More than 50 years have elapsed since Coupland made his classic observations on the ultrastructure of chromaffin granules in the rat adrenal medulla. Depending on the fine structure of secretory granules, two types of chromaffin cells can be distinguished: adrenaline-storing cells and noradrenaline-containing cells 1. It was generally accepted that the chromaffin cells of the adrenal medulla release their catecholamine content by exocytosis 1–3. This implies that the limiting membranes of chromaffin granules adhere to and fuse with the plasma membrane of the chromaffin cell, and that the granule content is subsequently secreted into the extracellular space. Recent studies have provided ultrastructural evidence for an alternative route of granule discharge from adrenomedullary cells, called piecemeal degranulation (PMD) 4–6. This consists of a particulate pattern of cell degranulation hitherto described in basophils, mast cells, and eosinophils 7–9. PMD basically differs from classical exocytosis because granules never fuse with the plasma membrane and never open to the cell exterior. In addition, secretory granules do not fuse with each other but empty in a particulate manner, leaving their individual structure intact. Viewed by electron microscopy, these organelles exhibit partial losses of content material, and at the end of the secretory process, granules transform into large containers devoid of secretory constituents or containing little amount of matrix remnants 10–13.
An asthmatic attack is directly due to the dysfunction of excitement and restraint of cholinergic nerves, adrenergic nerves, and their receptors. Owing to a lack of adrenergic nerves innervating human airway smooth muscles, the relaxation and constriction of airways are mainly regulated by catecholamine (epinephrine), which binds the adrenergic receptors of airway smooth muscles. Epinephrine is synthesized by the adrenal medulla, and then released into the circulation, and can effectively relieve the bronchoconstriction and other pathophysiologic reactions in asthma. Some data have indicated that epinephrine release is impaired in asthma. Meanwhile, others have reported that its concentration in the circulation cannot relieve the bronchoconstriction during an asthmatic attack 14,15.
It is also now recognized that glucocorticoids (GCS) are the most effective drugs for the long-term treatment of asthma. However, asthmatics still exhibit airway hyper-responsiveness (AHR) following even a prolonged treatment with GCS, suggesting an irreversible component to asthma that is not affected by treatment with these drugs 16. Furthermore, regular treatment of allergic rabbits with GCS failed to reverse baseline airway AHR, even though these drugs clearly inhibited allergen-induced exacerbations of AHR in the same species 17.
On the basis of the above-mentioned data, this study was designed to shed light on PMD as an alternative method for secretion in the normal adult rat chromaffin cells. Moreover, ultrastructural changes in the catecholamine granules as well as serum epinephrine and norepinephrine levels following chronic asthma were assessed. In view of the widespread use of systemic steroids in the treatment of asthma, it was necessary to determine whether these ultrastructural and serum changes will be corrected after dexamethasone (DEX) treatment.
Materials and methods
Experimental animals and design
Twenty-four adult male albino rats weighing 150–250g from the animal house of the Faculty of Pharmacy, Mansoura University, were used in this study. The rats were housed in single cages at 20°C on a 12-h light/dark cycle and had free access to food and water. The animals were divided into three equal groups at random: group A (control), group B (asthmatic nontreated rats), and group C (asthmatic rats treated with DEX). In rats of group B, the chronic asthma model was established by an intraperitoneal injection of 10% ovalbumin (OVA) (Winlab Company, Laboratory Chemicals, Leicestershire, UK.) on days 1 and 8 and then challenge with inhalation of 1% aerosolized OVA every other day for 8 weeks from day 15. The challenge was continued for 30min every time 18,19. Signs of bronchospasm were assessed by the presence of whistling wheeze, dyspnea, and acrycyanosis of the paws, limbs, ears, and tails. Sometimes, tachypnea and convulsions were also observed 20.
In animals of group C, chronic asthma was established by the same method and the rats were pretreated intraperitoneally with DEX at a dose of 2mg/kg 30min before each challenge 18–22. The control rats were intraperitoneally injected with equal amounts of isotonic saline at each time point of challenge 19.
