Gratzl, Manfred

Russell, J. T.

Thorn, N. A.

English

Universität München: Elektronischen Publikationen

EXPERIENTIA 
Managing editors: 
H. Mislin, Basel and Carona 
W.P. Koella, Basel 
Editors: 
H. Aebi, Bern 
D. Arigoni, Zürich 
R. Bachofen, Zürich 
P. S. Chen, Zürich 
A. Pletscher, Basel 
S. Weidmann, Bern 
R. Weil, Geneve 
VoL39 
1983 
Part II 
Birkhäuser Verlag 
Basel • Boston • Stuttgart 
Experieniia 39 (1983). Birkhäuser Verlag, CH-4010 Basel/Switzerland 1007 
Stimulation-dependent uptake of an extracellular marker to subcellular fractions of isolated neurohypophysial 
tissue 
M. Gratzl2, J.T. Russell3 and N. A. Thorn 
Institute ofMedical Physiology C, University of Copenhagen, The Partum Institute, Blegdamsvej 3c, DK-2200 Copenhagen N 
(Denmark), December 6, 1982 
Summary. Slices of ox neurohypophyses and groups of isolated rat neurointermediate lobes were incubated in a medium 
containing horseradish peroxidase (HRP) and stimulated by high K+ concentrations in the medium. After washing, the 
tissue was homogenized and subjected to subcellular fractionation in a Percoll/sucrose gradient. HRP was exclusively 
taken up by particles banding at a low density of the gradient. The HRP containing particles located to this region included 
vacuoles of a size comparable to secretory vesicles. 
Some essential facts are known about the mechanism by 
which the neurohypophysial hormones Vasopressin and 
Oxytocin are released. It has been reported that on Stimula-
tion of isolated rat neurohypophyses, vesicles containing 
Oxytocin and the binding protein (neurophysin) release 
their contents to the medium in a roughly stoichiometric 
way4, whereas axoplasma enzyme markers are not 
released5. These facts support the hypothesis that release 
occurs by exocytosis. Since the area of the cell membrane in 
the terminal dilatations is not increased after strong Stimu-
lation67 retrieval of membrane (vesicle membrane and/or 
cell membrane of a size similar to the total area of mem-
brane secretory vesicles incorporated) must take place after 
Stimulation. On the basis of morphological studies the 
prevailing hypothesis has been that secretory vesicles are 
retrieved as microvesicles89, although early histological 
investigations have provided evidence that membrane is 
retrieved as vacuoles similar in size to the secretory vesi-
cles1011. In recent years, an increase in the vacuole popula-
tion on Stimulation without increase in the microvesicle 
population has been shown in several morphometric 
studies612'14. 
Stimulation-dependent uptake of isotope-labeled extracel-
lular markers has been observed when whole rat neurohy-
pophyses were incubated in media containing such mark-
ers15. No studies have been reported on larger animals and/ 
or on attempts to isolate the vesicles retrieved after exocy-
tosis. Such isolation would be necessary to answer the 
question whether the vacuole membrane is similar to that 
of the secretory veiscle membrane or that of the cell 
membrane and might be of use in studies of the fate of the 
vacuoles. 
In the present work we report on stimulation-dependent 
uptake of HRP into subcellular fractions from rat and 
bovine neurohypophyses. As analyzed in density gradients 
the material containing the extracellular marker can be 
isolated from both species. 
Materials and methods. Isolated rat neurointermediate lobes. 
For each of 3 experiments, 60 isolated neurointermediate 
lobes from female Wistar rats (300 g b.wt) killed by 
decapitation, were divided into 2 groups of 30. Both groups 
were washed twice in 'controP medium without HRP 
(table 1). The 2 groups were then incubated in 'controP 
medium and 'Stimulation* medium, respectively, for 60 
1008 Experientia 39 (1983), Birkhäuser Verlag. CH-4010 Basel/Switzerland 
min. After this, both sets of neurohypophyses were washed 
with 15 changes of 4 ml control medium, containing no 
HRP, and 3 times with a Solution containing 10 mM N-Tris 
(hydroxymethyl) methyl-2-aminoethanesulphonic acid 
(TES), pH 7.0, 190 mM sucrose and 25 mM EGTA after 
which they were homogenized. HRP was from Sigma 
(type II). 
