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Heterogeneity studies of hamster calcitonin following acute exposure to cigarette smokeEvidence for monomeric secretion.

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THE ANATOMICAL RECORD 236:253-256 (1993)
Heterogeneity Studies of Hamster Calcitonin Following Acute
Exposure-to Cigarette Smoke: Evidence for Monomeric Secretion
ALI R. TABASSIAN, RICHARD H. SNIDER, JR.,ERIC S. NYLEN, MARIE CASSIDY,
AND KENNETH L. BECKER
Departments of Medicine and Physiology, George Washington University School of Medical
Sciences, and the Veterans Affairs Medical Center, Washington, D.C.
ABSTRACT
Various acute stimuli, including cigarette smoke, induce
hypercalcitonemia in man and hamsters. We have shown that this occurs
also in thyroidectomized subjects. In the present study we have further
explored this phenomenon of secretion from the lungs by studying, simultaneously, the HPLC characteristics of pulmonary tissue and serum in control hamsters and in animals immediately following short-term exposure to
cigarette smoke. In addition, we have studied the immunoheterogeneity of
lung calcitonin 24 hours following the acute exposure. Control lungs contained monomeric immunoreactive calcitonin (M-iCT),high molecular mass
iCT (H-iCT), and CT fragments. Immediately following smoke exposure,
there was an acute decrease of lung iCT by radioimmunoassay (RIA)which
consisted primarily of a decrease in M-iCT by HPLC. Simultaneously, the
iCT increase in the serum by RIA was shown by HPLC to involve M-iCT.
Twenty-four hours after smoke inhalation, the lung iCT by RIA and M-iCT
by HPLC had returned towards control levels. These findings document
the molecular characteristics of lung iCT following acute cigarette smoke
stimulation, and suggest that under certain circumstances M-iCT may be
actively secreted by the lung. It remains to be determined whether this type
of secretion reflects hemocrine or paracrine release and what the physiological role for such a secretion may be. o 1993 Wiley-Liss, Inc.
Key words: Hypercalcitonemia, Pulmonary neuroendocrine cells, Nicotine, RIA, HPLC, Hemocrine secretion, Paracrine secretion
Calcitonin from several species, including man, is
contained within the pulmonary neuroendocrine (PNE)
cells of the lungs (Becker et al., 1980; Becker and Gazdar, 1984). In vitro, we have reported that dissociated
hamster PNE cells in culture secrete monomeric immunoreactive calcitonin (iCT), which is the bioactive
form of CT (Nylen et al., 1987). In vivo, evidence from
our laboratory indicates a hemocrine response from
these cells when under the influence of various pharmacologic and pathophysiologic conditions (Becker et
al., 1981; Tabassian et al., 1988, 1989; O’Neill et al.,
1993). Thus, secretagogues, such as nicotine, acutely
increase serum iCT while simultaneously depleting
lung iCT; this occurs even in the absence of the thyroid
gland (Tabassian et al., 1990). The current study explores, simultaneously, the molecular heterogeneity of
both pulmonary and serum iCT of the hamster, before,
immediately following, and 24 hours after the shortterm exposure to cigarette smoke.
nicotine content of 2.45 mglcigarette were used (2R1
cigarettes, Tobacco and Health Research Institute,
Univ. of Kentucky, Lexington, KY). An Amicon LP-1
perfusion pump was utilized to advance the cigarette
smoke into the chamber at a constant flow rate. The
animals, which had been fasted for 24 hours prior to
exposure, were exposed to smoke from four of the standard research cigarettes during 30 minutes. Control
animals (n = 10) were sacrificed without smoke exposure; test animals were sacrificed immediately following the single smoke exposure (n = 12) and 24 hours
afterwards (n = 11).The hamsters were sacrificed by
the administration of a n overdose of sodium pentobarbital(250 mg/kg); this dose does not change iCT levels
(results not shown). This was followed by the removal
of blood via the right ventricle, and the dissection of
both lungs. The concentration of lung tissue iCT was
determined following fat and tissue extraction, CIS Sep
Pak cartridge (Millipore) purification, and lyophiliza-
MATERIALS AND METHODS
Six-week-old, male, inbred type CB hamsters
(Charles River, Wilmington, MA) were used in these
studies. The animals, four a t a time, were placed in a
7.5 liter smoking chamber as previously described
(Tabassian et al., 1988). Research cigarettes having a
0 1993 WILEY-LISS, INC
Received December 5, 1991; accepted June 5, 1992.
