Low activation threshold of juxtapulmonary capillary (J) receptors of the lung.

Respiratory reflexes arising from stimulating juxtapulmonary capillary (J) receptors by increasing doses of phenyl diguanide (PDG) were examined in 18 spontaneously breathing cats. In 60% an immediate and four-fold increase in breathing frequency (fR) was produced by doses as small as 5.1 ± µg/kg (range: 3.5-7.5) thus establishing that a significant increase in fR is produced by J receptors by stimulating them with minimal or threshold doses of PDG. In response to similar minimal doses of PDG J receptor afferent activity increased accompanied by acceleration of breathing rate. The response to supra threshold doses was either an apnoea followed by rapid shallow breathing (rsb) or to an apnoea preceded by rsb or only to rsb. Respiratory excursions counted from high-speed run records of intrapleural pressure revealed that the apnoeic response obtained in some cases was a phase of high-frequency breathing and not its suspension. These findings using a chemical stimulus demonstrate that J receptors, with some variability, have a very low threshold for stimulation resulting in notable respiratory acceleration. Thus their afferent output could increase significantly at low intensities of their physiological stimuli such as rise in cardiac output and incipient pulmonary congestion that are generated with mild exercise, to give rise to augmented breathing which is consequently seen.

Biodistribution of gold nanoparticles in mice and investigation of their possible translocation by nerve uptake around the alveolus.

The effect of nanoparticles in the environment on our health is a cause of concern. The greatest concern with respect to the biological effect of nanoparticles is that they remain in the body and invade tissues, overcoming the protective mechanisms of the body. It is generally believed that nanoparticles invading a living body move into the blood and are carried by the bloodstream to all organs. However, some studies have shown that the inhaled nanoparticles directly translocate to the central nervous system by nerve uptake. Here quantification of the amount of migration of nanoparticles to organs in short time spans (1, 3, and 6 hr) was attempted by animal experiments. Furthermore, the possibility of migration of nanoparticles through the nerves that project around the alveolus, including the nodose ganglion and dorsal root ganglion (DRG), was investigated. Gold (Au) nanoparticles (15 nm) were administered to mice by intratracheal instillation and tail vein injection. After tail vein injection, most nanoparticles were distributed in the liver. After intratracheal instillation, approximately 80% of detected nanoparticles remained in the lungs at 1 hr and were believed to be translocated to digestive organs, including the stomach and intestine, at 3 and 6 hr. With respect to quantification in ganglia, the levels in most samples were lower than the limit of quantification of inductively coupled plasma mass spectrometry (ICP-MS). However, Au nanoparticles were detected in DRG in only some samples of intratracheal instillation. Therefore, this suggests the possibility of translocation of nanoparticles to DRG via nerves.

Pulmonary neuroepithelial bodies are innervated by vagal afferent nerves: an investigation with in vivo anterograde DiI tracing and confocal microscopy.

The pulmonary airway and alveolar epithelia contain distinctly innervated clusters of basally granulated cells: the neuroepithelial bodies. In the past, morphological criteria and the results of selective vagotomy have led to the interpretation that their innervation is sensory. Consequently, they are regarded as receptor organs. As a further test of this hypothesis, the present investigation set out to label vagal sensory nerve fibres to the lungs by anterograde neural tracing, and to establish the relationship between these fibres and the neuroepithelial bodies. A fluorescent neural tracer was injected unilaterally into the left or right nodose ganglion of adult rats. After suitable survival times, thick frozen sections of lung tissue were studied with laser scan confocal microscopy. Sensory nerve fibres were seen to run in the airway walls and occasionally penetrated the epithelium, where they formed complex terminals. The resulting intraepithelial sensory end organs showed a close morphological resemblance to the neuroepithelial bodies. Subsequently, electron microscopic investigation of such identified structures revealed the typical ultrastructural characteristics of neuroepithelial bodies: corpuscular cells containing dense cored secretory vesicles and contacted by mitochondria-rich nerve endings. We conclude that anterograde tracing of sensory nerves from the nodose ganglion confirms the receptor nature of the pulmonary neuroepithelial bodies, which may correspond to a subpopulation of the irritant and C-fibre receptors.