Slices from ox neurohypophyses. Hypophyses from cows, 
aged 2-5 years, killed by shooting, were obtained at the 
Copenhagen Public Slaughterhouse, shortly after death of 
the animals. The neural lobes were carefully dissected free 
of pars intermedia. For each of 4 experiments 2 sets, each 
with 2 neural lobes, were cut longitudinally with a scalpel 
into 5 slices (thickness approximately 0.8 mm), and placed 
into 2 x 2 ml of control medium, not containing HRP (see 
table 1). The material was kept at 20 °C and transported to 
the laboratory within 20 min. Here, 1 group of slices was 
ineubated twice for 15 min in the control medium (table 1). 
The control medium in these studies contained 1 mM 
EGTA to reduce 'basal' release. For Stimulation studies, 
another group of slices was ineubated for 2 periods of 15 
min in Stimulation medium (table 1). After ineubation, 
both groups of slices were washed once in control medium, 
containing no HRP and 3 times for 1 min with a Solution 
containing 10 mM TES (pH 7.0), 190 mM sucrose and 
25 mM EGTA, after which they were homogenized. 
Homogenization, subfractionation, and other procedures 
used were carried out as described previously for ox 
neurohypophyses16. Briefly, the homogenate was centri-
fuged at 750 x gav 15 min at 4°C. The supernatant (S,) was 
centrifuged at 28,000 x gav for 15 min using a TI50 rotor in a 
preparative Beckman ultracentrifuge. The pellet (P2) was 
resuspended, mixed with a 50% Solution of Percoll® in 
homogenization medium and centrifuged at 60,000 (cow 
neurohypophyses) or 80,000 xg a v (rat tissue) for 30 min at 
4°C using a Beckman 40.2 rotor with the brake on. After 
centrifugation the tubes were fractionated through a needle 
in the bottom. The S2 supernatant was further centrifuged 
at 170,000 xg a v for 60 min, giving a pellet (P3) and a 
supernatant (S3)16. 
The HRP activity was determined in the gradient and other 
fractions using 3,3'-diaminobenzidine as a hydrogen 
donor17 with 0.2% Triton X-100 present in the assay. 
Results and discussion. Figure A shows the distribution of 
HRP in the gradients containing the material from stimu-
lated and non-stimulated rat tissue. In the fractions ob-
tained from stimulated neurohypophyses, a Symmetrie peak 
in the gradient contained more than twice as much HRP 
activity as the control. In 2 more experiments, the HRP 
activity was increased by a factor 2.2 and 3.3, respectively. 
Increased uptake of HRP into subcellular particles obvious-
ly had occurred. This stimulation-dependent uptake was 
abolished by lack of Ca2+ in the medium or by D600 (a 
methoxy derivative of Verapamil) which blocks stimulation-
induced release of Vasopressin18 (data not shown). It can be 
concluded that the extracellular marker used in these 
experiments is useful to mark newly formed particles not 
only in the tissue101115 but it is also kept within these 
struetures during homogenization, differential centrifuga-
tion and density gradient centrifugation. 
Although fresh rat neurohypophyses can easily be ob-
tained, a major problem in work with the gland from these 
animals is the minute amount of tissue ( < 1 mg/neural 
lobe) and difficulties in separating pars intermedia from 
the neural lobe. Therefore, biochemical analyses and sub-
fractionation studies have been carried out mainly with 
bovine neurohypophyses (150 mg/neural lobe), with the 
drawback that the hemorrhage during slaughtering stimu-
lates neurohypophyses to release hormone19. Despite this 
fact, we have found an increased uptake of HRP into 
previously characterized subcellular fractions from bovine 
neurohypophyses16 which have the advantage that they can 
be freed completely of tissue from the intermediate lobe. 
The distribution of HRP in fractions obtained from stimu-
lated and non-stimulated bovine neurohypophyses is 
shown in table 2. The high amount of enzyme activity 
observed in all supernatant fractions most likely results 
from incomplete washout of extracellular marker prior to 
subfractionation. We observed a small increase of HRP 
activity into the microsomal fraction (P3) and a larger 
increase in fraction P2. The latter subcellular fraction has 
been found to contain about 40-50% of the marker for 
secretory vesicles, mitochondria, microsomes, lysosomes 
and cell membranes16. The hormone containing material 
recovered in P2 can be separated from other struetures by 
density gradient centrifugation using Percoll®16. Using an 
identical procedure we found maximal HRP activity 
around fraction 23 in the gradients (see fig. B), weil 
separated from secretory vesicles (which form a band 
around fraction 7), and mitochondria (centered around 
fraction 13)16. In 3 experiments, besides the one shown in 
figure B, the uptake of HRP was increased by a factor of 
1.6, 1.6, and 1.3, respectively. However, the fractions con-
taining HRP activity are still contaminated by lysosomes, 
Table 1. Composition (mM) of media used for HRP uptake studies in rat neurointermediate lobes and in ox neurohypophysial slices. 
