1 Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, Kansas 66160; and 2 Hoffmann-La Roche, Nutley, New Jersey 07110-1199
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We investigated the effects of 1,25-dihydroxyvitamin
D3 [25(OH)2D3] on
tissue plasminogen activator (tPA) secretion from primary cultures of
rat heart microvascular cells. After an initial 5-day culture period,
cells were treated for 24 h with 1,25(OH)2D3
and several of its analogs. The results showed that
1,25(OH)2D3 induced tPA secretion at
1010 to
10
16 M. A less calcemic analog,
Ro-25-8272, and an analog that binds the vitamin D receptor but is
ineffective at perturbing Ca2+ channels, Ro-24-5531,
were ~10% as active as 1,25(OH)2D3. An analog that binds the vitamin D receptor poorly but is an effective Ca2+ channel agonist, Ro-24-2287, required
~10
13 M to induce tPA secretion.
Combinations of Ro-24-5531 and Ro-24-2287 were approximately
as potent as 1,25(OH)2D3. Treatment of the cells with BAY K 8644 or thapsigargin also increased tPA secretion, suggesting that increased cytosolic calcium concentration
([Ca2+]) induces tPA secretion. The results
suggested that the sensitivity of the tPA secretory response of
microvascular cells to 1,25(OH)2D3 was
due in part to generation of a vitamin D-depleted state in vitro and in
part to synergistic effects of 1,25(OH)2D3 on
two different induction pathways of tPA release.
heart; vitamin D; calcium; analogs
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
VITAMIN D3 through its hormonally active
metabolite 1,25-dihydroxyvitamin D3
[1,25(OH)2D3] is an essential
component of the systems that regulate bone calcification (36),
intestinal Ca2+ absorption (29), kidney function, and
parathyroid gland activity (10). 1,25(OH)2D3
may also be important to functions of the immune system, heart,
pancreas, brain, pituitary, cancer cells, and vascular endothelium (32,
48). Most effects of 1,25(OH)2D3 are thought to
result from its binding to a cytosolic/nuclear receptor, similar to
receptors of steroids, thyroid hormone, and vitamin A (14), which has a
dissociation constant (Kd) of
1010 to
10
11 M (40). The ligand-activated
vitamin D receptor is a heterodimer with the retinoid (RXR) receptor
that binds to vitamin D response elements in responsive genes (13).
Recent studies suggest that actions of 1,25(OH)2D3 may involve both genomic and nongenomic pathways (37). Nongenomic actions of 1,25(OH)2D3 are rapid and include a prolonged open time of voltage-gated Ca2+ channels and a shift in the threshold of activation toward the resting cell potential (7). Several cellular signal transduction pathways may be involved in nongenomic actions of 1,25(OH)2D3 such as those involving cAMP, protein kinase C, and inositol phosphate (12, 41, 46).
The vascular endothelium plays an essential role in the fibrinolytic system by virtue of the secretion of tissue plasminogen activator (tPA) (11, 44). tPA converts inactive plasminogen to the active serine protease plasmin that degrades the fibrin component of thrombi. The regulation of tPA secretion involves second messengers, such as calcium (42), protein kinase C, cAMP, and diacylglycerol (20), and may involve increased transcription of the tPA gene (38). Among the agents known to increase tPA secretion from the endothelium are thrombin and histamine (21), butyrate (27), phorbol-12-myristate-13-acetate (47), and retinoids (26).