Collection of blood sample
Twenty-four hours after the last challenge with OVA, blood was obtained from the ophthalmic venous plexus using a fine-walled Pasteur pipette. The rats of the three groups were anesthetized using halothane inhalation and the pipette was positioned at the inner corner of the eye beside the eyeball, and pushed gently but firmly along the side of the orbit to the ophthalmic venous plexus. The blood samples were then centrifuged at 1000 rpm and serum was stored at −20°C until biochemical analysis 23. Serum specimens were utilized for assay of epinephrine and norepinephrine levels using the enzyme-linked immunosorbent assay (ELISA) technique.
Bronchoalveolar lavage and lung tissue specimens
Immediately after obtaining blood samples, the animals of all groups were perfused through the heart apex with 100 ml of isotonic saline, followed by 250 ml of 2.5% glutaraldehyde in 0.1 mol/l cacodylate buffer (pH 7.3). The rats were then sacrificed and the right main-stem bronchus was occluded with a clamp and the left lung was lavaged three times through a tracheal cannula with 3 ml of sterile saline. The bronchoalveolar lavage fluid (BALF) was recovered manually by gentle aspiration with a disposable syringe after each infusion. The total cell count was estimated using a hemocytometer. After lavage, the right middle lung lobes from each rat were formalin fixed and embedded in paraffin. Tissue sections were stained with H&E 21,24.
Transmission electron microscopy
Immediately after sacrifice, the adrenal glands from the rats of all groups were removed and cleaned from adherent adipose tissue. The adrenal medulla was dissected into fine pieces, which were immersed in 2.5% gluteraldehyde in 0.1mol/l cacodylate buffer (pH 7.3) for 4h and then postfixed in 1% osmium tetroxide in 0.1mol/l cacodylate buffer (pH 7.3) for 2h. The specimens were dehydrated in ascending grades of alcohol, then subjected to two changes of propylene oxide, and finally embedded in epon. Semithin sections (1 μm thick) were stained with toluidine blue to select the proper sites for ultrathin sections (60–80 nm thick), which were cut, double stained with 2% uranyl acetate and lead citrate, and examined with a transmission electron microscope (TEM) 4,25.
Enzyme-linked immunosorbent assay
Epinephrine and norepinephrine levels in the serum were quantified using the ELISA technique with commercially available kits (CatCombi ELISA; RE59242, DRG international. Inc. USA). The reactions were read using an ELISA microplate reader (TLACOSR 496; Mabaret-Alasafra, Alexandria, Egypt) at 405 nm. The concentrations of epinephrine and norepinephrine were measured in pg/ml. Data were expressed as mean ± SD and analyzed statistically using Student's t-test. Values of P less than 0.05 were considered significant. The software package SPSS (Chicago, Illinois, USA) IBM was used for data analyses 24.
Quantitative evaluation of electron microscopic data
To assess whether PMD was a significant event in adrenomedullary chromaffin cells from asthmatic and DEX-treated rats, we performed a series of morphometric comparative analyses with the chromaffin cells from control animals. Ultrastructural quantitative investigations were conducted both in adrenaline-containing and in noradrenaline-containing cells and included determination of the total number of granules, number of resting granules, altered granules showing reduced or mobilized secretory components, and empty containers. Six adrenal chromaffin cells from the adrenal medulla of the animals of each group (three adrenaline cells and three noradrenaline cells) were selected at random and photographed at 8000× and 15000× magnification. Enlarged prints (18×13 cm) were analyzed for morphometric evaluation. The numbers were counted in groups of 30 low-magnification micrographs 26. Data entry and analysis was carried out using the software SPSS version 16. All data are expressed as mean ± SD. The one-way analysis of variance test with Tukey's post-hoc test were used. Values of P less than 0.05 were considered significant.
