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Solubility studies of the secretion granules of the guinea pig pancreas.

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XOLURTLITY STUDIES O F THE SE(>RETIOY
GBANULES O F THE GUINEA PIG
PANCREAS
8. H. BENBLEY
Hull Laboratory of Anatoniy, 1 A e University of Chicago
Woerner ('38) has described, for the first time, deg
0 ciierative changes in the alpha cells of the island of Langcrlians,
following continuous intravenous injection of dextrose i n
guinea pigs.
These changes occurred when moclerate
aiuounts of sugar were injected and there was no resulting extensive hyperglycemia or glycosuria. However, these aiiirnals
exhibited evidences of increased metabolic rate with extensive
loss of weight aud there were coincident f a t t y changes in the
mitochondria of t,he acinar cells of the pancreas.
It occurred to us o n thc basis of these findings that there
might be some relation between exliaustion of the alpha cells
and increased fat metabolism under conditions of increased
oxidation of sugar. In order to test this hypothesis it
seemed desirable to obtain an extract of the alpha cells.
Consequeiitly the following solubility studies were undertaken.
1. The pancreases froin three guinea pigs, whose pancreatic
ducts had bccii ligated for 3 weelis, were frozen i n liquid
isoperitaiie and dehydrated iii ~ a c u oa t -40°C. Pieces of this
tissue were treated with twelve organic aiid ninc aqueous
solrents and one piece TI-^ put directly into formol-Zenker
for a control. The organic solvents used were: methyl
alcohol, ethyl alcohol, butyl alcohol, iso-butyl alcohol, propyl
alcohol, iso-propyl alcohol, iso-amyl alcohol, ally1 alcohol,
ethylene dichloride, etliyleiie glycol, methyl cellosolve, and
butyl cellosolvc.
131
THI A N A T O M I C A L XECORD, VOL. 72, NO. 2
OCTOBER, 1938
132
S. H. BENSLEY
The tissue in the organic solvents was subjected lo negative pressure, t o extract tlie air from thc tissue, and allowed
to remain in the solvcnt at room temperature for 24 hours.
Thc pieces wcre then cleared in oil of bergamot, embedded
in paraffin and sectoned at 5 to 8 p. One series of sections was
mordanted with 1%osniic acid and stained with aniline acid
fuchsin and methyl green ; another was mordarited with 2.5%
aqueous solution of potassium hichromate and stained with
neutral gentian.
The aqueous solrents used were : distilled water, 0.85%
sodium chloride, 10% sodium chloride, 0.1% acetic acid, 0.1%
sodium hydroxide, 2.5% glycerine, 5% dextrose, 10% succharose and 1%urea. The pieces of tissuc were put into the
aqueous solvents a t room temperature f o r 1 hour. They arid
the control piecc wcrc then transferred to formol-Zenkcr,
subjected to negative pressure, and then left in the fixative
f o r 24 hours. Subsequently the tissues were washed, dehydrated, cleared, embedded i n paraffin, sectioned and stained
by the two methods used, f o r the tissues treated with organic
solvents.
With freezing and drying in vacua, the restricted permeabilities of the cell membranes are abolished and solvents
can readily penetrate the cell. Moreover, the solvents are not
diluted by water in the cell. By this method, then, the action
of the pure solvent on the intraeellular structures can be det ermined directly.
The action of these solvents may be briefly summarized :
1. The pure organic solvents used, with the exception of
etliylcne glycol, modified but did not remove the alpha, beta
or zymogen granules. Ethylene glycol removed them all.
2. Of the aqueous solvents, distilled water, normal salt
solution, 10c/b sodium chloride and 5% dextrose removcd the
alpha granules ; 0.1% sodium hydroxide removed both alpha
and beta granules; 1%urea modified both alpha and beta
granules ; 5% dextrose nioclified the beta granules.
Since the zymogen granules may almost completely disappear following ligation of the duct, the absence of zymogen
SOLUBILITIES O F SECRETION G R A N U L E S
133
granules in the aqueous series could not be interpreted a s
solution of them by the solvent.
11. IVhole fresh guinea pig pancreas was extracted for 24
hours at refrigeration temperature 1) with distilled water,
2 ) with normal salt solution and 3 ) first with an equal volume
of 95% alcohol for 12 hours aiid then with distilled water f o r
24 hours. Pieces of each were then fixed f o r 24 hours in
formol-Zenker, washed, debydrated, cleared, embedded i n
paraffin, sectioned and stained with aniline acid fuchsin,
methyl green and with neutral gentian.
Examination of the sections revealed: A ) The alpha
granules were removed with both water. and iiorinal salt
solution. B) The beta, zymogen and hlankowski granules
aiid ewii tlie mitochondria were resistant to both. C ) After
ion fii-st with alcohol and then with water, only the
Xaiikowski granules remained. F r o m this we may deduce
that the alpha granules treated with alcohol, to wliich they a r e
resistant, may be subsequently removed by water.
111. The reaction of pieces of fresh pancreas to various
aqueous solvents was observed directly uiicler the oil immersion lens of the microscop.
The identification of the zymogenic and Manko-cvski cells
i n fresh tissue is easy with the dark-adapted eye, because of
the size, refraction and distribution of the characteristic
granules. The zymogen granules a r e the largest, most highly
rcfractile and occiii’ grouped, for the most part, at thc free
pole of the cell. The Mankowski granulcs a r e smaller, highly
refractile and a r e distributed in the base or throughout the
cell.