Vascular endothelial cells contain the vitamin D receptor (32), but the
function(s) of 1,25(OH)2D3 in the vasculature
have not been examined. We decided to test the hypothesis
that 1,25(OH)2D3 could regulate the
release of PA from cultured rat heart microvascular cells. This
was based on our observation that 1,25(OH)2D3
induces PA secretion from bovine parathyroid cells (2) and the
demonstration by Fukomoto et al. (18) that
1,25(OH)2D3 increases the release of PA
activity from bone cells. In the present study, we show that
1,25(OH)2D3 and several of its synthetic
analogs stimulate the secretion of tPA. Under the conditions of our
experiments, 1,25(OH)2D3 increased tPA
secretion within 24 h at concentrations of
1016 M and above. By comparison,
responses to 1,25(OH)2D3 in other cultured
target cells have been observed at hormone concentrations of
10
11 to
10
9 M (1, 2, 25); the concentration of
the free secosteroid in the circulation in vivo is 1-2 × 10
13 M (45). In addition to the
beneficial effects of preculture in lipid-depleted medium, our
experiments using synthetic analogs of
1,25(OH)2D3 suggest that the high level of
sensitivity to 1,25(OH)2D3 may have resulted in
part from synergy between two different mechanistic pathways of vitamin
D action.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials
Human 52 kDa urokinase, human 70 kDa tPA, goat anti-human tPA IgG, human Glu-type plasminogen, H-D-norleucyl-hexahydrotyrosyl-lysine-p-nitroanilide diacetate salt, human 2-thrombin, and plasminogen-free fibrinogen were purchased from American Diagnostica (Greenwich, CT). BAY K 8644 was purchased from Calbiochem (La Jolla, CA). Thapsigargin, amiloride, lipid-depleted controlled process serum replacement-1 (CPSR-1), glucose, MOPS, insulin, creatine, taurine, carnitine, SDS, and routine chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Acrylamide/bisacrylamide, ammonium persulfate, and glycine were purchased from Bio-Rad (Richmond, CA). Fetal bovine serum was purchased from Hyclone (Logan, UT). Purified collagenase, papain, and elastase were obtained from Worthington Biochemical (Lakewood, NJ). 1,25(OH)2D3, 25-hydroxy-16,23-diene-vitamin D3 (Ro-24-2287), 1,25-dihydroxy-16-ene-26,27-hexafluoro-vitamin D3 (Ro-24-5531), and 1,25-dihydroxy-16-ene-24-oxo-vitamin D3 (Ro-25-8272) were obtained from Hoffmann-La Roche (Nutley, NJ).Cell Preparation and Treatment
Female Sprague-Dawley rats (retired breeders) were anesthetized with xylazine and ketamine. Hearts were excised and briefly perfused with Ca- and Mg-free Hank's balanced salt solution (HBSS) to remove red cells. Rat heart microvascular cells were isolated by the method of Nishida et al. (35), with minor modifications. Contamination by mesothelial and endocardial endothelial cells was minimized by devitalizing the endocardial and epicardial surfaces with 70% ethanol and dissecting away the atria, valves, right ventricle, and one-third to one-fourth of the left ventricular pericardial wall. The remaining tissue was minced. The tissue fragments from 2 or 3 hearts were digested in ~30 ml of Leibovitz's L15 medium, containing 250 U/ml of purified bacterial collagenase, 5 U/ml of papain (unactivated), and 0.8-1 U/ml (0.25 mg/ml) of elastase with continuous tumbling at 37°C. The tissue fragments were gently triturated with a 10-ml serological pipette every 20-30 min. After 2-3 h, the digest was filtered through a 70-µm Falcon cell strainer (Becton-Dickinson Labware, Franklin Lakes, NJ) and centrifuged at low speed for 10 min to sediment the cells. The pellet was suspended in HBSS and then washed by sedimentation for 5 min at the same speed. The wash was repeated twice more. The final pellet of microvascular segments and cells was suspended in modified Leibovitz's L15 medium containing 11 mM glucose, 20 mM MOPS, 4 × 10PA Activity Assay
For assay of cellular PA, cell extracts were freeze-thawed three times before use; media samples were assayed directly. PA activity was assayed colorimetrically, essentially according to Campbell et al. (8) by use of human tPA as a standard. Briefly, 50 µl of media or diluted cell extracts were incubated at 37°C in 96-well plates with (final concentrations) plasminogen (10 µg/ml), alkali-denatured fibrinogen (170 µg/ml) (39), and the chromogenic peptide substrate of plasmin H-D-norleucyl-hexahydrotyrosyl-lysine-p-nitroanilide diacetate salt (170 µM). The samples were buffered with 0.1 M tricine buffer (pH 8.3). All assay mixtures contained Triton X-100 at a final concentration of 0.5%. The color development at 405 nm was read periodically on a plate reader (Titertech Multiscan Plus, Labsystems, Helsinki, Finland). The PA activity was calculated by comparison with tPA standards and was expressed as milliunits of PA, or milliunits of PA secreted per day.Characterization of PA Activity
