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Annual report : National Institute of Environmental Health Sciences (Volume 1981) online

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steps in the formation of PG's from AA.



635



PHS-6040
(Rev. 2-81)



ZOl ES 80008-07 LP FT

PROJECT DESCRIPTION

METHODS EMPLOYED : Prostaglandin (PG) thromboxane (TX) and hydroxy fatty acid
(HFA) synthetase activity was measured in vitro using the microsomal protein
from a variety of tissues and organs as an enzyme source. C-arachidonic acid
(AA) or prostaglandin endoperoxides were incubated at 37°C for various times and
under several conditions. After incubation, the PG and TX were removed by solvent
extraction, separated by thin-layer chromatography or high pressure liquid chroma-
tography, and estimated by liquid scintillation techniques.

An isolated perfused rat, guinea pig, or rabbit lung was used to examine the up-
take, metabolism, and efflux of prostaglandins (PG's) and their metabolites from
lung tissue. The isolated perfused lung was designed to permit infusion of a
constant concentration of PG's and perfusion with drug- free perfusate. PG metabo-
lites were isolated from the perfusate by extraction and separated by thin-layer
chromatography techniques. The unidirectional flux of PG into the lung was
measured by extrapolation of the net uptake velocity of PG to zero time. PG and
TX from either incubation mixtures or perfusate of lung were also measured by
radioimmunoassays.

MAJOR FINDINGS AND PROPOSED COURSE : A rapid and simple method based on HPLC was
developed for the separation of PG's. This system was expanded to permit further
separation of HFA. We are currently developing a new method which will separate
leukotrienes (LT). Using this system we have studied the formation of AA meta-
bolites in a variety of test animals and in human lung. Using either AA or PGH-p,
the human lung made PGI2 and TXAo in approximate equal amounts. Rat lung was
very similar to human lung in producing PG's from PGH2. Guinea pig lung pro-
duced mainly TXA2 and was least like human lung. Except for guinea pig lung, con-
version of AA to PG's was very low and appears to be due to inactivation of cyclo-
oxygenase during tissue preparation.

On incubation of human lung with AA the major product was^an unknown HFA. The
formation of this HFA was increased by the presence of Ca and ionophore in the
incubation system. HPLC analysis indicated that the HFA was not a
mono- hydroxy la ted HFA nor 8,15-diHETE. The unknown may be 5,12-diHETE the degrada-
tion product of LT's. Further studies are in progress to determine the structure
of this HFA. In addition, we are now studying the conversion of PGH2 to PG's
using isolated veins, arteries and bronchi from human lung to investigate
regional difference in AA metabolism.

We have also studied the mechanisms involved in the oxidation of AA to PG's.
Using ESR spin trapping techniques, we have identified a fre.i radial involved in
the oxidation of AA to PG's in ram seminal vesicle microsomes. Our data suggest
that the trapped free radical is carbon-centered free radical at C-11 and is pro-
bably the first intermediate formed during the oxidation of arachidonic acid to
PG's. We are currently using purified cyclo-oxygenase to further study the
process.



636



ZOl ES 80008-07 LPFT

SIGNIFICANCE TO BIOMEDICAL RESEARCH AND THE PROGRAM OF THE INSTITUTE : PG's and
HFA have a large diversity of physiological effects. Alterations in PG control
of cellular events may be related to transport of PG's across cell membranes.
The lung is an important site for the synthesis and metabolism of PG's, altera-
tions in the PG biosynthesis, release, transport and metabolic systems may be
related to toxic effects of exposures to pollutants and induction of lung diseases.
The lung makes a variety of PG and HFA but little is known of the cells respons-
ible for biosynthesis. This information appears to be important for the eluci-
dation of the role of PG in pulmonary disease.

PUBLICATIONS

Crutchley, D. , Boyd, J., and Eling, T. E.: Potentiation of TXB release from
guinea pig lung during anaphylaxis following exposure to 100% 0,. Amer. Rev.
of Resp. Dis. 121: 695-699, 1980. '^

Eling, T., Warnock, R., Dick, D., and Tainer, B.: Separation of PG, TX, HFA
and arachidonic acid by high pressure liquid chromatography. Prost. and Med.
5: 345-355, 1980.