Capsaicin-activated bronchial- and alveolar-initiated pathways regulating tracheal ciliary beat frequency.

We questioned whether the prolonged stimulation of ciliary beat frequency (CBF) to a short exposure of low-dose capsaicin (Wong et al. J. Appl. Physiol. 68: 257-2580, 1990) could be due to the activation of indirect pathways involving neural reflexes initiated independently in the bronchi and alveoli. Tracheal CBF (CBFtr) was measured temporally in anesthetized groups of 10 dogs by means of heterodyne-mode correlation analysis laser light scattering. To elucidate the site of the afferent neural stimulation and the efferent mediators affecting the ciliated epithelium, capsaicin (3 nM) aerosol was delivered for 4 min, either predominantly to the bronchi or to the alveolar regions, with use of pulsed aerosol techniques. This resulted in 13 pg of bronchial (85%) and 10 pg of alveolar (96%) capsaicin deposited, which caused marked stimulation of CBFtr with maxima at 7 and 35 min, respectively. Prior administration of aerosolized indomethacin to the bronchi or aerosolized cromolyn to the alveoli inhibited the bronchial and alveolar responses, respectively. Prior administration of aerosolized hexamethonium to the tracheal lumen blocked the stimulatory CBFtr responses from both capsaicin challenges. Ipratropium or propranolol aerosols delivered to the tracheal lumen also inhibited these responses. It is proposed that these pathways comprise one set of sensitive mechanisms to ensure a prolonged stimulation of CBF to effect the removal of secretions and the irritant from the lungs.

Ultrastructure and innervation of neuroepithelial bodies in the lungs of newborn cats.

Neuroepithelial bodies (NEB) occur throughout the airway mucosa and alveolar parenchyma of kitten lungs. In the bronchi, they are often situated on top of a cartilage plate. They form compact corpuscles containing 10-20 corpuscular cells and appear covered with a layer of flattened Clara cells. Kitten NEB are occasionally observed to display mitosis of the corpuscular epithelial cells. A prominent blood capillary lies at their basal pole. The corpuscular cells contain numerous dense core vesicles (DCV), whose number and diameter remain unchanged with age. Kitten NEB are innervated by nerve fibres that "loop" through the corpuscle and form morphologically afferent as well as efferent nerve endings. The nerve endings display afferent synaptic junctions with the corpuscular cells and sometimes run in clusters, so that they contact each other. Many nerve endings undergo spontaneous degeneration. We conclude that kitten NEB are well adapted to function as chemoreceptors and as endocrine or paracrine organs. Their chemoreceptor activity could be modulated by axon reflexes since their afferent nerve endings are often continuous with the efferent ones, as well as by interneural modulation since nerve endings often form clusters. In addition, kitten NEB innervation appears to involute rapidly soon after birth. This may indicate that their chemoreceptor function is only of primary importance during gestation and at birth. However, the secretory function of kitten NEB, as evidenced by the unchanged numbers and dimensions of their DCV, seems to remain steady throughout life.

Tachykinin-, calcitonin gene-related peptide-, and protein gene product 9.5-immunoreactive nerve fibers in alveolar walls of mammals.

The presence and distribution of the presumed pan-neural marker protein gene product 9.5 (PGP)- and peptide-immunoreactive (ir) nerve fibers in alveolar walls of various species was investigated by light microscopic single and double staining immunohistochemistry. PGP-, tachykinin (TK)-, and calcitonin gene-related peptide (CRGP)-ir fibers were sparsely distributed in a similar pattern in alveolar walls of all species investigated. No vasoactive intestinal peptide-, peptide histidine isoleucine-, galanin-, and opioid-ir nerve fibers could be detected. PGP-ir fibers outnumbered those staining for TKs and CGRP. There was partial coexistence of PGP and TK as well as of TK and CRGP. PGP-, TK-, and CGRP-ir fibers were in close spatial relationships to the cells building up the alveolar walls and to alveolar capillaries. The function of PGP is unknown. TK- and CGRP-ir nerves in alveolar walls may be sensory and function as chemo-, stretch-, and/or immuno-receptors. TKs and CGRP released from alveolar fibers may influence the alveolar epithelium and the various non-epithelial alveolar cells, including immune cells. The alveolar TK and CGRP innervation may be of unrecognized importance in physiological and pathophysiological regulation of lung functions.

