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Monoamine-containing cells in the fetal and newborn guinea-pig pancreas
Vol . 6, pp . 1647-1652, 1967 . LIFE SCIENCES Printed in Crest Britain.
Pergamon Press Ltd .
MONOAMINE-CONTAINING CELLS IN THE FETAL AND NEWBORN GUINEA-PIG PANCREAS
Lennart Cegrell
Institute of Anatomy and Histology, University of Lund, Lund, Sweden
(Received 3 April 1967 ; in final form 22 May 1967) With the histochemical method of Falck and Hillary, which makes it possible to localize some monoamines on the cellular level, Falck and Hellman (1) demonstrated a monoamine in the insulin-producing H-cells in the islets of Langerhans in the adult guinea-pig pancreas . The fluorescence characteristics suggested that the amine might belong to the tryptamine group . Further investigations, however, have revealed that in the fetal and prenatal stage the conditions differ considerably from the adult stage with respect to the monoamine stores .
Material and Methods Guinea-pig pancreas from 20 fetuses aged 30 to 65 days and from 12 newborn animals (leas than 24 hours old) were used . The pencreae tissue was freeze-dried and processed for fluorescence microscopy according to Falck and Owman (2) . The cellular fluore-
1648
PANCREAS CELLS
Vol . 6, No . 15
stance wee analyzed in a Leitz micro-spectrograph . (This procedure is described in a forthcoming paper : Ehinger and Falck, to be published) . The islet cells were classified according to Hellman and Hellerström (3) : 1) aldehyde-fuchsin positive, insulin-producing H-cells, 2) silver-paaitive A l -cells with unknown function, 3) AZcello presumed to produce glucagon . In order to identify the fluorsscent cells, three micrographs of one and the same section were obtained first in the fluorescence microscope and than in the light microscope after silver-impregnation and subsequent aldehydefuchsin staining, respectively . Janninga
modification (4) of the
aldehyde-fuchsin staining was used since this proouces an excellent result on freeze-dried material .
Rea~lte and Çomment~ The 30-day old guinea-pig fetuses reveal no observable specific (i .e, formaldehyde-induced) fluorescence ; but from about the 35-day stage, a weak fluorescence appears in a few cells . The number of fluorescent cell$ and their fluorescence intensity successively tnereaae until pertus . Parallel with this, there is an increase in the ingrowth of bdrenergic nerves . The pancreas of a 65-day old fetus contains numerous single fluorescent cells scattered in the exocrine tissue ; in the islets, nearly all the cells fluoresce . A similar picture is obtained in the newborn guinea-pig pancreas . The fluorescence is confined to small cytopleemic granules, its colour lying in the green to yellow range as studied in the routine microscope (2) . Microspectrographic analyses revealed that cells with a clearly green fluorescence have
PANCREAS CELLS
Vol . 6, No . 15
1649
FA F ~ max
0.5
400
500
600 mN
FIG . 1 y-exiss relative fluorescence intensity . Excitation and emission and of a clearly green spectra of an edrenergic nerve ( fluorescent cell ( """""" ) maxima . a t 425 and 490 mp . Uncorrected instrument values . excitation and emission peaks et wave-lengths similar to adrenergic nerves (Fig . 1) ; cells with clearly yellow fluorescence have similar emission characteristics as enterochromaffin cells (Fig . 2) ; these findings indicate the ~+resence of a catechol derivative and a tryptamine derivative, respectively . Many cells, however, appear as intermediates between the green and the yellow cells with respect to the emitted light . This third population of fluorescent cells shows, in the microspectrograph emission, curves whose maxima vary
1650
PANCREAS CELLS
Vol. 6, No . 15
FA FA max
0.5
400
500
FIG .
600 mN
2
y-axis : relative fluorescence intensity . Emission spectra of a clearly green fluorescent cell (- " - " - ; max at 490 mk), of a clearly yellow fluorescent cell ( + ; max at 545 mk), of an enterochromaffin cell (------ ; max at 545 mp) and of one cell of the third population ( """""" ; max at 520 mu) . Uncorrected instrument values .
between those of the catecholamine and the 5-hydroxytryptamine spectra
(Fig .
2) . Whether this finding reflects the concomitant
occurrence of a catechol derivative and a tryptamine deriv-~tive in one and the same cell or the presence of a new type of amine is not yet established . may be correct .