Eling, T. , Tainer, B., Ally, A. and Warnock, R.: Separation of arachidonic
acid metabolites by HPLC. Method in Enzymology (in press) 1981.

Eling, T. and Ally, A.: Pulmonary biosynthesis and metabolism of prostaglandins
and related substances. Bull. Europ. Physicol. Resp. (in press) 1981.

Mason, R., Kalyanaraman, B., Tainer, B., Eling, T. E.: A carbon centered free
radical intermediate in prostaglandin synthetase oxidation of arachidonic acid.
J. Biol. Chem. 255: 5019-5022, 1980.



637



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PROJECT NUMBER (Do NOT use this space)



U.S. DEPARTMENT OF

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PROJECT NUMBER



Z01 ES 80029-05 LPl-T



PERIOD COVERED

October 1, 1980 to September 30. 1981



TITLE OF PROJECT (80 characters or less)

Investigations of Human Pulmonary Diseases



NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT



PI:
Others :



G. E. R. Hook
L. B. Gilmore



Head, BPG
Biologist



LPFT
LP FT



NIEHS

NIE;,S



COOPERATING UNITS (If any)

A. Spock, M.D.

Department of Pediatrics, Duke University



lab/branch
Laboratory of Pulmonary Function and Toxicology



SECTION

Biochemical Pathology Group



INSTITUTE AND LOCATION

NIEHS5 NIHs Research Triangle Park, North Carolina 27709



TOTAL MANYEARS;



1.0



PROFESSIONAL:



0.5



OTHERt



0.5



CHECK APPROPRIATE BOX(ES)
D (a) HUMAN SUBJECTS

n (al) MINORS n (a2) INTERVIEWS



D (b) HUMAN TISSUES



[)} (c) NEITHER



ine key'

Identification and characterization of disease- re la ted pulmonary components could
provide information concerning the disease process and also function as markers
for the diagnosis and monitoring of the disease. Current attention has focused
on the multilamellated myelin- like structures present in the alveoli and airways
of patients with pulmonary alveolar proteinosis . The objectives of this study
have been to elucidate the composition, structure and origins of this unusual
material .



638



PHS-6040
(Rev. 2-81)



ZOl ES 80029-05 LPFT



PROJECT DESCRIPTION

METHODS EMPLOYED : Bronchoal veolar lavage effluents from patients with pulmonary
alveolar proteinosis were supplied by the Department of Pediatrics at Duke
University Medical Center. These lavage effluents were obtained as a by-product
of the therapy essential to the well being of the patients.

Insoluble materials were sedimented by centrifugation and then separated from the
soluble phase for examination under the electron microscope following dehydration
and embedding.

MAJOR FINDINGS AND PROPOSED COURSE : A major component of the insoluble materials
present in lavage effluents from the lungs of patients with alveolar proteinosis
consists of multi-lamellated myelinlike structures (MS) which appear to form extra-
cellularly in the alveoli and airways of patients with this disease. Ihe MS con-
sist of trilaminated membranes whose thickness varies from 85 to 100 A. These
membranes are separated from each other by a region of amorphous material between
150 A and 300 A wide. Digestion of the structures with proteases results in
destruction of the amorphous material indicating that it must consist primarily of
protein. Digestion of the structures with phospholipase C results in the destruc-
tion of the membranous constituents indicating that the membrane must be com-
posed of phospholipid. The structures are also severely disrupted by chaotropic
agents such as potassium thiocyanate and potassium iodide, suggesting that the
structures are held together by hydrophobic interactions and not by covalent bonds.

These myel in-like structures will be further investigated as to their protein and
lipid composition and compared with normal constituents of extracellular lining.