Calcitonin gene-related peptide and its mRNA in pulmonary neuroendocrine cells and ganglia.

The occurrence of calcitonin gene-related peptide (CGRP) and it's mRNA was studied in lungs of rats and piglets using in situ hybridization with two synthetic oligonucleotide probes followed by immunocytochemistry (ICC). CGRP mRNA was present in pulmonary neuroendocrine cells (PNEC) of both the solitary type and cluster type (neuroepithelial body; NEB) at all levels of the airway epithelium from bronchi to alveoli. The distribution of labelled cells was similar to that previously described with ICC. The 44-mer probe provided stronger hybridization signal than the 34-mer and the two combined increased labelling slightly. Formalin fixation reduced labelling and tended to increase background. Labelling for CGRP mRNA was evenly distributed over the cytoplasm, whereas CGRP-like immunoreactivity (LI) usually was of highest intensity toward the base of the PNEC, suggesting basal accumulation of synthesized peptide. CGRP-LI was also observed in occasional rat ganglia and in some, but not all, piglet ganglia. These local neurons may contribute to the CGRP fibers of airways and vasculature, and could theoretically bridge their dendrites and axons between NEB and the effector organ (e.g. artery or arteriole) thus accomplishing a function similar to the postulated axon reflex.

Unilateral diaphragmatic paralysis inhibits postnatal lung growth in the piglet.

To test the hypothesis that regional growth of lung parenchyma depends on regional distending pressure, left cervical phrenectomy was done in ten 2-month-old piglets. The unilateral diaphragmatic paralysis reduced mean transpulmonary pressure in the left hemithorax from 5.5 +/- 1.0 (means +/- SD) to 2.5 +/- 1.2 cm H2O (p less than 0.01, n = 5). When five of the piglets were killed 48 h later, wet lung weight, total protein content, and nucleic acid content did not differ from values in the five sham operated controls. The five remaining phrenectomized piglets were compared to their five sham-operated controls 7 days after surgery. Wet weight of the left lung was reduced by 29% (p less than 0.01) and DNA content was reduced by 18% (p less than 0.05). Wet weight of the right lung, contralateral to the paralyzed hemidiaphragm, was reduced by 11% (p less than 0.05). At this time, body weight, bilateral transpulmonary pressure, and ratios of total protein/DNA and RNA/DNA in lung tissue did not differ from the sham-operated controls. These results suggest that regional growth of lung parenchyma by cell proliferation adjusted to changes in regional transpulmonary pressure caused by the unilateral phrenectomy.

[Characteristics of the secretory activity of the respiratory cells of mouse lungs after partial chemical sympathectomy]. / Osobennosti sekretornoi aktivnosti kletok respiratornogo otdela legkikh myshei posle chastichnoi "khimicheskoi simpatéktomii".

Partial "sympathectomy" in the neonatal BALB mice was achieved by the treatment with guanethidine. The number of neurons in the stellate ganglion decreased to 20% of the control values and remained constant throughout the subsequent period of 1 month. Partial "chemical sympathectomy" did not affect the postnatal growth and development of the lungs. Partial "chemical sympathectomy" significantly increased the number of secreting cells in bronchiolar and alveolar regions. Secretory activity of the alveolocyte population, type two, synthetizing and secreting surfactant also increased. It has been concluded that the partial "chemical sympathectomy" stimulated the alveolar surfactant secretion.

[Role of the pulmonary mechanoreceptor apparatus in molding the natural respiration of the frog Rana temporaria]. / Rol' mekhanoretseptornogo apparata legkikh v formirovanii estestvennogo dykhaniia u liagushki Rana temporaria.