Some results Recently,
suggest that the first alternative
a catechol derivative, which
spectro-
Vol . 6, No . 15
PANCREAS CELLS
1651
photofluorimetrically behaves like dopamine, has been found in the pancreas from guinea-pig fetuses 50 to 65 days old . The amount of this substance varies widely (0 .12 - 4 .9 kg/g) but seems to correspond roughly to the variations in green fluorescence found histochemically (5, 6) . Further, the impregnation with silver and subsequent staining with aldehyde-fuchsin reveal that the Al -cells do not fluoresce but that the H-cells, together with many ailver-negative and aldehyde-fuchsin negative cells, display a specific fluorescence in the islets, indicating a monoamine store not only in the H-cells but also in the A 2 -cells . There seems to be no doubt that, at least in the H-cells, the fluorescence varies from green to yellow . In some animals, the catecholemine containing cells dominate the picture ; in others, most of the islet cells show the peculiar intermediate emission spectra . Thus, if one is to accept that the aldehyde-fuchsin positive cells represent a uniform cell type, i .e . the insulin producing H-cells, the moat reasonable presumption seems to be that these cells store both dopamine and e tryptamine derivative in varying proportions . The tryptamine may well be 5-hydroxytryptamine since preliminary studies have shown that this substance is present in the newborn guinea-pig pancreas at which stage very few 5-hydroxytryptamine-containing enterochro maffin celle hava appeared in the epithelium of the pancreatic ducts (7) . Some of the fluorescent cells which appeared scattered in the exocrine parenchyma are aldehyde-fuchsin positive, but none are silver-positive . On the other hand, silver-positive cells occur scattered in the parenchyme, thus adding further evidence that this celltype does not store an amine capable of condensing
PANCREAS CELLS
1652
Vol . 6, No . 15
with formaldehyde to fluorescent derivative . The identity of the remaining acettered cells is not yet established . Thus, in the fetal end newborn guinea-pig pancreas a monoeminergic mechanism operates both in the H-cells and in the A 2cells but not in the A1 -cells .
This work was supported by grants from Nordiak Inaulinfood and the Faculty of Medicine, University of Lund, Sweden .
~f erençgs 1.
H . Falck and H . Hellman, Acta Endocrinol . ~5, 133 (1964) .
2.
H . Falck and Ch . Owman, Acta Univ . Lund Sectio II, no . 7 (.1965),
3.
H . Hellman and C . Helleratrôm, Z . Zollforsch . 52, 278 (1960) .
4.
H .M . Jenninga, J . histochem . cvtochem . 1~3, 328 (1965) .
5.
L . Cegrell, H . Falck and E . Rosengran, Communic . Deot . Anat . Univ . Lund 4, 1 (1966) .
6.
L . Cegrell, H . Falck and E . Rosengran, to be published (1967) .
T.
L . Cegrell and H . Falck, to be published (1967) .
Pergamon Press Ltd .
MONOAMINE-CONTAINING CELLS IN THE FETAL AND NEWBORN GUINEA-PIG PANCREAS
Lennart Cegrell
Institute of Anatomy and Histology, University of Lund, Lund, Sweden
(Received 3 April 1967 ; in final form 22 May 1967) With the histochemical method of Falck and Hillary, which makes it possible to localize some monoamines on the cellular level, Falck and Hellman (1) demonstrated a monoamine in the insulin-producing H-cells in the islets of Langerhans in the adult guinea-pig pancreas . The fluorescence characteristics suggested that the amine might belong to the tryptamine group . Further investigations, however, have revealed that in the fetal and prenatal stage the conditions differ considerably from the adult stage with respect to the monoamine stores .
Material and Methods Guinea-pig pancreas from 20 fetuses aged 30 to 65 days and from 12 newborn animals (leas than 24 hours old) were used . The pencreae tissue was freeze-dried and processed for fluorescence microscopy according to Falck and Owman (2) . The cellular fluore-
1648
PANCREAS CELLS
Vol . 6, No . 15
stance wee analyzed in a Leitz micro-spectrograph . (This procedure is described in a forthcoming paper : Ehinger and Falck, to be published) . The islet cells were classified according to Hellman and Hellerström (3) : 1) aldehyde-fuchsin positive, insulin-producing H-cells, 2) silver-paaitive A l -cells with unknown function, 3) AZcello presumed to produce glucagon . In order to identify the fluorsscent cells, three micrographs of one and the same section were obtained first in the fluorescence microscope and than in the light microscope after silver-impregnation and subsequent aldehydefuchsin staining, respectively . Janninga
modification (4) of the
aldehyde-fuchsin staining was used since this proouces an excellent result on freeze-dried material .