Fibrin zymography. Single-step fibrin zymography for the media was carried out according to modifications of the method of Heussen and Dowdle (22). Fibrinogen (90 µg/ml), plasminogen (3.375 µg/ml), and thrombin (0.3 U/ml) were combined with acrylamide/bisacrylamide before gel formation or electrophoresis. By this means fibrinogen was converted to fibrin and copolymerized with the acrylamide in the electrophoretic gel. Samples were separated on 9% SDS-acrylamide-separating gels. After electrophoresis, gels were extracted by 2.5% Triton X-100 in water for 2 h with three changes to remove SDS. To express PA activity, the fibrin-containing gels were incubated in 0.1 M tricine buffer (pH 8.3) at 37°C for 15 h. The proteins were then fixed in 10% TCA and then extracted with 7% ethanol. The gels were stained for 1 h with Coomassie Brilliant Blue R250 and then destained with 7% acetic acid. They visualized PA activity as clear bands in the gels caused by plasmin-mediated digestion of the fibrin. Zymograms were scanned using a Hewlett-Packard Scanjet II CX/T scanner.
Inhibitor studies. The samples were incubated with a blocking antibody against human tPA or with amiloride. The mixtures were then assayed for PA activity or subjected to zymography to determine the nature of the PA(s) in the samples. The anti-tPA antibody was used at concentrations from 3 to 30 µg/ml. Amiloride was used at a concentration of 100 µM.
Statistical Analysis
The data were expressed as averages ± SE and were analyzed for significant differences between the means of experimental and control samples using Student's t-test. ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Rat heart microvascular preparations consisted of capillaries and some single cells. These cells were slow to migrate and flatten onto cultureware, but after 3 days in medium containing 20% fetal bovine serum, they formed semiconfluent monolayers. Two days after substitution of 2% lipid-depleted serum replacement for the fetal bovine serum, some cells retracted into cell clumps that remained attached to the plate.
The freshly isolated microvascular cells contained high levels of PA
activity. After 24 h in culture, however, 80 to >90% of the activity
had disappeared (Fig. 1A). After 2 days of culture, the cellular levels of PA activity stabilized at 10%
of the amounts in the freshly dispersed cells. In contrast to the rapid
changes in cell content of PA, the secretory rates of PA under control conditions were stable throughout the periods of culture. After the
cellular levels had stabilized on day 2, the cells secreted ~1-2 times the cellular content each day, indicating that PA
synthesis was an active process (Fig. 1B). The PA activity lost
from the cells in the first 24 h of culture was not recovered in the
media.
|
Nature of Secreted PA Activity
Effects of anti-human tPA serum on secreted PA activity.
The antiserum was incubated with samples of culture media from control
cells and from those treated with 1,25(OH)2D3
or isoproterenol. PA assays were subsequently performed. The results
(Table 1) indicated that the majority of
the activity in the media from control incubations, as well as that
from incubations after 1,25(OH)2D3 or
isoproterenol treatment, was neutralized by 30 µg/ml of anti-human tPA antibody. In addition, the degree of inhibition by the antibody was
greater in the 1,25(OH)2D3- or
isoproterenol-treated groups, suggesting that most of the PA secreted
in response to treatment with these agents was tPA. The action of
amiloride, a specific inhibitor of human urokinase, was also tested.
Amiloride inhibited the PA activity in media, but zymography studies
showed that it inhibited rat tPA as well as urokinase and thus was not
sufficiently selective to permit conclusions as to the contribution of
urokinase to the PA activity of rat cell culture media (data not
shown).
|
Zymography.