In acute experiments on the frog Rana temporaria, studies have been made of the effect of afferent impulsation in pulmonary mechanoreceptors on respiration pattern. Simultaneous recordings were made of total afferent activity in the pulmonary branch of the vagal nerve and of the activity of motor respiratory nerves as an index of respiration pattern. It was shown that to the end of ventilation period, the level of afferentation decreases up to a threshold value which favours the onset of inspiration. Artificial decrease of afferentation level by the increase in CO2 content in the lung or by novocain application to the latter always resulted in extra-inspiration. On vagotomized frogs, it was found that maximum level of afferent influences is necessary for the onset of expiration.

[Receptor apparatus of the bronchioles and pulmonary alveoli]. / Retseptornye apparaty bronkhiol i legochnykh al'veol.

Applying the method of silver impregnation in modification by Bielschowsky--Gros and Lavrentiev, two main types of sensitive neural terminals have been revealed in alveolar parenchyma: 1) sensitive neural terminals of bushy type, situating in the connective tissue of the alveolar walls, immediately under flat epithelium covering them; 2) sensitive neural apparatuses with a complex structure, protruding into the alveolar lumen. They are seen as an accumulation of epithelial cells, among them fine branches of myelin neural fibres (neuroepithelial bodies) terminate. Owing to space arrangement of the terminal branchings in the first type terminals, it is possible to consider them as mechanoreceptors accepting impulses which are connected with certain shifts in the pulmonary parenchyma during the process of respiration. Peculiar structure of the second type sensitive apparatuses (neuroepithelial bodies) and their arrangement in the pulmonary alveolar lumen suggest their connection with changes in the alveolar air composition. They are considered to be airreceptors.

Innervation of alveolar walls in the human lung: an electron microscopic study.

Unmyelinated nerve fibres, probably sensory in type, are present in the human alveolar wall. It is possible that these fibres may be associated with juxta-capillary receptors which have been identified on physiological grounds in animals.

Innervation of rabbit fetal lungs.

Nerve fibers, autonomic ganglia, and neuroepithelial bodies of the lungs of rabbit fetuses, 17 to 31 days gestational age, were studied with neurohistological techniques including silver impregnation, acetylcholinesterase histochemistry, and glyoxylic-acid-induced histofluorescence for monoamines. The silver impregnation method showed that nerve fibers and ganglia accompanied the bronchi and large pulmonary blood vessels to enter the developing lungs by the 17th day of gestation. Cholinergic and adrenergic nerves began to appear in the walls of the bronchi on the 21st day. The developing pulmonary arteries had accompanying adrenergic nerves on the 25th day. Acetylcholinesterase-positive parasympathetic ganglia were seen on the 27th day. Silver-impregnated nerve fibers in the developing alveolar walls and pleura were found on the 25th day. Neuroepithelial bodies and specialized single cells which were argyrophilic, acetylcholinesterase-positive, and fluorescent could be demonstrated in 19--21-day-old and older fetuses; and some of these structures were innervated by sensory and autonomic motor fibers. These observations indicated that nervous tissue and neuroepithelial bodies appeared in the lungs during the glandular stage of the lung development and that differentiation of adrenergic and cholinergic nerves began in the late glandular stage.

Distribution and ultrastructural characteristics of Feyrter cells in the rat and hamster airway epithelium.

Feyrter cells were found both singly and in groups at all levels of the respiratory tract studied in both rats and Syrian golden hamsters. Particularly large and prominent groups of Feyrter cells were present at bronchiolar bifurcations and bronchiolar-alveolar junctions. Single Feyrter cells were also found throughout the respiratory tract, but their distribution appeared entirely random. In all cases examined the groups of Feyrter cells were overlaid by intermediate and serous secretory epithelial cells. Occasionally small areas of the Feyrter cells were exposed to the airway lumen. Small canaliculi-like intercellular spaces were present between adjacent Feyrter cells. Single unmyelinated axons which contained neurotubules, mitochondria, and vesicles were occasionally seen in close association with Feyrter cells.

Ultrastructural evidence for the innervation of human pulmonary alveoli.