All media were kept at 37 °C and gassed with 0 2 
Neurointermediate lobes 
Control NaCl(140) KCl (5) MgS04(1.5) Stimulation - (45) KCl (100) - (1.5) 
Neurohypophysial slices 
Control CholineCl(150) - (0) MgCl2 (2) 
Stimulation - (90) - (56) - (2) 
EGTA(1) 
(1) 
pH 7.0 
CaCl2(2.5) Hepes(lO) 
(2.5) -
pH 7.3 
( l ) Hepes(lO) 
(4) 
Dextrose (10) 
(10) 
Dextrose (10) 
(10) 
HRP (5 mg/ml) 
(5 mg/ml) 
(5 mg/ml) 
(5 mg/ml) 
Table 2. Distribution of horseradish peroxidase in bovine neurohypophysial fractions separated by differential centrifugation 
Values in percent of homogenate 
Pi s, 
P2 s2 P3 s3 
Control 
Stimulation 
13.6+3.0 94.9+3.1 
12.0±1.6 82.5 + 6.6 
1.8 
2.8 
;±o.2 
;±o.3 
79.9 ±4.6 
68.5 ±3.9 
0.9 ±0.2 
1.3 + 0.2 
71.4 + 2.3 
63.2 ±3.4 
100% equals the total peroxidase activity in the homogenate of two neurohypophyses stimulated by 56 mM K+ (104.7± 19.8 umoles/min) 
or in nonstimulated neurohypophyses (137.3 ± 23.8 jimoles/min). Values are means of 5 experiments± SD. 
Experientia 39 (1983), Birkhäuser Verlag, CH-4010 Basel/Switzerland 1009 
microsomes, and cell membrane fragments16. In thin sec-
tion electronmicrographs of the HRP containing fractions 
recovered from the gradients the mentioned impurities 
were also observed, in addition to vacuoles which con-
tained reaction product if the material was preincubated 
with 3,3'-diaminobenzidine and H 2 0 2 (not shown). 
The distribution within the gradients for rat and ox neural 
lobes were very similar. The peak in the rat experiments 
(using 80,000 x gav for 30 min) was at fraction 18, in the ox 
experiments (using 60,000 x gav for 30 min) it was at frac-
tion 23. 
The exclusive uptake of HRP into the material banding at 
low density of the gradient argues against a non-specific 
A) 
Fractions 
Figure A. Distribution on a Percoll/sucrose gradient of HRP taken 
up by subcellular fractions of stimulated ( • ) and non-stimulated 
(O) rat neurointermediate lobes. Fractions were collected from the 
bottom. 
4 r B) 
°0 10 20 30 
Fractions 
Figure B. Distribution on a Percoll/sucrose gradient of HRP taken 
up by subcellular fractions of stimulated ( • ) and non-stimulated 
(O) bovine neurohypophysial slices. 
adsorption of HRP to membranes. If non-specific adsorp-
tion had occurred, enzyme activity should also have been 
observed in the secretory vesicle fraction which is centered 
around fraction 7 of the gradient used. 
There was some uptake of HRP to non-stimulated rat 
neurohypophyses and a considerable uptake by non-stimu-
lated ox neural lobe slices, even if they had been ineubated 
in a control medium with a very low concentration of free 
Ca2+ . This is probably due to the fact that even in the 
control circumstances there is some Stimulation of the 
tissue. 
The increased uptake of marker into fractions from bovine 
neurohypophyses stimulated with potassium was smaller 
than observed in experiments with rat neural lobes. This is 
probably due to the massive release of hormone induced by 
bleeding of the animals during slaughtering. However, an 
increased uptake of HRP could still be observed, which 
encourages further work on the isolation of these particles. 
The demonstration in the present experiments that HRP 
containing particles could be isolated after Stimulation adds 
independent support to the hypothesis that release occurs 
by exoeytosis. 