SIGNIFICANCE TO BIOMEDICAL RESEARCH AND THE PROGRAM OF THE INSTITUTE : Many
human pulmonary diseases have been described but unfortunately the diagnosis of
these diseases is not usually made until the disease is well advanced. X-ray
methods generally are not capable of detecting pulmonary diseases except in the
advanced states and even then are often incapable of distinguishing between
diseases. Methods for the diagnosis of pulmonary disease and the detection of pul-
monary damage in the earliest possible stages are needed.

Our studies indicate that the myelin-like material which accumulates in the lungs
of patients with pulmonary alveolar proteinosis is closely related to tubular
myelin as found in the lungs of normal humans. Apparently, in the lungs of
patients the tubular myelin structure is assembled in an aberrant manner.

PUBLICATIONS

Nadeau, D. , Reasor, M. J., and Hook, G. E. R. : Extracellular alkaline phosphatase
from alveolar secretions of patients with pulmonary alveolar proteinosis. Can. J.
Biochemistry 59: 290-300, 1981.



639



smTthsqnIan science information exchange

PROJECT NUMBER (Oo NOT use this space)



U.S. DEPARTMENT OF

HEALTH AND HUMAN SERVICES

PUBLIC HEALTH SERVICE

NOTICE OF

INTRAMURAL RESEARCH PROJECT



PROJECT NUMBER



ZOl ES 80032-08 LPFT



PERIOD COVERED . , „„,

October 1, 1980 to September 30, 1981



TITLE OF PROJECT (80 charactops or less)

The Composition and Origins of the Acellular Lining Layer of the Lung



NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI: G. E. R. Hook Head, BPG LPFT NIEHS

Others: J. W. Spalding Research Biologist LPFT NIEH?

L. B. Gilmore Biologist LPFT NIEHS

S. Douthit Bio. Lab. Technician LPFT NIEHS



COOPERATING UNITS (if any)

M, Ortner, Ph.D.

Laboratory of Environment of Biophysics



lab/branch
Laboratory of Pulmonary Function and Toxicology



SECTION

Biochemical Pathology Group



INSTITUTE AND LOCATION

lljJEHS, NIH, Research T,ri.annlp Par k . N orth Carolina 27709 ,



NIEHS

TOT At MA



MAflYEARS:'



3.0



PROFESSIONAL:



1.5



1.5



CHECK APPROPRIATE BOX(ES)
D (a) HUMAN SUBJECTS

g (al) MINORS D (a2) INTERVIEWS



D (b) HUMAN TISSUES



H (c) NEITHER



SUMMARY OF WORK (200 words or X«ss - underline keywords)

The alveoli and distal airways of the lung are lined with an acellular layer of
material which is essential for the maintenance of normal pulmonary functions
such as gas exchange. The composition and origins of the acellular lining are
being investigated. Current attention has been directed towards: (1) the
biosynthesis and secretion of pulmonary surfactant and (2) tho nature of the cytc
plasmic organelles known as lamellar bodies. The objectives of this investigation
are as follows: (1 ) to further develop methodology for the isolation of lamellar



bodies from the lungs of rabbits (2) characterize the lamellar bodies according
to phospholipid, protein and enzymic components, (3) to elucidate processes in
volved in the formation of the extracellular lining from secreted lamellar bodies



640



PHS-60M
(Rev. 2-81)



ZOl ES 80032-08 LPFT

PROJECT DESCRIPTION

METHODS EMPLOYED : Acellular lining material is obtained by lavaging the lung
of rabbits via the trachea. Lamellar bodies from the cytoplasm of Type II cells
are isolated on discontinuous sucrose gradients using differential centrifugation.
Enzyme and protein analyses are carried out using polyacryl amide gel electrophoresis.

MAJOR FINDINGS AND PROPOSED COURSE : Lamellar bodies of the alveolar Type II cells
are storage sites of pulmonary surfactant. These structures are secreted into the
alveoli where the surface active phospholipids help stabilize the distal regions of
the lungs.