Electron Microscopic observations of the biopsied human pulmonary alveoli showed the occurrence of unmyelinated axons in the interstitium near the type I pneumocytes. These axons very likely have sensory functions.

Histological and histochemical studies on the innervation of the respiratory organs of Calotes versicolor Daud. (Agamidae, Lacertilia).

A study on the innervation of the respiratory organs of Calotes versicolor has been made using cholinesterase and silver reduction techniques. The tracheal and bronchial plexuses have been described. A number of nerve terminations along with synapses are reported. ChE activity has been observed on the periphery of the alveoli which is believed to be of importance in controlling the acetylcholine level, acting as a general tissue enzyme to prevent excessive accumulation of acetylcholine. Vascular nerve supply has been found to be largely of vagal origin.

[Relationship between hypercapnic and hypoxic stimuli of respiration during muscular activity]. / Vzaimodeistvie giperkapnicheskogo i gipoksicheskogo stimulov dykhaniia pri myshechnoi rabote.

Pulmonary ventilation (V) and alveolar gas composition (PACO2, PAO2) were studied in 12 healthy men who performed gradual muscular work under conditions of controlled hypercapnia, hypoxia, hyperoxia or their combinations. The respiratory response was estimated by absolute values of ventilation at the given PACO2 value and by its rise by 1 mm Hg of increased PACO2 (delta V/delta PACO2) under rest and under transitional and steady-state exercise. The exercise on-switch was accompanied by displacement to the top and an increased slope of the response curve (delta V/delta PACO2) not related to the work load. These changes suggest multiplicative interaction of the neurogenic and hypercapnic drives in the load switch-on. During steady-state exercise an important role of the hypoxic drive was revealed: hypoxemia induced a shift of the delta V/delta PACO2 response curve to a higher level, especially with the great work load. Thus the positive interaction between the hypercapnic and hypoxic respiratory drive augments with muscular exercise.

Influence of sympathetic nervous system on surfactant production. An ultrastructural study.

The influence of the sympathetic nervous sytem on pulmonary surfactant was investigated in an ultrastructural study of normal and chemically sympathectomized rat lungs. Chemical sympathectomy was accomplished by administration of 6- hydroxydopamine (6-OHDA). The 6-OHDA did not have any systemic influence on the experimental animals. These animals grew normally and were killed at intervals of three, four, six, and seven weeks. The mean weight of the controls at three weeks was 41.75 +/- 3.12; at four weeks, 77.00 +/- 2.68; at six weeks, 169.25 +/- 11.29; and at seven weeks, 182.25 +/- 4.94. The mean weight of the 6-OHDA treated group at three weeks was 48.50 +/- 1.04; at four weeks, 76.25 +/- 1.93; at six weeks, 171.75 +/- 3.40; and at seven weeks, 180.25 +/- 2.36. The body/ lung weight ratio was about the same in both groups. In the control animals, the alveolar capillary membranes were intact, the alveoli were well expanded, and epithelial cells appeared normal. The cytoplasm of the granular II pneumocytes contained their characteristic osmiophilic lamellated bodies as well as multiple transition forms between the multivesicular and lamellated bodies. It is these lamellated bodies that are the suspected site of surfactant production in the lung and they appear to migrate towards the alveolar lumen. In addition to the lamellated bodies, multiple lattice-like tubular myelin figures were abundant in the alveoli of the normal rat lung. Following sympathectomy, structural alterations occurred within the cytoplasm of the granular II pneumocytes, involving mainly the lamellated bodies. There was a decrease of osmiophilic content as well as loss of organization of the bodies. This change was quantitated by scoring the degree of osmiophilia in the control and sympathectomized animals. The mean score in the control animals was 4.43 +/- 0.03 S.E., whereas in the sympathectomized group it was 3.77 +/- 0.16 S.E. It was concluded that the lamellated bodies, the site of phospholipid synthesis in the granular II pneumocyte, arise from the multivesicular bodies and are extruded into the alveolus. They then participate in the formation of the alveolar acellular lining layer. Sympathectomy alters the structure of the lamellated bodies, thereby influencing the development of the surfactant system.