The identification of the alpha and beta cells is somewhat
more difficult in fresh tissue. A superficial island is located
under low power by the slightly different color which the
rich blood supply gives it. Under oil immersion, when the eye
is dark adapted, the cells may be differentiated by the following characteristics. The beta cells which make up the
majority of the cell content of the island a r e relatively small
and well filled with tiny highly refractile granules. The
134
S. H. BENSLEY
nuclei are f o r the most part spherical a i d (when the chromatin niay be visualized) have a considerable content of
chromatin rather regularly arranged. The clear Golgi net
may sometimes be visualized between the refractile granulcs.
K h e n the beta granules are dissolved the cytoplasm appears
optically homogeneous.
The alpha cells are larger than the beta cells and tend to
be elongated. The granules are larger, riot very refractile
aiid appear watery. The nuclei are oval, pale and poor in
chromatin, with a prominent nucleolus. When the alpha gmnulcs are dissolved the cytoplasm becomes highly vacuolatcd.
The followiiig observations were recorded.
1. Distilled water and normal salt solution rapidly remove
the alpha granules while the beta, zyinogen and Mankowski
gimiules remain. On standing at room temperature there is a
gradual dccrcnient in the beta and zynogeu granules, especially in distilled water.
2. Glycerine penetrates the cells slowly and the zymogen
granules arc lost. No changes are observed in the alpha and
beta granules.
3. Dilute sodium hydroxide rapidly and dramatically removes the alpha, beta and zymogen granules. The 3Iankow&i granules are somewhat modified.
4. Tt'ith dilute acetic acid, the alpha arid beta granules are
preserved but with stronger acid the granules are riot distinguishable and the cells show a heavy precipitate. The
zymogen granules are preserved in both cases.
TESTS O F THE SOLUTION O F THE A1,PHA GRANIJLES
On the basis of these findings fresh pancreas was ground
up with sand and extracted with distilled water, the whole
centrifuged aiid re-centrifuged at high speeds four time3
and the supernatant fat skimmed off. The sediment and llie
suspensj on w3i-e examined by the stained smear method.
Beta-like granules were observed in the suspension arid illcreasingly in the sediment. The filial suspension was filtered
through a Berkefeld filter and collected into a sterile flask.
SOLUBILITIES O F SECRETIOK GRANTTTlES
135
The total sediment, when extracted i n ‘70% alcohol, the extract evaporated and the residue taken up in Ringer’s solution and injected intravenously into a fasted rabbit, produced
a €all in blood sugar equivalcrit to about that produced by
1unit of insulin.
Preliminary chemical studies of the filtered extract showed :
pII of about 7 ; a very fairit Millon reaction ; fine precipitation
with ethyl alcohol, ether and acetic acid ; n o precipitation with
salt solutions or dextrose.
Intravenous injection of the filtered extract produced n o
immediate toxic symptoms in the guinea pig, and a fall i n
blood sugar only within the range of experimental error’.
From these ohservations 1F-e may concludc that :
1. Alpha granules of the guinea pig pancreas a r e removed
by water, normal salt, 10% salt, 5% dextrose mid tliliite
sodium hydroxide solutions and a r e resistant to the pure
organic solvents (m.ith the exception of ethylene glycol) a i d
to dilute acetic acid.
2. Beta granules a r e rcmovcd by dilute sodium hydroxide,
and ‘ioyb alcohol and a r e resistant to water, normal salt solution and dilute acetic acid.
3. Zymogen granules a r e removed by dilute sodium hydroxide, alcohol, glycerine and a r e resistant to water, normal
salt solution and acetic acid.
4. Mankomski granules a r e resistant to all the aqueous
solvents so f a r tested and to alcohol.
These properties of the secretion granules correspond t o
those described by Lane (’07) with the exception of the reaction of ilie alpha and beta granules t o acetic acid. Lalie
believed that both types of granules were soluble in acetic
acid. His negative findings may be due to a loss of staining
capacity of tlie granules following fixation in acetic acid, or
to chaiiges i n the cells produced by too great a coiicentratiori
of the acid. Certainly, after fixation hy Bensley’s aceticosmic-bichromate fluid both granules may be well preserved
and stained by the aniline acid fuchsin-methyl green method.
The properties of the Mankowski granules correspond to
those described by Bensley ( ’14PI5).
136
6. H. BENSLEY
Because the alpha granules are so labile in water aiid salt
solutions, it becomes apparent that no inethods of supravital
staining o r perfusion with these fluids can be adequate to
demonstrate these granules. And i n order t o study experimental changes in the granule content of the alpha cells, ercry
effort must be made t o fix the tissue immediately.
The author wishes t o express her gratitude to Prof. R. R.
Rensley f o r his keen interest, his confirmation of observations and his technical and chemical assistance; to Dr. h’. TJ.
Hoerr for his assistance with the freezing-drying proccdurc ;
and to Dr. C. ,4.Woerner f o r blood sugar deteriniiiatioris.
LITERATURE CITED
BENSLEY,R. R. 1914-1915 Structure and relationship of thc islets of
Langerhans. The Harvey Lectures, Series X, p. 373.
LANE,hl. A. 1907 The cytological character of the arcas of Langerhans. Am.
J. Anat., vol. 7, pp. 409-424.
WOERNER,C’. A . 1938 Studies of the islands of Langrrhans after continuous
i n t r n e n o u s injection of dextrose. Anat. Xec., 701. 71, pp. 33-57.
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