Lamellar bodies were isolated from homogenized lungs of rabbits using a method
developed in this Laboratory involving the use of discontinuous sucrose gradients.
The isolated lamellar bodies appeared similar to those found in the cytoplasm of
Type II cells insofar as their appearance under the electron microscope is
concerned. The lamellar bodies retained their lamellae, perilamellar membrane
and amorphous core. The structures were analyzed according to their phospholipid
and fatty acid composition. Analysis of the structures by isopycnic centrifuga-
tion indicated that NADPH cytochrome C reductase detected in the lamellar bodies
may be a component and not a microsomal contaminant as previously assumed.

The membranous components of the extracellular lining have been isolated from
pulmonary lavage effluents from the rabbit. The fluidity characteristics of
these membranes have been studied using electron paramagnetic resonance (EPR)
and the probe 5-doxylmethyl stearate. These studies indicate that the extracellular
membranes are highly fluid structures. However, compared with the extracellular
membranes, the major constituent of those membranes dipalmitoyl phosphatidyl choline
(DPPC) is not very fluid at physiological temperatures. The fluidity of DPPC is
modified considerably by the presence of other phospholipids.

These studies of lamellar bodies and pulmonary surfactant will continue. Lamellar
bodies will be further characterized as to their enzymic components and ability
to synthesize phospholipids. The fluidity properties of the membranous constituent
of the extracellular lining will be further studied and attempts will be made to
identify the active constituent responsible for modifying the fluidity of DPPC.

SIGNIFICANCE TO BIOMEDICAL RESEARCH AND THE PROGRAM OF THE INSTITUTE : The
acellular lining of the lung is vital for the maintenance of normal lung functions
such as gas exchange. Inhaled toxicants such as the oxidant gases (e.g., ozone),
particulate materials (e.g., silica) and chemicals (e.g., paraquat) appear to
affect the acellular lining both qualitatively and quantitatively. The involve-
ment of the acellular lining in the progression and mediation of some pulmonary
diseases such as alveolar proteinosis appears certain. Unfortunately, the
mechanisms which underlie these pulmonary diseases and agent-induced lung damage
are not known. Elucidation of the biochemical processes which contribute to the
formation of pulmonary surfactant and the acellular lining are a necessary step
in the understanding of the disease process.



641



ZOl ES 80032-03 LPFT



PUBLICATIONS

Bell, D. Y., Haseman, J. A., Spock, A., McLennan, G., and Hook, G. E. R. : Plasma
proteins of the bronchoalveolar surface of the lungs of smokers and nonsmol;ers.
Am. Rev. Resp. Dis. (in press) 1981.

Gilmore, L. B. and Hook, G. E. R. : Quantitation of proteins in polyacryl amide gels
by the elution of Fast Green FCF. J. Biochem. Biophys. Methods (in press) 1981



642



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PROJECT NUMBER (Do NOT use this space)



U.S. DEPARTMENT OF

HEALTH AND HUMAN SERVICES

PUBLIC HEALTH SERVICE

NOTICE OF

INTRAMURAL RESEARCH PROJECT



PROJECT NUMBER



ZOl ES 80033-05 LPFT



PERIOD COVERED



OrtnhPr 1, IQfiO tn .September 30. 1981



TITLE OF PROJECT (80 characters or less)

Neuroendocrine (Small Granule) Cells of the Lung



NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT



PI:


R. P. Di Augustine


Head, EG


Others:


D. Vembu


Staff Fellow




I. Linnoila


Staff Fellow



LPFT
LPFT
LP



NIEHS
NIEHS
NCI



COOPERATING UNITS (if any)



Histology Laboratory, TRTP, NIEHS



lab/branch
Laboratory of Pulmonary Function and Toxicology



SECTION

Endocrinology Group



NSTITUTE AND LOCATION

NIEHS, NIH, Research Triangle Park, North Carolina 27709



TOTAL MANYEARS:



3.5



PROFESSIONAL:



3.0



CHECK APPROPRIATE BOX(ES)
D (a) HUMAN SUBJECTS

D (al) MINORS n (a2) INTERVIEWS



OTHERj



0.5



D (b) HUMAN TISSUES



^ (c) NEITHER



SUMMARY OF WORK (200 words or less - underline keywords)