1 Acknowledgments. The authors thank Mrs. M. Elis and B. 
Lynderup for excellent technical assistance and help during 
preparation of the manuscript. This work was supported by the 
Deutsche Forschungsgemeinschaft (G. 681/2-1), the Danish 
Medical Research Council, the NOVO Foundation, the Nor-
disk Insulin Foundation and the P. Carl Petersen Foundation. 
2 Present address: Abteilung Klinische Morphologie. Universität 
Ulm, Ulm. FRG. 
3 Present address: Section on Functional Neurochemistry, Nat. 
Inst. Child Health & Human Development, Bethesda, Md, 
USA. 
4 Matthews, E.K., Legros, J.J., Grau, J.D., Nordmann, J.J., and 
Dreifuss, J. J., Nature New Biol. 241 (1973) 86-88. 
5 Edwards, B.A., Edwards, M.E., and Thorn, N.A., Acta en-
doer. 72(1973)417-424. 
6 Morris, J.F., and Nordmann, J.J., Neuroscience 5 (1980) 639-
649. 
7 Pickering, B.T., in: Essays in Biochemistry, vol. 14, pp.45-81. 
Eds P.N. Campbell and W.N. Aldridge. Academic Press, 
London 1978. 
8 Nagasawa, J., Douglas, W.W., and Schulz, R.A., Nature 227 
(1970) 407-409. 
9 Nagasawa, J.. Douglas, W.W., and Schulz, R.A., Nature 232 
(1971) 341-342. 
10 Gerschenfeld, H.M., Tramezzani, J.H., and De Robertis, E., 
Endocrinology 66 (1960) 741-762. 
11 Castel, M., Gen. comp. Endoer. 22 (1974) 336-337. 
12 Lescure, H., and Nordmann, J.J., Neuroscience 5 (1980) 651-
659. 
13 Nordmann, J.J., and Morris, J.F., Nature 261 (1976) 723-725. 
14 Theodosis, D.T., Dreifuss, J.J., Harris, M.C., and Orci, L., J. 
Cell Biol. 70(1967) 294-303. 
15 Nordmann, J.J., Dreifuss, J.J., Baker, P.F., Ravazzola, M., 
Malaisse-Lagae, F., and Orci, L., Nature 250 (1974) 155-157. 
16 Gratzl, M., Torp-Pedersen, C , Dartt, D., Treiman, M., and 
Thorn, N.A. , Hoppe-Seylers Z. physiol. Chem. 361 (1980) 
1615-1628. 
17 Herzog, V., and Fahimi, H.D., Analyt. Biochem. 55 (1973) 
554-562. 
18 Russell, J.T., and Thorn, N.A., Acta endoer. 76 (1973) 471-
487. 
19 Weinstein, H., Berne, R.M., and Sachs, H., Endocrinology 66 
(1960) 712-718. 
0014-4754/83/091007-03$ 1.50 + 0.20/0 
©Birkhäuser Verlag Basel, 1983 
6 


Stimulation-dependent uptake of an extracellular marker to subcellular fractions of isolated neurohypophysial tissue

Open Access LMU ( Ludwig-Maximilians-Univ. München)

EXPERIENTIA Managing editors: H. Mislin, Basel and Carona W.P. Koella, Basel Editors: H. Aebi, Bern D. Arigoni, Zürich R. Bachofen, Zürich P. S. Chen, Zürich A. Pletscher, Basel S. Weidmann, Bern R. Weil, Geneve VoL39 1983 Part II Birkhäuser Verlag Basel • Boston • Stuttgart Experieniia 39 (1983). Birkhäuser Verlag, CH-4010 Basel/Switzerland 1007 Stimulation-dependent uptake of an extracellular marker to subcellular fractions of isolated neurohypophysial tissue M. Gratzl2, J.T. Russell3 and N. A. Thorn Institute ofMedical Physiology C, University of Copenhagen, The Partum Institute, Blegdamsvej 3c, DK-2200 Copenhagen N (Denmark), December 6, 1982 Summary. Slices of ox neurohypophyses and groups of isolated rat neurointermediate lobes were incubated in a medium containing horseradish peroxidase (HRP) and stimulated by high K+ concentrations in the medium. After washing, the tissue was homogenized and subjected to subcellular fractionation in a Percoll/sucrose gradient. HRP was exclusively taken up by particles banding at a low density of the gradient. The HRP containing particles located to this region included vacuoles of a size comparable to secretory vesicles. Some essential facts are known about the mechanism by which the neurohypophysial hormones Vasopressin and Oxytocin are released. It has been reported that on Stimula-tion of isolated rat neurohypophyses, vesicles containing Oxytocin and the binding protein (neurophysin) release their contents to the medium in a roughly stoichiometric way4, whereas axoplasma enzyme markers are not released5. These facts support the hypothesis that release occurs by exocytosis. Since the area of the cell membrane in the terminal dilatations is not increased after strong Stimu-lation67 retrieval of membrane (vesicle membrane and/or cell membrane of a size similar to the total area of mem-brane secretory vesicles incorporated) must take place after Stimulation. On