Læknablaðið - 15.11.1990, Blaðsíða 60

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LÆKNABLAÐIÐ
12,4 mmol; trypsin, 104,0 KIU) as compared with the
fasting levels alone (P<0,05) (see above). A dominant
period of about 60 min. was still detected but second
periods of~45 min. and~95 min., respectively, were
also observed. The peak power and the total power of
the biliary secretion signals were reduced. Reinfusion of
aspirated duodenal fiuid into the intestine (jejunum) led
to a further decrease in peak power and totai power of
the biliary signals. Trypsin secretion into the duodenum
revealed mainly insignificant changes in peak power and
total power upon hormone stimulation despite a definite
increase in total amount of trypsin secreted. - The results
indicate that parenteral ceruletide/secretin stimulation has
a stabilizing effect on biliary secretion in man, and that
reinfusion of aspirated duodenal fluid into the intestine
during experiments further stabilizes the secretory output.
pge2 or pgd, suppress vldl
TRIÁCYLGLYCEROL SECRETION IN CULTURED
RAT HEPATOCYTES, AND WITHOUT CAUSING
AN INCREASE IN cAMP OR KETONE BODIES
PRODUCTION
Ó.G. Björnsson, S.M. Bartlett, G.F. Gibbons.
Metabolic Research Laboratory, Nuftield Department
of Clinical Medicine, Radcliffe Infirmary, Oxford,
U.K.
Several types of prostaglandins are produced by the
liver (Kupffer cells and endothelial cells) (Biochem
Biophys Acta 1988; 959: 143-52). The liver is also a
main organ for prostaglandin metabolism (J Biochem
1984; 96: 429-36), and recent studies suggest that
prostaglandins (of D. E or F series) may also regulate
hepatic metabolism (inhibit epinephrine or glucagon-
stimulated glycogenolysis or fatty acid oxidation, and
cAMP accumulation) (Biochem Biophys Acta 1988;
958: 179-87, Biochem J 1990; 267: 59-62). We have
studied the effect of PGE2 or PGD, on hepatocellular
triacylglycerol synthesis and VLDL triacylglycerol
secretion in cultured rat hepatocytes. Hepatocytes from
Wistar rats fed on carbohydrate rich 41B Dixon diet
were isolated under sterile conditions by the collagenase
method of Berry and Friend and cultured in Waymouth’s
medium as described before (Biochem J 1988; 249: 37-
43). High rates of cellular triacylglycerol synthesis and
VLDL triacylglycerol secretion were achieved for at least
3 days by culturing the cells in medium supplemented
with dexamethasone and triacylglycerol precursors
(pyruvate, lactate, oleate); VLDL triacylglycerol secretion
was 169±32/ig mg'1 protein (day 1); 217±33/ug mg"1
(day 2); 241±39/ig mg"1 (day 3); SEM, n = 5-6. During
the 3 days of culture, PGE2 (5xlO"7 M-5xl0’5 M)
suppressed VLDL triacylglycerol secretion in a dose-
dependent manner (max. 47% of control, P<0,05, n=16;
compared with max. suppression to 66% of control by
glucagon (10 7 M), P<0,05). The suppressive effect of
PGE, and glucagon in combination was similar to the
effect of PGE, alone (max. 41% of control). However,
the stable PGÉ, analogue 16,16-dimethyl-PGE, (5xl0 6
M) suppressed VLDL triacylglycerol secretion to max.
33% (n = 6) of control, (compared with only 61% by
PGE2 at 5x 10’6 M), indicating a considerable breakdown
of natural PGE2 during the experiments. PGD, (5x 10’5
M) suppressed VLDL triacylglycerol secretion to 64%
of control (n = 5). Neither prostaglandins (E, or D,)
nor glucagon increased hepatocellular net synthesis of
triacylglycerol in the present studies as estimated on
incorporation of 5H from oleate into triacylglycerol).