Samples of culture media and cell extracts were subjected to
electrophoresis in fibrin- and plasminogen-containing polyacrylamide gels followed by zymography. The zymography conditions were optimized for maximal sensitivity to PA activity but were not suitable for quantitative comparisons, although comparisons between the intensities of experimental zymography bands after scanning generally confirmed the
results of activity assays. The appearance of all bands was dependent
on the presence of plasminogen in the gels, indicating that the bands
represent PA activity. On these gels three activity regions were
consistently observed (Fig. 2) that were
similar to patterns observed previously (1, 2). Both native human 70 kDa tPA and corresponding areas of lanes representing the PA of
conditioned media displayed a zone of activity containing sub-bands. The band multiplicity probably reflected selective adsorption of
fibrin-binding regions of the human and rat tPAs to the immobilized fibrin in the gels. This zone was shown by densitometry to become more
intense after treatment of cells with
1,25(OH)2D3 or isoproterenol (e.g., Fig. 2,
lanes 5 and 6 vs. lane 3). Second, media and
cell extract samples contained a sharp band of activity that migrated more rapidly than did human 52 kDa urokinase; the intensity of this
band was not observed to be affected by experimental treatments. Third,
a sharp but not intense band of activity was observed that migrated
more slowly than did the tPA standard.
|
|
Effects of 1,25(OH)2D3 and
1,25-dihydroxy-16-ene-24-oxo-vitamin D3 on cellular PA
activity and PA secretion.
We tested the effects of different concentrations of
1,25(OH)2D3 and of a less calcemic analog,
1,25-dihydroxy-16-ene-24-oxo-vitamin D3 (28). The amount of
PA activity in cell lysates from the culture wells was unaffected by
any of the treatments. The results with conditioned media
(Fig.4) showed that rat heart microvascular cells under the conditions of these experiments increased their PA
secretory rates in response to very low concentrations of the test
compounds. Significant increases in PA secretion were consistently observed at 1016 M
1,25(OH)2D3. Maximal induction usually occurred
between 10
11 and
10
14 M secosteroid. The degree of
increase of secretion resulting from 24-h treatment with maximally
effective concentrations of either hormone varied between experiments
from ~50% to a maximum of approximately fivefold. The maximal degree
of secretory induction was often equivalent to that attained by
treatment of the cells with 10
6 M
isoproterenol (Table 1), which was used as a positive control for
inducibility. In multiple experiments, there were parallels in
magnitude between the effects of isoproterenol and
1,25(OH)2D3 suggesting that the
interexperiment variability in degree of inducibility of PA secretion
by 1,25(OH)2D3 did not reflect altered
sensitivity to the secosteroid, but rather variations in the ability of
the cells to increase their production and secretion of PA above their baseline rates.
|
Effects of two analogs of 1,25(OH)2D3 with
different affinities for the vitamin D receptor.
The great sensitivity of the microvascular cells to
1,25(OH)2D3 compared with the
(Kd) for its receptor of
1010 to
10
11 M suggested that the hormone might
induce PA secretion from rat cardiac microvascular cells by more than
one mechanism and that the mechanisms might interact to maximize
hormone sensitivity. To examine this hypothesis, we tested the
effects of two 1,25(OH)2D3 analogs, added
either individually or mixed. The first,
1,25-dihydroxy-16-ene-23-yne-hexafluoro-vitamin D3 (Ro 24-5531) interacts strongly with the
vitamin D receptors of chick intestine and ROS 17/2.8 cells and is
~0.01% as active as 1,25(OH)2D3 in
stimulating transmembrane Ca2+ influx into ROS 17/2.8 cells
(15, 25). The second, 25-hydroxy-16,23E-diene-vitamin D3
(Ro 24-2287) binds the vitamin D receptor only ~1/1,000 as well
as does 1,25(OH)2D3 (9, 16) but is a stronger
activator of Ca 22+ channels than is
1,25(OH)2D3 itself (25). Experiments were performed in which cells were treated with different
concentrations (from 10
13 to
10
16 M) of
1,25(OH)2D3 of each analog, individually or
with equimolar mixtures of the analogs at the same concentrations.
|
Effects of BAY K 8644 and thapsigargin.
