Læknablaðið - 15.12.1986, Page 74

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LÆKNABLAÐIÐ
25(OH)D3 measured in 36 healthy Icelanders, matched
for sex, age and seasonal variations in vitamin levels,
served as a control, and bone specimens (n = 36)
obtained from similarly matched males who had died
suddenly without evidence of metabolic bone disease.
Compared with the matched control group (necropsies)
osteoid bone volume was increased in the resected
subjects (0.46% ±0.07, SEM vs. 0.18% ±0.03, control,
p<0.01). During the 4 months of treatment with
vitamin D and calcium, bone osteoid volume decreased
to 0.15% ±0.03, p<0.05. Calcified bone volume
remained unchanged during treatment (17.9% ±1.4 vs.
15.8% ± 1.0, 4 months), and was not different from the
controls (17.0% ±1.1). Histological evidence of
osteoporosis was found in 5 resected subjects. During
treatment serum conc. of 25(OH)D3 increased from
54.6±6.2 nmol/1 (SEM) (beginning) to 73.4±7.0
nmol/1 (day 10) (p<0.01) to 77.1 ±6.7 nmol/1 (day
120), respectively (control, 56.8 ±4.1 nmol/1). Alkaline
phosphatase fell from 87.0±6.9 U/1 (day 0) to
70.0±6.2 U/1 (day 10) to 61.5±4.3 U/1 (day 120)
(p<0.01). Serum conc. of calcium (corrected for
albumin), inorganic phosphate and 25(OH)D3 were low
in several individuals at the beginning, and in 11 out of
32 resected subjects serum total protein was below
normal values (<65 g/1). In addition, total protein
levels were just above the lower normal margin (66-70
g/1) in 14 subjects. Protein remained low in these
individuals throughout the study but calcium and
25(OH)D3 levels increased upon treatment. Histological
evidence of osteomalacia was found in Icelandic males
following partial gastrectomy. Low serum calcium and
25(OH)D3 levels were found in several of the resected
subjects and most of them had low total protein. The
osteomalacia responded to vitamin D and calcium
treatment.
ESTIMATION OF CYTOSOLIC FREE CALCIUM
(Ca!+) BY FLUORESCENCE INDICATORS, QUIN-2
AND FURA-2: Studies in mycocytes
Ó.G. Björnsson, J.R. Williamson. Department of
Biochemistry and Biophysics, University of
Pennsylvania, Philadelphia, U.S.A.
Quin2 and Fura2 are fluorescent Ca2* sensitive
indicators which can be loaded into cells by incubating
the cells with membrane-permeant ester derivative of
these indicators, Quin2/AM and Fura2/AM (JBC; 260:
3440-50, 1985). Cytosolic esterases split off the ester
groups, and trapped membrane impairment Quin2 and
Fura2 tetra anions bind Ca2+. Subsequently, changes in
fluorescence can be used to monitor changes in (Ca2*),.
Due to stronger fluorescence (higher fluorescence
quantum yield), weaker affinity for Ca2+ (higher Kd) and
better selectivity against other divalent cations, Fura2
has been claimed to be superior to Quin2 in measuring
Ca2+ in adherent cells, bulk tissues and single cells. We
used these indicators to measure changes in (Ca2+)j
induced by electrical depolarization in cardiac myocytes
in cell suspensions. Varying esterloading and the effects
of compounds assumed to raise or lower
(Ca2+), were studied and comparison made between the
two markers. Myocytes (rat) were isolated by
collagenase in calcium free Krebs-Henseleit buffer.
After repleting intracellular Ca2+ pools, cells were
loaded with varying amounts of Quin2/AM (0.1-5
nmol/mg drw) or Fura2/AM (0.01-5 nmol/mg drw) and
fluorescent measurements were carried out in dual
channel fluormeter. Synchronous cell contractions were
observed upon electrical stimulation and these
paralleled with transient changes in Quin2-Ca2+ or
Fura2-Ca2+ fluorescence. In Quin2 loaded cells (Ca2+),
levels rose from 54.6 ±7.2 nM (SEM, n= 16) (resting) to
107.3 ± 16.6 nM upon electrical stimulation, and a
further rise to 154.0±46.5 nM was observed in the
presence of isoprolerenol (1 x 10 6 M) (n = 7). Addition
of the adenylate cyclase inducer forskolin, or the
dihydropyridine calcium agonist BAY-K 8644, increased
(Ca2+), further. Adenosine or prostacyclin, effective
inducers of adenylate cyclase in vascular smooth
muscle, had no effect on calcium in the myocytes. The
sulfidopeptide leukotriene L I D.. suppressed (Ca:') in
stimulated Quin2 loaded cells: control 88.3 ±6.5 nM
(SEM, n = 4), LTD4 1 x 10"7 M. 76.2±10.7 nM, LTD4
5 x 10-7 M, 71.2±4.7 nM, LTD4 1 x 10~6 M, 56.9±5.2
nM, LTD4 5 x 10-6, 50.1 ±1.7 nM. Followed by
addition of isoproterenol this suppression was
overcome. In Fura2 loades cells resting (Ca2+); levels
were 71.1 ±14.7 nM (SEM, n = 12), and electrically
stimulated (Ca2+), 135.9±27.1 nM. These figures were
not significantly different from the corresponding
figures obtained from Quin2 loaded cells. In a separate
series of Fura2 loaded cells isoproterenol of conc. as low
as 1 x 10 ’ or 1 x 10 “ increased electrically stimulated
(Ca2+), (128% and 146% respectively, n = 4), and
addition of forskolin (1 x 10’6 M) increased (Ca2+),
further (183% of control). LTD4 (1 x 10"7 M, 5x 10"7
M, and 1 x 10“6 M) suppressed (Ca2+)i to 77.5%, 67.9%
and 60.7% of control, respectively. Fluorescence
transients upon electrical stimulation were detected at as
low loading of Quin2/AM and Fura2/AM as 0.5
nmol/mg drw and 0.05 nmol/mg drw, respectively.
Maximum transients for Fura2/AM were observed at 1
nmol/my drw. At Fura2/AM loading as high as 5
nmol/mg drw the fall in fluorescence after electrical
stimulation was small and the fluorescence peak signal
became »blunted«. In Quin2 loaded cells fluorescence
did never return completely to basal (resting) levels after
stimulation at any loading of Quin2/AM tested. -The
calcium-sensitive fluorescent indicators Quin2 and
Fura2 were used to measure conc. of cytosolic free
calcium in cardiac myocytes. Similar resting and
stimulated levels of free calcium were obtained in Quin2
and Fura2 loaded cells. However, the fluorescence
pattern depended on which indicators were used and the
amount of indicator used. In Quin2 loaded cells, and in
cells loaded with Fura2 of high conc., the fluorescence
peak »blunted« and the resting levels became high after
stimulation, presumably due to buffering effects of the
indicators.