The epithelium of the lung of mammals is known to contain at least two principal
classes of neuroendocr i ne- 1 i ke , or small-granule cells , those which exist as
solitary cells and those which exist in an organoid structure referred to as
the neuroepithelial body . (1) In one study, quantitation of the solitary neuro-
endocrine cells of the cricoid epithelium of the guinea-pig larynx by electron
microscopy revealed a homogeneous population consisting of 4-9% of the total epi
thelial cells. The neuroendocrine cells identified in the tracheal epithelium
had the same ultrastructural properties as those observed in the cricoid. (2)
Continued efforts to identify known polypeptide hormones in the trachea by immuno
cytochemical techniques revealed no positive cells. (37 Tracheal neuroendocrine
cells, identified by argyrophilia in light microscopy, did not incorporate H-
thymidine into cell nuclei until approximately 3 days after administration of
tracer, as demonstrated by autoradiography. This finding supports the notion that
lung neuroendocrine cells are derived from a separate cell population. (4) The
hyperplasia of lung neuroendocrine cells observed in hamsters treated with die-
thyl nitrosamine appears to originate with cells of the neuroepithelial body or



Us progenitor .

PHS-6040
(Rev. 2-81)



643



Z01 ES 80033-05 LPFT



PROJECT DESCRIPTK



METHODS EMPLOYED : General histological procedures were used for fixation and
staining of tissue sections for Tight and electron microscopy. The Grimelius
method was used to detect argyrophilic cells. Most of the antisera to various
polypeptide hormones were obtained from rabbits following intermittent subcutaneous
injections of the peptide coupled to albumin or hemocyanin; other antisera were
obtained from the Pituitary Agency, individual investigators, or commercial labora-
tories. The immunoperoxidase-bridge technique was employed for immunohistocheii ical
localization of polypeptide hormones. Details of the methods for dissociation of
lung cells and cell culture are described in last year's report. Other methods
used were autoradiography, radioimmunoassay, and gel filtration column
chromatography.

MAJOR FINDINGS AND PROPOSED COURSE : The present program consists of investiga-
tions of solitary neuroendocrine- like cells of the pulmonary epithelium and the
response of these cells to a respiratory carcinogen, diethylnitrosamine (DEN).

Neuroendocrine-like Cells of the Normal Pulmonary Epithelium . This investigation
essentially consists of two major parts. In the first, attempts were made to
identify by electron microscopy neuroendocrine-like (NEC), or small-granule, cells
in the upper respiratory tract of the guinea pig. We were especially interested in
examining whether more than one subpopulation of NECs existed. In the present
studies we confined our examinations to the epithelium of the cricoid region of the
larynx and the tracheal epithelium. We also wanted to quantitate the number of
NECs in this region of the airways.

We were readily able to identify NECs in the cricoid so this region was used
initially for a systematic quantitative study. Solitary NECs were readily identi-
fied by the presence of unique small membrane-encapsulated granules (mean diam. :
131 ± 28 nm, SD). The ultrastructural characteristics of the NECs were homo-
geneous throughout the various regions of the cricoid sampled, suggesting a single
population. Quantitation of NECs by electron microscopy was made by sequentially
surveying cells along the basement membrane. NECs constituted 4-9% of the total
cricoid epithelial cells in 2 days - to 7- week old guinea pigs. A representative
quantitation is shown in Table 1. Only solitary NECs were observed and no specific
concentration of these cells occurred in any region of the mucosal surface of the
cricoid.

Animals treated with 5-hydroxytryptophan had cricoid cells with yellow fluorescence
by the formaldehyde-induced fluorescence procedure, indicating that these NECs can
synthesize serotonin.

Similar small-granule cells were identified by electron microscopy in the upper,
middle, and lower regions of the trachea. Quantitation of these cells in the
tracheal epithelium is now i.i progress for the epithelium covering the 3rd, 18th,
and 34th cartilage rings.