the basis of morphological studies the prevailing hypothesis has been that secretory vesicles are retrieved as microvesicles89, although early histological investigations have provided evidence that membrane is retrieved as vacuoles similar in size to the secretory vesi-cles1011. In recent years, an increase in the vacuole popula-tion on Stimulation without increase in the microvesicle population has been shown in several morphometric studies612'14. Stimulation-dependent uptake of isotope-labeled extracel-lular markers has been observed when whole rat neurohy-pophyses were incubated in media containing such mark-ers15. No studies have been reported on larger animals and/ or on attempts to isolate the vesicles retrieved after exocy-tosis. Such isolation would be necessary to answer the question whether the vacuole membrane is similar to that of the secretory veiscle membrane or that of the cell membrane and might be of use in studies of the fate of the vacuoles. In the present work we report on stimulation-dependent uptake of HRP into subcellular fractions from rat and bovine neurohypophyses. As analyzed in density gradients the material containing the extracellular marker can be isolated from both species. Materials and methods. Isolated rat neurointermediate lobes. For each of 3 experiments, 60 isolated neurointermediate lobes from female Wistar rats (300 g b.wt) killed by decapitation, were divided into 2 groups of 30. Both groups were washed twice in 'controP medium without HRP (table 1). The 2 groups were then incubated in 'controP medium and 'Stimulation* medium, respectively, for 60 1008 Experientia 39 (1983), Birkhäuser Verlag. CH-4010 Basel/Switzerland min. After this, both sets of neurohypophyses were washed with 15 changes of 4 ml control medium, containing no HRP, and 3 times with a Solution containing 10 mM N-Tris (hydroxymethyl) methyl-2-aminoethanesulphonic acid (TES), pH 7.0, 190 mM sucrose and 25 mM EGTA after which they were homogenized. HRP was from Sigma (type II). Slices from ox neurohypophyses. Hypophyses from cows, aged 2-5 years, killed by shooting, were obtained at the Copenhagen Public Slaughterhouse, shortly after death of the animals. The neural lobes were carefully dissected free of pars intermedia. For each of 4 experiments 2 sets, each with 2 neural lobes, were cut longitudinally with a scalpel into 5 slices (thickness approximately 0.8 mm), and placed into 2 x 2 ml of control medium, not containing HRP (see table 1). The material was kept at 20 °C and transported to the laboratory within 20 min. Here, 1 group of slices was ineubated twice for 15 min in the control medium (table 1). The control medium in these studies contained 1 mM EGTA to reduce 'basal' release. For Stimulation studies, another group of slices was ineubated for 2 periods of 15 min in Stimulation medium (table 1). After ineubation, both groups of slices were washed once in control medium, containing no HRP and 3 times for 1 min with a Solution containing 10 mM TES (pH 7.0), 190 mM sucrose and 25 mM EGTA, after which they were homogenized. Homogenization, subfractionation, and other procedures used were carried out as described previously for ox neurohypophyses16. Briefly, the homogenate was centri-fuged at 750 x gav 15 min at 4°C. The supernatant (S,) was centrifuged at 28,000 x gav for 15 min using a TI50 rotor in a preparative Beckman ultracentrifuge. The pellet (P2) was resuspended, mixed with a 50% Solution of Percoll® in homogenization medium and centrifuged at 60,000 (cow neurohypophyses) or 80,000 xg a v (rat tissue) for 30 min at 4°C using a Beckman 40.2 rotor with the brake on. After centrifugation the tubes were fractionated through a needle in the bottom. The S2 supernatant was further centrifuged at 170,000 xg a v for 60 min, giving a pellet (P3) and a supernatant (S3)16. The HRP activity was determined in the gradient and other fractions using 3,3'-diaminobenzidine as a hydrogen donor17 with 0.2% Triton X-100 present in the assay. Results and discussion. Figure A shows the distribution of HRP in the gradients containing the material from stimu-lated and non-stimulated rat tissue. In