Address reprint requests to Dr. K.L. Becker, Endocrinology, Veterans Affairs Medical Center, 50 Irving Street, N.W., Washington, DC
20422.
254
A.R. TABASSIAN E T AL.
tion following techniques we have previously described
(Snider et al., 1977; Becker et al., 1979). Pooled lung
extracts from controls, from immediate post-exposure,
and from 24 hours post-exposure groups were analyzed
by HPLC to determine the effects of the acute smoke
exposure on the heterogeneity pattern of lung tissue
CT; and pooled sera from controls and animals immediately post-exposure were also studied.
High performance liquid chromatography (HPLC)
analysis is performed at room temperature on a n AxxiChrom 4.6 x 100 mm C,, reversed-phase column (3
particle size) with 3-step linear gradients using mobile
phase A (trifluoroacetic acid [ l giL1 in 0.1 M
[NH,],SO,) and mobile phase B (trifluoroacetic acid [ l
giL] in acetonitrile) a t pH = 2.8 (flowrate = 1mLimin
with a starting pump [Waters 6000A pumps1 pressure
of 2,600 psi): In step 1, 100% mobile phase A is passed
through the column prior to the injection and for 2
minutes after the injection. Fifty to two hundred microliters (adjusted to maintain nearly equal total
amounts of iCT) of tissue extract was injected into the
column, using a WISP 710B auto-injector (Waters). In
step 2, mobile phase B is increased from 0 to 20% during 5 minutes. In step 3, mobile phase B is increased
from 20 to 40% during 50 minutes. A Frac-100 (Pharmacia) fraction collector is used to collect 60 1-minute
(1mL) aliquots. These aliquots are lyophilized and reconstituted in 0.3 mL of 0.2% HSA for radioimmunoassay (RIA) as previously reported using a C-terminal
recognizing antiserum (Ab IV) (Snider e t al., 1977).
Occasionally, when RIA with two different antisera is
desired, the 60 aliquots are split into two equal portions prior to lyophilization and RIA. The CT concentration is plotted against fraction number or elution
time.
Each column was calibrated by running synthetic
CT, CT fragments, and other polypeptide markers
through the column.
80
2
0
&
d:
70
60
50
P
0
40
I
n
a
30
20
10
0
z
70
5
60
0
5
10
15
20
25
30
MINUTES
35
)
45
50
5
10
15
20
25
30
MINUTES
35
40
45
50
5
10
15
20
25
30
MINUTES
50
:
0
40
0
30
20
10
0
RESULTS
Figure 1 shows the effect of acute smoke exposure on
the iCT heterogeneity pattern of the lung tissue extracts of control, immediate post-exposure, and 24
hours post-exposure animals. Immediately after smoke
exposure, the relative concentration of the monomericiCT (M-iCT) fraction (retention time of 40 minutes)
decreased, when compared to controls. By 24 hours after exposure, there was a rebound increase in the relative concentration of the M-iCT fraction to a level
higher than controls. The relative concentration of the
high molecular weight-iCT (H-iCT fraction with retention time of 30 minutes) decreased slightly after exposure to smoke. Twenty-four hours after exposure, however, there was no rebound increase in the H-iCT
fraction as was observed for M-iCT. Interestingly, it
appeared that the relative concentration of the iCT
fragments (retention times of less than 30 minutes)
increased immediately after exposure to smoke; 24
hours after exposure, the relative concentrations of the
iCT fragments decreased to levels comparable to controls.