Total white cell count of bronchoalveolar lavage fluid
Total cell counts were significantly increased in the asthmatic group as compared with the control and DEX-treated groups (P < 0.05) (Fig. 1).
Serum levels of epinephrine and norepinephrine
The serum levels of epinephrine and norepinephrine in asthmatic rats were assayed by ELISA. Compared with the control rats, the levels of epinephrine and norepinephrine were decreased in asthmatic rats (P < 0.05). Treatment with DEX led to a significant increase in serum epinephrine and norepinephrine levels (P < 0.05), when compared with the asthmatic group (Figs 2a and b).
Histology of the lung tissues
H&E-stained paraffin sections of the lung tissue of asthmatic rats revealed the presence of congested blood vessels, perivascular edema, and heavy infiltration with inflammatory cells. Alveolar lumens contained extravasated blood and were infiltrated with inflammatory cells. Bronchiolar epithelial cells were partially detached. However, the control lung tissue presented clear alveolar lumens with no apparent inflammatory cellular infiltrate. Comparatively, these changes were reduced after DEX treatment, group C. The lung tissue exhibited little inflammatory cells, clear alveolar lumens, and unaffected bronchiolar epithelium (Figs 3–5).
Transmission electron microscopy of catecholamine granules in the control rat adrenal medulla
Groups of adrenaline-secreting and noradrenaline-secreting cells, separated by connective tissue septa, were found (Fig. 6).
The adrenaline-containing chromaffin cells exhibited several rounded or oval (150–300nm) granules. The majority of granules appeared electron dense, with a few moderate electron-dense ones. PMD manifested as budding from the original granules with tail-like projections and numerous small cytoplasmic vesicles less than 100 nm in diameter were recognized (Fig. 7).
Noradrenaline-containing chromaffin cells presented a large number of granules that were variable in size and shape: rounded, oval, or irregular, with characteristic asymmetrical contents. Some granules presented eccentric osmiophilic densities and most granules were subdivided into electron-dense and electron-lucent compartments. As compared with adrenaline-secreting cells, signs of PMD were manifested to a large degree. Partially empty containers with little osmiophilic residual contents, tail-like projections, and altered granules with eroded or mobilized constituents were observed. In addition, numerous small cytoplasmic vesicles were detected (Figs 8a–c).
Transmission electron microscopy of catecholamine granules in the adrenal medulla of the asthmatic nontreated rats (group B)
The adrenaline-containing chromaffin cells exhibited relatively fewer granules that were widely separated from each other. Fewer numbers of resting granules with an electron-dense core were recognized. Signs of PMD were more evident as manifested by an apparent increase in the number of altered granules with eroded or mobilized constituents, partially empty granules with little residual contents, and dilated empty containers. The mobilized constituents were observed throughout the cytoplasm and near the cell border (Figs 9 and 10). Similarly, features of PMD were markedly evident in noradrenaline-secreting cells as compared with the control: fewer number of resting granules, increased number and size of empty containers, and numerous altered granules. In addition, aggregates of mobilized constituents were observed throughout the cytoplasm (Figs 11 and 12).
Transmission electron microscopy of catecholamine granules in the adrenal medulla of the asthmatic rats treated with dexamethasone (group C)
The number of granules in adrenaline-containing chromaffin cells was relatively unaffected. The cells exhibited nearly the same number as the control. The majority of granules were resting, with no apparent empty containers (Figs 13 and 14). Similarly, noradrenaline-containing chromaffin cells exhibited almost the same number of granules as the control. The majority were resting, with electron-dense and electron-lucent compartments. Signs of PMD manifested as budding, altered granules, few empty containers, and numerous small cytoplasmic vesicles were recognized (Figs 15 and 16).
Quantitative evaluation of electron microscopic data
See Tables 1 and 2 for morphometric study of adrenaline-secreting and noradrenaline-secreting cells.
The total numbers of both adrenaline and noradrenaline granules in asthmatic rats were significantly lower than the control and DEX treated rats. While, signs of PMD were significantly higher in group B than the control and group C (Tables, 1 and 2).