the fractions ob-tained from stimulated neurohypophyses, a Symmetrie peak in the gradient contained more than twice as much HRP activity as the control. In 2 more experiments, the HRP activity was increased by a factor 2.2 and 3.3, respectively. Increased uptake of HRP into subcellular particles obvious-ly had occurred. This stimulation-dependent uptake was abolished by lack of Ca2+ in the medium or by D600 (a methoxy derivative of Verapamil) which blocks stimulation-induced release of Vasopressin18 (data not shown). It can be concluded that the extracellular marker used in these experiments is useful to mark newly formed particles not only in the tissue101115 but it is also kept within these struetures during homogenization, differential centrifuga-tion and density gradient centrifugation. Although fresh rat neurohypophyses can easily be ob-tained, a major problem in work with the gland from these animals is the minute amount of tissue ( < 1 mg/neural lobe) and difficulties in separating pars intermedia from the neural lobe. Therefore, biochemical analyses and sub-fractionation studies have been carried out mainly with bovine neurohypophyses (150 mg/neural lobe), with the drawback that the hemorrhage during slaughtering stimu-lates neurohypophyses to release hormone19. Despite this fact, we have found an increased uptake of HRP into previously characterized subcellular fractions from bovine neurohypophyses16 which have the advantage that they can be freed completely of tissue from the intermediate lobe. The distribution of HRP in fractions obtained from stimu-lated and non-stimulated bovine neurohypophyses is shown in table 2. The high amount of enzyme activity observed in all supernatant fractions most likely results from incomplete washout of extracellular marker prior to subfractionation. We observed a small increase of HRP activity into the microsomal fraction (P3) and a larger increase in fraction P2. The latter subcellular fraction has been found to contain about 40-50% of the marker for secretory vesicles, mitochondria, microsomes, lysosomes and cell membranes16. The hormone containing material recovered in P2 can be separated from other struetures by density gradient centrifugation using Percoll®16. Using an identical procedure we found maximal HRP activity around fraction 23 in the gradients (see fig. B), weil separated from secretory vesicles (which form a band around fraction 7), and mitochondria (centered around fraction 13)16. In 3 experiments, besides the one shown in figure B, the uptake of HRP was increased by a factor of 1.6, 1.6, and 1.3, respectively. However, the fractions con-taining HRP activity are still contaminated by lysosomes, Table 1. Composition (mM) of media used for HRP uptake studies in rat neurointermediate lobes and in ox neurohypophysial slices. All media were kept at 37 °C and gassed with 0 2 Neurointermediate lobes Control NaCl(140) KCl (5) MgS04(1.5) Stimulation - (45) KCl (100) - (1.5) Neurohypophysial slices Control CholineCl(150) - (0) MgCl2 (2) Stimulation - (90) - (56) - (2) EGTA(1) (1) pH 7.0 CaCl2(2.5) Hepes(lO) (2.5) -pH 7.3 ( l ) Hepes(lO) (4) Dextrose (10) (10) Dextrose (10) (10) HRP (5 mg/ml) (5 mg/ml) (5 mg/ml) (5 mg/ml) Table 2. Distribution of horseradish peroxidase in bovine neurohypophysial fractions separated by differential centrifugation Values in percent of homogenate Pi s, P2 s2 P3 s3 Control Stimulation 13.6+3.0 94.9+3.1 12.0±1.6 82.5 + 6.6 1.8 2.8 ;±o.2 ;±o.3 79.9 ±4.6 68.5 ±3.9 0.9 ±0.2 1.3 + 0.2 71.4 + 2.3 63.2 ±3.4 100% equals the total peroxidase activity in the homogenate of two neurohypophyses stimulated by 56 mM K+ (104.7± 19.8 umoles/min) or in nonstimulated neurohypophyses (137.3 ± 23.8 jimoles/min). Values are means of 5 experiments± SD. Experientia 39 (1983), Birkhäuser Verlag, CH-4010 Basel/Switzerland 1009 microsomes, and cell membrane fragments16. In thin sec-tion electronmicrographs of the HRP containing fractions recovered from the gradients the mentioned impurities were also observed, in addition to vacuoles which con-tained reaction product if the material was preincubated with 3,3'-diaminobenzidine and H 2 0 2 (not shown). The distribution within the gradients for rat and ox neural lobes were very similar. The peak in the rat experiments (using 80,000 x gav for 30 min) was at fraction 18, in the ox experiments (using 60,000 x gav for 30 min) it was at frac-tion 23. The exclusive uptake of HRP into the material banding at low density of the gradient argues against a non-specific A) Fractions Figure A. Distribution on a Percoll/sucrose gradient of HRP taken up by subcellular fractions of stimulated ( • ) and non-stimulated (O) rat neurointermediate lobes. Fractions were collected from the bottom. 