption while DA promotes vasodilation and natriuresis. In 15 patients investigated for renin-mediated hypertension measurements of plasma renin activity (PRA), NA and DA concentrations were made in arterial and renal venous blood from both kidneys before and after acute stimulation of renin release by i.v. dihydralazine. Nine patients had unilateral renin secretion and were classified as renin-positive, while the remaining six patients were renin-negative. Renin-positive patients had higher arterial and renal venous PRA, NA and DA levels than the negative ones. In the renin-positive group V-A differences for NA and DA were present on both sides despite unilateral secretion of renin. NA but not DA levels were higher in the renin-secreting kidney, which can partly be explained by the reduced plasma flow to the involved kidney. After dihydralazine the arterial NA and DA rose similarly in renin-positive and renin-negative patients, while PRA rose only in the renin-positive cases. In the renin-positive patients where stimulation of renin secretion caused a marked increase of the PRA gradient on the affected side only, renal gradients for NA and DA increased bilaterally. The increase in DA was more pronounced than that of NA yielding a rise in DA/NA ratio on the affected side. Arterial PRA was positively correlated to the plasma concentrations of NA and DA. V-A differences for PRA and NA or DA were positively correlated on the involved renin-secreting side. In summary, patients with renin-dependent hypertension have elevated plasma NA and DA concentrations. Stimulation of renin release by dihydralazine increases the DA/NA ratio in arterial and renal venous blood indicating release of 'precursor dopamine' from noradrenergic fibres and/or activation of dopaminergic nerves. There seems to be a relationship between renal nerve activity and renin release in renin-dependent hypertension.     

Relationship between the overflow of endogenous and radiolabelled noradrenaline from canine blood perfused gracilis muscle

The effects of sympathetic nerve stimulation (SNS) on the overflow of endogenous noradrenaline (NA) and on vasoconstrictor responses were studied in blood perfused canine gracilis muscle in situ. A conventional tracer technique with 3H-labelled NA (3H-NA) was used in parallel. At rest there was a net extraction of endogenous NA and adrenaline across the tissue. The SNS evoked overflow of endogenous NA was frequency-dependent and logarithmically correlated to the vasoconstrictor responses. The neuronal uptake inhibitor desipramine doubled the SNS induced overflow of endogenous NA without enhancing the vasoconstrictor responses. A further fourfold increase in NA overflow was caused by a dose of the alpha-blocker phenoxybenzamine which reduced the vasoconstrictor responses by 50-75%. Less than 10% of the spontaneous 3H efflux was recovered as unmetabolized 3H-NA, whereas virtually all 3H overflow evoked by SNS was 3H-NA. The fractional release of NA or 3H-NA per nerve impulse increased with increasing frequencies of SNS under all conditions studied. Although there was a preferential release of the newly stored radiolabelled transmitter, results concerning endogenous NA and 3H-NA overflow were qualitatively similar, also under conditions with marked changes in transmitter overflow. Endogenous NA gave a more reproducible index of transmitter overflow than did 3H-NA and, in particular, total 3H. The overflow of endogenous NA closely reflects SNS evoked neuronal release of NA in blood perfused skeletal muscle and seems more suitable than conventional radiotracer techniques for studies of NA release under in vivo conditions.     

Modulation of NA-synthase activity in rat cortex using NA measurement with ultramicro carbon electrode following topical applications of pharmacological agents