Figure 2 contrasts the effect of acute smoke exposure
on the serum iCT heterogeneity pattern before and immediately following smoke exposure. Control hamster
serum contained both M-iCT and H-iCT a t relatively
2
70
6
60
0
50
6
.-
40
0
30
20
10
0
Fig. 1. HPLC of iCT of hamster lung. A: Control animals. B: Immediately following acute smoke exposure. C: Twenty-four hours following smoke exposure.
equal proportions. Immediately after exposure to
smoke, however, the relative concentration of the
M-iCT fraction increased, indicating that acute smoke
255
MONOMERIC SECRETION AFTER SMOKE EXPOSURE
TABLE 1. The relative changes in serum and lung iCT
(control = 100%)as determined by RIA following
acute and 24 hours after exposure to cigarette smoke
z
70
60
.
0
LL
50
Grour,
Control
Acute
24 hours
Serum
*
100 33
194 ? 14*
161 2 17"
Lunp
100 ? 21
81 21*
101 ? 36
*
"P < 0.05.
t-
40
0)
H-iCT, as well as traces of iCT fragments. After exposure to cigarette smoke, the levels of M-iCT decreased
20
compared with H-iCT (Fig. 1).Since the total amount
of iCT applied to the HPLC column was equal in all
10
cases, the levels of M-iCT and H-iCT could be evalu0
ated in terms of both relative and absolute concentra5
10 15 20 25 30 35 40 45 50
tions. Thus, the relative a s well as the absolute (Table
MINUTES
1) concentrations of M-iCT decreased within the lung
after acute smoke inhalation, while the relative and
1
2
0
'
absolute concentrations of CT fragments increased and
B
those of H-iCT decreased, albeit less than M-iCT. This
indicated depletion of M-iCT from the lung, perhaps
100-1
due to hemocrine secretion into the circulation or, alternatively, overflow paracrine secretion. The HPLC
profile of hamster serum iCT before and after acute
P 80-'
cigarette smoke inhalation supported this hypothesis.
G
Following cigarette smoke inhalation, the relative con60-/
centration of the M-iCT increased dramatically com0
pared to control levels (Fig. 2). Indeed, when absolute
2 40-'
concentrations of serum iCT are considered, it becomes
apparent that nearly 100% of the increase in serum
iCT is solely attributable to M-iCT.
20
Interestingly, 24 hours after smoke inhalation, the
relative and absolute concentrations of M-iCT increased beyond those present in control lungs; frag0
5
10 15 20 25 30 35 41
45
50
ment iCT levels decreased below control levels and
MINUTES
H-iCT levels remained slightly below control levels.
Fig. 2.HPLC of iCT of hamster sera. A Control animals. B: Imme- The excess M-iCT may reflect de novo synthesis of this
peptide. While it is known that both M-iCT and H-iCT
diately following acute smoke exposure.
can be metabolized into fragments, there is currently
no data regarding interconversion of H-iCT into
M-iCT.
exposure caused the release of M-iCT into the serum.
In the present study, we did not utilize thyroidectoSince the relative concentration of the M-iCT fraction mized animals. Hence, although there clearly was a
decreased in the lung, these results suggest a prefer- marked depletion of pulmonary M-iCT, some of the inential release of M-iCT from the lung into the blood.
creased serum M-iCT could have emanated from the
Table 1summarizes the effect of cigarette smoke ex- thyroid gland. In this respect, our prior published studposure on iCT levels in the serum and lung a s deter- ies may cast some light. In hamsters, we have demonmined by RIA. The serum iCT increased significantly strated that intact animals have a 33% depletion of
following the acute exposure and increased levels per- their total lung iCT and a 100% increase in their serum
sisted a t 24 hours. The lung levels of iCT, however, iCT following acute exposure to cigarette smoke
initially decreased, followed by a return to control lev- (Tabassian et al., 1988,1990).Nicotine, which we have
els.
found to be a secretagogue of iCT, also causes pulmonary depletion of pulmonary iCT and increases of seDISCUSSION
rum iCT when injected subcutaneously into intact
Previously, we have demonstrated that various acute hamsters. Importantly, nicotine induces the same efstimuli, including cigarette smoke, induce hypercalci- fects in thyroidectomized animals. Cigarette smoke (a
tonemia (Tabassian et al., 1988). In the current study, known source of nicotine) increases serum iCT in the
this increase persisted at 24 hours post-exposure. Con- thyroidectomized human (Tabassian et al., 1988). On
comitantly with the acute hypercalcitonemia, there is a the other hand, we have found that the intravenous
depletion of total pulmonary iCT, which then returns infusion of calcium, a known secretagogue of iCT in the
human, is without effect in thyroidectomized humans
to normal values by 24 hours (Table 1).