There was an old general consensus that adrenal chromaffin cells discharge their granule content by exocytosis 1–3. However, the present study was in agreement with recent investigations 4–6 that provided further ultrastructural evidence for the hypothesis that these cells may express PMD as an alternative secretory pathway. Features of PMD manifested as budding, tail-like projections, altered granules with eroded contents, dilated partially empty granules, and large completely empty containers were observed. Cytoplasmic changes consisted of a rich supply of membrane-bound small-sized electron-dense vesicles that were either free in the cytoplasm or located close to the cell border. These signs were more prominent in noradrenaline-containing than adrenaline-containing chromaffin cells of the control rats.
This kind of cell secretion has classically been described in basophils, mast cells, and eosinophils 7–9. The authors explained how closed granules eventually release their constituents and have proposed the ‘shuttling vesicle’ theory. According to this interpretation, vesicles containing bits of granule materials would bud from the perigranule membrane. They then move through the cytoplasm and fuse with the plasma membrane. Thus, small quanta of secretory components flow into the extracellular space. The biological significance of this secretory process may be to extrude little amount of highly active and potentially harmful molecules, such as biogenic amines, in a slow and long-lasting manner. Thereafter, to maintain granule size, endocytotic vesicles would be retrieved from the plasma membrane and shuttle back to fuse with the granule membrane. If the rate and amount of vesicle traffic are balanced, granules would empty in a piecemeal manner but maintain a relatively constant size. If, however, the inward flow of endocytotic vesicles exceeds the outward flow of exocytotic vesicles, the granule chamber would become enlarged. The latter event might explain the occurrence of large-sized partially empty granules and empty containers. Moreover, the increased frequency of budding morphologies and small-sized cytoplasmic vesicles points in favor of an enhanced vesicle movement from the granule compartment to the plasma membrane and vice versa. This coupled process of vesicles moving back and forth between the granule compartment and the plasma membrane would affect mobilization and extrusion of granule constituents as well as insertion of new membrane material into the perigranule membranes, leading to both granule emptying and increased granule dimension 7–9. However, the inward flow of endocytotic vesicles was not demonstrated in this study and should be further studied.
The finding of PMD in medullary chromaffin cells raises some important questions as to the role of this secretory model in the physiological processes of catecholamine release, both in basal conditions and during stress-induced reactions. The adrenal medulla is considered the fast-acting arm of a stress response 27. Experimental conditions that generate acute stress in animals are accompanied by a rapid increase in the serum of both adrenaline and noradrenaline 28,29. Ultrastructural examinations of adrenal medullary glands under such conditions have demonstrated that the chromaffin granules move toward the peripheral cytoplasm and augment granule density beneath the plasma membrane, suggesting the ready release of granule material 30. Exocytosis is considered a rapid means of granule content discharge, and is therefore well suited to sustain a rapid adaptive reaction. By contrast, PMD is indicative of a slow, chronic release of secretions from secretory granules. As a consequence, the question arises as to whether PMD in the adrenal medulla is a physiological part of resting (stress-free) conditions or is indicative of chronic stress. Accordingly, the ultrastructural features of chromaffin cells in experimentally induced chronic asthma were investigated in the current study.
The chronic asthma model was established by the rat's general appearance, a significant increase in the leukocytic count in the BALF, and by histological changes in the lung tissue. These changes included congested blood vessels, perivascular edema, and inflammatory cellular infiltrate. This was consistent with previous data 18,22.
TEM of the adrenomedullary chromaffin cells revealed that the total number of adrenaline and noradrenaline granules was significantly reduced in asthmatic rats (group B) as compared with the control. This was associated with a significant increase in the size of the granules. Despite the prominent features of PMD observed in both adrenaline-storing and noradrenaline-storing cells, the serum epinephrine and norepinephrine levels were significantly decreased in asthmatic rats as compared with the control. This is consistent with previous published data 14,15,31. This might indicate that under conditions of bronchial asthma, the chromaffin cells cannot synthesize new granules to compensate the augmented secretory activity and granule loss. This finally leads to reduced serum levels of catecholamines.