However, suppression of VLDL triacylglycerol secretion
was followed by an accumulation of triacylglycerol
within the cells (35% increase by PGE,). Glucagon
increased ketone bodies output (0,534±0,05/tmol ml "1
vs. 0,384±0,04/tmol ml"1 control, P<0,05), while PGE,
alone or glucagon and PGE, in combination did not
increase ketone bodies production. Glucagon increased
cAMP levels (max. 269 pmol mg’1 protein by 30 min.,
29 pmol mg 1 by 3 h; down to resting levels by 24 h),
while PGE, or PGD, had no effect on cAMP levels. -
PGE, or PGD, suppress VLDL triacylglycerol secretion
in cultured rat hepatocytes and caused accumulation of
triacylglycerol within the cells. In contrast to glucagon
which also reduced VLDL triacylglycerol secretion, PGE,
or PGD, did not increase cellular levels of cAMP nor did
they cause /3-oxidation of fatty acids as estimated by an
increase in ketone bodies production.
BUFFERING OF CYTOSOLIC FREE Ca2+ BY
FURA-2 IN ELECTRICALLY STIMULATED
CARDIAC VENTRICULAR MYOCYTES OF THE
RAT, AND ESTIMATION OF Ca2+ MOBILIZED
INTO THE CYTOSOLIC COMPARTMENT
Ó.G. Björnsson, I.R. Siemens, J.R. Williamson.
Department of Biochemistry and Biophysics,
University of Pennsylvania, Phiadelphia, PA 19104,
USA.
Fura-2 is a fluorescent Ca2+ chelator (J Biol Chem
1985; 260: 3440-50) which is widely used to monitor
changes in conc. of cytosolic free Ca2+, [Ca2*], in living
cells. However, the Kj of fura-2 for Ca'+ is in the same
range as [Ca2+]|, thus fura-2 acts as a Ca2+ buffer. The
buffering effects of fura-2 on [Ca 2+]j were studied in
cardiac ventricular myocytes in suspensions. Increasing
the fura-2 load from 72 to 632 pmol mg’1 drw (~36 to
316/iM) had no detectable effect on the rate of rise of
the electrically induced [Ca2+]j transient (to5=15,5±0,8
ms. vs 16,0±0,6 ms., SEM, respectively, P>0,05),
whereas the time of decline towards basal levels was
increased from 172,6±6,7 to 319,4±23,7 ms., and peak
[Ca2+]j decreased from 234±17 nM to 110±8 nM,
SEM, n=36. Resting [Ca2+]j fell from 84±6 nM to 46±5
nM (P<0,05). Fura-2-bound Ca2+ rose from 19,1 ± 1,3
to 31,8± 1,8 pmol mg’1 drw. (n = 30) upon electrical
stimulation in myocytes loaded with low amounts of fura-
2 (36,l±l,8/iM), indicating that~44% of cytosolic fura-2
was bound to Ca+’ (comparative value for fura-2 levels of
315,9±30,8/tM was~27%, n=8). From the [Ca+2]j, Ca+2
bound to fura-2 and Ca+2 bound to cytosolic proteins, the
amount of Ca+2 mobilized into the cytosol was estimated
at 53±7 pmol mg’1 drw. per electrical stimulation in
cells incubated in buffer alone, compared with 145±28
pmol mg’1 drw. and 88± 10 pmol mg 1 drw. in cells
pretreated with isoproterenol (ÍO'6 M) or Bay K 8644
(5x 10’6 M), respectively (in the case of Bay K 8644,
resting [Ca+2]j levels were increased). Comparative values
for nifedipine (10'5 M), ryanodine (5 x 10 6 M) or TMB-
8 (ÍO'5 M) pretreated cells were 21±2, 19±1, and 36±3
pmol mg 1 drw., respectively (n = 5 - 10). In the presence
of EGTA, [Ca+2]0 < 100 nM, caffeine (10'2 M) induced
Ca+2 release was estimated at 129± 12 pmol mg'1 drw.
(further release of 116± 15 pmol mg 1 drw. was obtained
by ionomycin 10'’ M). - The data show that the chelator