In this study, HPLC analysis demonstrated that con- (Silva et al., 1978).
The nicotine from inhaled cigarette smoke enters the
trol, untreated hamster lung contained both M-iCT and
a
s
30
256
A.R. TABASSIAN ET AL.
blood circulation nearly instantaneously and reaches
most body tissues. If our studies in the human are applicable, our current findings in the hamster suggest
that the nicotine content of cigarette smoke, plus perhaps other constituents or secondary effects of cigarette
smoke, may preferentially affect the lungs. Nevertheless, any definitive conclusions concerning whether the
increased serum iCT also may reflect a thyroidal iCT
contribution requires further studies using thyroidectomized animals.
In summary, the present HPLC study provides further support for our observations of secretion of pulmonary CT under certain circumstances, and documents
that the acute increase of serum iCT monomer occurs
concurrently with a depletion of lung iCT monomer,
and that the serum and lung levels again return towards baseline 24 hours later. Hopefully, further studies of the dynamics of CT release from the lung may
provide further insights into a possible role of CT in
pathologic states, both within the lung and beyond the
confines of that organ; and also may determine
whether such a secretion occurs physiologically.
LITERATURE CITED
Becker, K.L., and A.F. Gazdar 1984 The pulmonary endocrine cell and
the tumors to which it rise. In: Comparative Respiratory Tract
Carcinogenesis. H.M. Reznick-Schuller, ed. CRC Press, Boca Raton, Florida, vol. 11, 161-187.
Becker, K.L., R.H. Snider, C.F. Moore, K.G. Monaghan, and O.L.
Silva 1979 Calcitonin in extrathyroidal tissues of man. Acta Endocrinol. (Copenh.) 92746-751.
Becker, K.L., D. Nash, O.L. Silva, R.H. Snider, and C.F. Moore 1981
Increased serum and urinary calcitonin levels in patients with
pulmonary disease. Chest, 79:211-216.
Becker, K.L., K.G. Monaghan, and O.L. Silva 1980 Immunocytochemical localization of calcitonin in Kulchitsky cells of human lung.
Arch. Pathol. Lab. Med., 104:196-198.
Nylen, E.S., R.I. Linnoila, R.H. Snider, A.R. Tabassian, and K.L.
Becker 1987 Comparative studies of hamster calcitonin from pulmonary endocrine cells in vitro. Peptides, 8:977-982.
ONeill, W.J., M.H. Jordan, M. Lewis, R.H. Snider, Jr. C.F. Moore, and
K.L. Becker 1993 Serum calcitonin may be a marker for inhalational burn injury. J . Burn Care Rehab., 13:in press.
Silva, O.L., L.A. Wisneski, J . Cyrus, R.H. Snider, C.F. Moore, and
K.L. Becker 1978 Calcitonin in thyroidectomized patients. Am. J.
Med. Sci., 275:159-164.
Snider, R.H., O.L. Silva, C.F. Moore, and K.L. Becker 1977 Immunochemical heterogeneity of calcitonin in man: Effects on radioimmunoassay. Clin. Chem. Acta, 76:l-14.
Tabassian, A.R., E.S. Nylen, A.E. Giron, R.H. Snider, M.M. Cassidy,
and K.L. Becker 1988 Evidence for cigarette smoke-induced calcitonin secretion from lungs of man and hamster. Life Sci., 42:
2323-2329.
Tabassian, A.R., E.S. Nylen, R.I. Linnoila, R.H. Snider, M.M. Cassidy,
and K.L. Becker 1989 Stimulation of pulmonary neuroendocrine
cells and their associated peptides by repeated exposure to cigarette smoke. Am. Rev. Respir. Dis., 140:436-440.
Tabassian, A.R., E.S. Nylen, L. Lukacs, M.M. Cassidy, and K.L.
Becker 1990 Cholinergic regulation of hamster pulmonary neuroendocrine cell calcitonin. Exp. Cell. Res., 16:267-277.
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