The finding of a homogeneous population of cricoid NECs at the frequency reported
provides a convenient system to assess their biological properties and eventually
identify their humoral components. In one proposed study, we plan to examine



644



ZOl ES 80033-05 LPFT

whether these NECs respoijicl by degranulation or loss of serotonin to hyperpolariz-
ing concentrations of [K ] as other NECs do. We speculate that a unique neuro-
peptide may be co- stored with serotonin in the lung solitary NECs.

TABLE 1

Quantitation of cricoid epithelial NECs in a 7 week-old guinea pig by electron
microscopy. A 2 mm- thick section was made by slicing the cricoid transversely
with a razor. The section was then divided in half by a section along the sagit-
tal plane. Each of these halves was then divided equally by a longitudinal sec-
tion. Finally, each of the resulting four cricoid pieces, as designated below,
was examined for epithelial NECs.

Regio" Total cells Neuroendocrine

examined counted cells



Rl 70 5

LI 96 4

R2 51 2

L2 88 2

305 ^13"

= 4% NEC



R = right
L = left

1 = ventral epithelial surface

2 = dorsal epithelial surface

In a second part of this work we exhaustively examined solitary NECs in the
tracheal mucosa by histochemistry for immunolocalization of known polypeptide
hormones. No positive cells could be identified in the epithelium. Other details
are given in last year's report.

Cytokinetic analysis of the argyrophilic cells using "^H-thymidine indicated no
labeled nuclei (> 200 argyrophilic cells examined) after a 60 min-pulse. Auto-
radiographic analysis after 3 days did reveal labeled nuclei which suggests that
the tracheal NECs derive from some separate population of epithelial cells
(basal cells).

As with the cricoid epithelium, we propose to establish by EM whether a single
population of NECs exists in the tracheal epithelium.

Proliferation of endocrine- like cells injungs of hamsters treated with diethyl-
nitrosamine (DEN ). Details of the experimental design were covered in last year's
report. The results of the study demonstrated in vivo hyperplasia of apparent
neuroepithelial bodies and subsequent culture of neuroendocrine- like cells.
Studies of the incorporation of H-thymidine into NEBs of treated and untreated
hamsters have been carried out independently by Dr. Ilona Linnoila, formerly in
the Endocrinology Group and now with the National Cancer Institute.

645



ZOl ES 80033-05 LPFT

SIGNIFICANCE TO BIOMEDICAL RESEARCH AND THE PROGRAM OF THE INSTITUTE : Identifica-
tion of the biological properties of lung epithelial neuroendocrine cells and
their humoral constituents should ultimately allow us to establish what function
these cells have in the normal lung. Some lung-associated abnormalities may be
initiated by improper levels of a regulatory peptide synthesized by a
lung neuroendocrine cell. The experimental induction of lung neuroendocrine cell
hyperplasia may indicate that certain lung tumors, namely bronchial carcinoid
tumors and small cell carcinomas, may be preferentially formed by exposure to the
nitrosamines in comparison to other classes of chemical carcinogens. Identi i i cation
of lung-specific polypeptides should conceivably serve as a valuable plasma
marker for early neoplastic responses of lung neuroendocrine cells.

PUBLICATIONS

Linnoila, R. E., Nettesheim, P. and DiAugustine, R. P.: Lung endocrine-like
cells in hamsters treated with di ethyl nitrosamine: Alterations in vivo and in
cell culture. Proc. Natl . Acad . Sci . (in press) 1981.



646



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U.S. DEPARTMENT OF


PROJECT NUMBER




1 HEALTH AND HUMAN SERVICES










PUBLIC HEALTH SERVICE










NOTICE OF










INTRAMURAL RESEARCH PROJECT


ZOl ES 80035-05 LPFT |


PERIOD COVERED






October 1, 1980 to


September 30, 1981




TITLE OF PROJECT (80 characters op less)




Co-oxidation of Xe


nobiotics by the Prostaglandin and Thromboxane Synthetase


NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER |


PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT



Online LibraryNational Institute of Environmental Health ScienceAnnual report : National Institute of Environmental Health Sciences (Volume 1981) → online text (page 57 of 92)