Rea~lte and Çomment~ The 30-day old guinea-pig fetuses reveal no observable specific (i .e, formaldehyde-induced) fluorescence ; but from about the 35-day stage, a weak fluorescence appears in a few cells . The number of fluorescent cell$ and their fluorescence intensity successively tnereaae until pertus . Parallel with this, there is an increase in the ingrowth of bdrenergic nerves . The pancreas of a 65-day old fetus contains numerous single fluorescent cells scattered in the exocrine tissue ; in the islets, nearly all the cells fluoresce . A similar picture is obtained in the newborn guinea-pig pancreas . The fluorescence is confined to small cytopleemic granules, its colour lying in the green to yellow range as studied in the routine microscope (2) . Microspectrographic analyses revealed that cells with a clearly green fluorescence have
PANCREAS CELLS
Vol . 6, No . 15
1649
FA F ~ max
0.5
400
500
600 mN
FIG . 1 y-exiss relative fluorescence intensity . Excitation and emission and of a clearly green spectra of an edrenergic nerve ( fluorescent cell ( """""" ) maxima . a t 425 and 490 mp . Uncorrected instrument values . excitation and emission peaks et wave-lengths similar to adrenergic nerves (Fig . 1) ; cells with clearly yellow fluorescence have similar emission characteristics as enterochromaffin cells (Fig . 2) ; these findings indicate the ~+resence of a catechol derivative and a tryptamine derivative, respectively . Many cells, however, appear as intermediates between the green and the yellow cells with respect to the emitted light . This third population of fluorescent cells shows, in the microspectrograph emission, curves whose maxima vary
1650
PANCREAS CELLS
Vol. 6, No . 15
FA FA max
0.5
400
500
FIG .
600 mN
2
y-axis : relative fluorescence intensity . Emission spectra of a clearly green fluorescent cell (- " - " - ; max at 490 mk), of a clearly yellow fluorescent cell ( + ; max at 545 mk), of an enterochromaffin cell (------ ; max at 545 mp) and of one cell of the third population ( """""" ; max at 520 mu) . Uncorrected instrument values .
between those of the catecholamine and the 5-hydroxytryptamine spectra
(Fig .
2) . Whether this finding reflects the concomitant
occurrence of a catechol derivative and a tryptamine deriv-~tive in one and the same cell or the presence of a new type of amine is not yet established . may be correct .
Some results Recently,
suggest that the first alternative
a catechol derivative, which
spectro-
Vol . 6, No . 15
PANCREAS CELLS
1651
photofluorimetrically behaves like dopamine, has been found in the pancreas from guinea-pig fetuses 50 to 65 days old . The amount of this substance varies widely (0 .12 - 4 .9 kg/g) but seems to correspond roughly to the variations in green fluorescence found histochemically (5, 6) . Further, the impregnation with silver and subsequent staining with aldehyde-fuchsin reveal that the Al -cells do not fluoresce but that the H-cells, together with many ailver-negative and aldehyde-fuchsin negative cells, display a specific fluorescence in the islets, indicating a monoamine store not only in the H-cells but also in the A 2 -cells . There seems to be no doubt that, at least in the H-cells, the fluorescence varies from green to yellow . In some animals, the catecholemine containing cells dominate the picture ; in others, most of the islet cells show the peculiar intermediate emission spectra . Thus, if one is to accept that the aldehyde-fuchsin positive cells represent a uniform cell type, i .e . the insulin producing H-cells, the moat reasonable presumption seems to be that these cells store both dopamine and e tryptamine derivative in varying proportions . The tryptamine may well be 5-hydroxytryptamine since preliminary studies have shown that this substance is present in the newborn guinea-pig pancreas at which stage very few 5-hydroxytryptamine-containing enterochro maffin celle hava appeared in the epithelium of the pancreatic ducts (7) . Some of the fluorescent cells which appeared scattered in the exocrine parenchyma are aldehyde-fuchsin positive, but none are silver-positive . On the other hand, silver-positive cells occur scattered in the parenchyme, thus adding further evidence that this celltype does not store an amine capable of condensing
PANCREAS CELLS
1652
Vol . 6, No . 15
with formaldehyde to fluorescent derivative . The identity of the remaining acettered cells is not yet established . Thus, in the fetal end newborn guinea-pig pancreas a monoeminergic mechanism operates both in the H-cells and in the A 2cells but not in the A1 -cells .
This work was supported by grants from Nordiak Inaulinfood and the Faculty of Medicine, University of Lund, Sweden .
~f erençgs 1.
H . Falck and H . Hellman, Acta Endocrinol . ~5, 133 (1964) .
2.
H . Falck and Ch . Owman, Acta Univ . Lund Sectio II, no . 7 (.1965),
3.
H . Hellman and C . Helleratrôm, Z . Zollforsch . 52, 278 (1960) .
4.
H .M . Jenninga, J . histochem . cvtochem . 1~3, 328 (1965) .
5.
L . Cegrell, H . Falck and E . Rosengran, Communic . Deot . Anat . Univ . Lund 4, 1 (1966) .
6.
L . Cegrell, H . Falck and E . Rosengran, to be published (1967) .
T.
L . Cegrell and H . Falck, to be published (1967) .