The aim of the study was to document NA, the active product of brain NA-synthase known as NO-synthase in rat cortex. NA measurements in brain extracellular fluid were performed using an ultramicro carbon electrode (0.5-2 microm) with differential pulse voltammetry. The ultramicro carbon electrode was inserted at a fixed depth (125 microm) into the frontal cortex. A constant level of neuronal NA (0.66 mM) was found in rat cortex during a few hours in the basal state. Topical applications of competitive inhibitors of brain NO-synthase (L-NNA, L-NMA, D-arginine, 1 mg ml(-1)) and a NO-donor (SNAP, 1 mg ml(-1)) resulted in a complete disappearance of NA. Simultaneous in vivo measurements of L-NNA, an electroactive inhibitor (-1.2 V vs. Ag/AgCl), and NA (-1.66 V vs. Ag/AgCl) allowed the following of the diffusion of this neuronal specific inhibitor and the simultaneous inhibition of NA synthesis. Topical addition of acetylcholine (10 microM) produced a NA increase, while bradykinin, adenosine, and hydrogen peroxide (10 microM each) resulted in the disappearance of NA. Topical addition of radical oxygen species (ROS) scavengers (oxy-hemoglobin, methylen blue, ascorbic acid and cystein, l mg ml(-1)) had no influence on NA concentrations which remained at a constant level in brain cortex. These preliminary results indicated that NA is continuously produced at a high level by neurons. Acetylcholine and vasodilatators modulated neuronal NA synthesis after topical application, but ROS had no effect.     

On the Noradrenaline Loading in Axonal Amine Storage Granules in Rat Crushed Sciated Nerves1

The increase of NA in rat sciatic nerve above a crush was investigated. The transported amounts of NA did not increase quite linearly with time, but more NA was found above the crush at 6 and 12 h than would be expected from the 3 h value. One possible reason for this phenomenon—an increased NA loading of the accumulated axonal granules—was investigated by 2 types of double crush experiments. One type involved simultaneous double crushes 1-1.5 cm apart. The increase in NA in the isolated segment 6 h after crushing indicated that the axonal amine storage granules had increased their NA load by about 70%. In the second type (“delayed double crushes”) a distal crush was made 6 h before a second crush, 1-1.5 cm proximal to the first crush. 1–12 h after the second high crush the NA content of the isolated segment was assayed. The results indicated an increased NA content in the axonal granules of 75% already 3–4 h after the isolation of the segment, remaining constant up to 9 h after the second crush. The results indicate that axonal storage granules may increase their NA content by a factor of about 2 (1.7) while being transported distally in the axons. This information together with the information from the preceding article of a mobile NA fraction of 45%, was used to calculate the rate of transport of NA granules proximal to a crush. The value obtained was 9 mm/h which is in good agreement with the value obtained for transport distal to a crush (8 mm/h) in the preceding article.     

Spontaneous and neurally evoked release of labelled noradrenaline from rabbit olfactory bulbs in vivo

1. Rabbit olfactory bulbs were labelled with either [(3)H]noradrenaline (NA) and [(14)C]urea, or [(14)C]NA and [(3)H]inulin. The labelled substances released were collected by a modified cortical cup technique and estimated by liquid scintillation spectrometry.2. In many experiments only total radioactivity originating from labelled NA was measured. In such cases the term NA radioactivity is used.3. The spontaneous release of both NA radioactivity and marker radioactivity followed a multiphasic course. After 140 min the rate of efflux of NA radioactivity was significantly slower than that of the labelled marker.4. Stimulation of one medial olfactory tract, one lateral olfactory tract or the surface of one bulb resulted in a selective increase in release of NA radioactivity. The size of the increase was dependent on the intensity, frequency and duration of stimulation.5. There was a characteristic delay in the increased release of NA radioactivity following electrical stimulation. Acute lesioning of the olfactory tracts caudal to the stimulating electrode not only abolished this delay, but also resulted in a larger increase in release following stimulation.6. The radioactivity attributable to unchanged NA was initially 91%, but decreased to 66% after 4(1/2) hr of sample collection. Stimulation did not affect significantly the relative amounts of NA and metabolites released.7. Replacement of calcium in the Krebs solution by magnesium did not affect measurably the spontaneous release of NA radioactivity. However, after 1 hr; stimulation failed to increase the efflux of NA radioactivity.8. It is suggested that the results provide further evidence for a role for NA in synaptic transmission in the olfactory bulbs.