Some studies have shown that plasma/serum epinephrine concentrations in asthmatic patients do not change after an exercise load, which was believed to induce an asthmatic attack. An equal exercise load does increase the epinephrine concentration in the control group 32,33. This indicates that the epinephrine release is impaired in asthma. It was postulated that, in chronic asthma, due to the homology between the adrenal medulla and sympathetic nerves, nerve growth factor may trigger the transition of chromaffin cells to sympathetic neurons. Consequently, the endocrine functions of the adrenal medullary chromaffin cells are impaired. Thereby, the composition of adrenal medullary hormones, and their synthesis, release, and uptake are altered and can lead to a decrease in epinephrine in the circulation 31.
GCS therapy is one of the most effective anti-inflammatory treatments available for asthma. This is likely to be due to multiple effects on the inflammatory response, including reduced production of cytokines by lymphocytes 34 and reduced expression of adhesion molecules, such as intercellular adhesion molecule-1, by vascular endothelial cells and airway cells 35. GCS have been shown to reduce antigen-induced infiltration of eosinophils, lymphocytes, and neutrophils 36 in Brown Norway rats and to reduce the numbers of eosinophils and mast cells in airway mucosal biopsy specimens from human participants with mild atopic asthma 37. However, it was observed that some asthmatics still exhibit AHR following even prolonged treatment with GCS, suggesting an irreversible component to asthma that is not affected by treatment with these drugs 16. Therefore, in the present study, DEX was injected before each challenge to clarify the possible protective effect of steroid therapy on the chromaffin cells of asthmatic rats.
As compared with the asthmatic nontreated rats, fewer leukocytes were detected in BALF. A previous investigation 38 has supported this finding and added that both inhaled and systemic GCS have been shown to block the antigen-induced increase in the number of BALF eosinophils. Serum epinephrine and norepinephrine levels were corrected to a large degree as compared with the asthmatic nontreated rats. In addition, there were no remarkable changes by TEM in the chromaffin cells in the number and size of granules, and features of PMD. These biochemical and ultrastructural data after DEX treatment are well in agreement with observations of previous investigators 39,40. They attempted to correlate between corticosterone and epinephrine secretion under basal and stress conditions and reported that a common mechanism links the secretion of these hormones, even though the adrenal medulla and cortex have different embryological origins. This mechanism is made possible by the intra-adrenal portal vascular system, which provides the medulla with uniquely high concentrations of corticosterone. These high concentrations induce activation of the medullary enzyme phenylethanolamine-N-methyltransferase, which controls the synthesis of epinephrine from norepinephrine. They added that after stress, adrenocorticotrophic hormone levels are elevated and exogenous DEX suppresses endogenous corticosterone and phenylethanolamine-N-methyltransferase production. Nonetheless, the authors suggested that serum catecholamine levels increase possibly due to direct neural stimulation, which may override the hormonal regulation of epinephrine synthesis during stress.
From the results of the current work, it could be concluded that there were features of PMD in the control adrenal chromaffin cells of adult rats and were augmented after experimentally induced chronic asthma. This indicates that this pattern of secretory activity is considered the principal method of secretion of the chromaffin cells under basal and chronic stress conditions. Despite the prominent features of PMD, serum epinephrine and norepinephrine levels were significantly decreased. This may indicate that the chromaffin cells are incapable of synthesizing new granules to compensate the increased granule loss in the condition of chronic asthma. These ultrastructural and biochemical changes are found to be corrected with DEX.
According to these biochemical and ultrastructural data, DEX can be considered a strong treatment modality in chronic asthma due to its effect on epinephrine and norepinephrine synthesis, secretion, and release.
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Conflicts of interest
There is no conflict of interest to declare.
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