4 r B) °0 10 20 30 Fractions Figure B. Distribution on a Percoll/sucrose gradient of HRP taken up by subcellular fractions of stimulated ( • ) and non-stimulated (O) bovine neurohypophysial slices. adsorption of HRP to membranes. If non-specific adsorp-tion had occurred, enzyme activity should also have been observed in the secretory vesicle fraction which is centered around fraction 7 of the gradient used. There was some uptake of HRP to non-stimulated rat neurohypophyses and a considerable uptake by non-stimu-lated ox neural lobe slices, even if they had been ineubated in a control medium with a very low concentration of free Ca2+ . This is probably due to the fact that even in the control circumstances there is some Stimulation of the tissue. The increased uptake of marker into fractions from bovine neurohypophyses stimulated with potassium was smaller than observed in experiments with rat neural lobes. This is probably due to the massive release of hormone induced by bleeding of the animals during slaughtering. However, an increased uptake of HRP could still be observed, which encourages further work on the isolation of these particles. The demonstration in the present experiments that HRP containing particles could be isolated after Stimulation adds independent support to the hypothesis that release occurs by exoeytosis. 1 Acknowledgments. The authors thank Mrs. M. Elis and B. Lynderup for excellent technical assistance and help during preparation of the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (G. 681/2-1), the Danish Medical Research Council, the NOVO Foundation, the Nor-disk Insulin Foundation and the P. Carl Petersen Foundation. 2 Present address: Abteilung Klinische Morphologie. Universität Ulm, Ulm. FRG. 3 Present address: Section on Functional Neurochemistry, Nat. Inst. Child Health & Human Development, Bethesda, Md, USA. 4 Matthews, E.K., Legros, J.J., Grau, J.D., Nordmann, J.J., and Dreifuss, J. J., Nature New Biol. 241 (1973) 86-88. 5 Edwards, B.A., Edwards, M.E., and Thorn, N.A., Acta en-doer. 72(1973)417-424. 6 Morris, J.F., and Nordmann, J.J., Neuroscience 5 (1980) 639-649. 7 Pickering, B.T., in: Essays in Biochemistry, vol. 14, pp.45-81. Eds P.N. Campbell and W.N. Aldridge. Academic Press, London 1978. 8 Nagasawa, J., Douglas, W.W., and Schulz, R.A., Nature 227 (1970) 407-409. 9 Nagasawa, J.. Douglas, W.W., and Schulz, R.A., Nature 232 (1971) 341-342. 10 Gerschenfeld, H.M., Tramezzani, J.H., and De Robertis, E., Endocrinology 66 (1960) 741-762. 11 Castel, M., Gen. comp. Endoer. 22 (1974) 336-337. 12 Lescure, H., and Nordmann, J.J., Neuroscience 5 (1980) 651-659. 13 Nordmann, J.J., and Morris, J.F., Nature 261 (1976) 723-725. 14 Theodosis, D.T., Dreifuss, J.J., Harris, M.C., and Orci, L., J. Cell Biol. 70(1967) 294-303. 15 Nordmann, J.J., Dreifuss, J.J., Baker, P.F., Ravazzola, M., Malaisse-Lagae, F., and Orci, L., Nature 250 (1974) 155-157. 16 Gratzl, M., Torp-Pedersen, C , Dartt, D., Treiman, M., and Thorn, N.A. , Hoppe-Seylers Z. physiol. Chem. 361 (1980) 1615-1628. 17 Herzog, V., and Fahimi, H.D., Analyt. Biochem. 55 (1973) 554-562. 18 Russell, J.T., and Thorn, N.A., Acta endoer. 76 (1973) 471-487. 19 Weinstein, H., Berne, R.M., and Sachs, H., Endocrinology 66 (1960) 712-718. 0014-4754/83/091007-03$ 1.50 + 0.20/0 ©Birkhäuser Verlag Basel, 1983 6 

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Stimulation-dependent uptake of an extracellular marker to subcellular fractions of isolated neurohypophysial tissue

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