The demonstrated abilities of 1,25(OH)2D3 to
activate Ca2+ channels (25) suggested that one mechanism by
which 1,25(OH)2D3 might induce PA secretion is
by the elevation of cytosolic [Ca2+]. To test
this hypothesis, we determined the effects on PA secretion of several
agents known to activate Ca2+ channels or to increase
cytosolic [Ca2+]. Table
3 shows that the calcium channel agonist
BAY K 8644 (2.8 M) increased PA secretion by 43% above control levels
(P < 0.001), similar to the effects of
1,25(OH)2D3 and the positive control
isoproterenol. The effects of BAY K 8644 were not, however, additive to
the effects of 1,25(OH)2D3 when the two agents
were added to the same sample wells.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The results of this study showed that, in lipid-depleted rat heart microvascular cells, 1,25(OH)2D3 induced the secretion of tPA from adult rat heart microvascular cells at sub-fM concentrations. We showed further that the high sensitivity of the cells to 1,25(OH)2D3 may be due in part to the secosteroid acting through more than one cellular pathway. It is likely that one induction pathway involves binding to the vitamin D receptor, but the manner(s) in which the 1,25(OH)2D3-bound receptor might induce tPA production and release have not yet been determined. In addition, the results are consistent with the notion that cytosolic [Ca2+] may be a major factor in one pathway.
The Microvascular Cell Culture System
The experimental system used in these studies is adult rat heart microvascular cells in short-term primary culture. It is comprised primarily of microvascular endothelial cells, and smaller percentages of vascular smooth muscle cells, interstitial cells (fibrocytes), and (vascular) pericytes (6). Our initial decision to study this cell preparation was based on the hypothesis that PA secretion would originate from endothelial cells in the cultures, but that responses to 1,25(OH)2D3 might require cooperative interactions between different cell types, because, for example, both endothelial cells and vascular smooth muscle cells contain vitamin D receptors (31). If pure cell populations or established endothelial cell lines were examined, therefore, physiologically relevant responses to 1,25(OH)2D3 might not be generated and observed. We performed one preliminary study with a rat endothelial cell line (graciously supplied by Dr. Clement Diglio, Wayne State University School of Medicine, Detroit, MI) and observed that the cells did not increase their rate of tPA secretion in response to 1,25(OH)2D3, isoproterenol, or thapsigargin. The results indicated that the ability to respond to 1,25(OH)2D3 might be lost from endothelial cells as a function of increased numbers of passages in culture.We performed additional preliminary studies (to be presented separately) that have supported the hypothesis that endothelial cells at low passages increase their rates of tPA secretion in response to 1,25(OH)2D3. We isolated purified rat heart microvascular endothelial cells and examined the effects of 1,25(OH)2D3 on PA secretion from cultures after three to four passages. The results showed that 1,25(OH)2D3 induced secretion of PA from the cultured endothelial cells. Second, we examined the effects of 1,25(OH)2D3 on fourth-passage newborn human dermal microvascular endothelial cells (purchased from BioWhittaker, San Diego, CA). PA secretion was increased by treatment with 1,25(OH)2D3 in these cells also. In summary, studies with the cell culture models described above led us to conclude that 1,25(OH)2D3 induced PA secretion from rat vascular endothelial cells and that no interactions with other cell types were required to generate the results.
Cell PA Content and Secretion
Freshly isolated microvascular cell preparations lost most of their cellular PA activity within 24 h of culture, but this phenomenon seemed unrelated to the rates of PA secretion that remained constant from the first time tested until the end of the culture and experimental periods (Fig. 1B). The initial rapid loss of PA activity, therefore, did not appear to result from secretion into the medium, and the fate of that enzyme is not known. It is possible that the cellular stores of PA in vascular endothelial cells in vivo represent the ability to secrete tPA rapidly in response to secretagogues such as histamine and thrombin. The loss of cellular PA in culture would be expected to correlate with a loss in the ability of the cells to secrete PA in response to acute stimuli. Our preliminary studies (data not shown) and the results of others (42) support the notion that acute secretory responses are compromised in cultured endothelial cells.One of the problems encountered in the study of the responses of rat heart microvascular cells to 1,25(OH)2D3 and other inducers of tPA secretion was that the degree of increase in induction of secretion varied from experiment to experiment. This variability can be observed in Tables 2-4 and Fig. 4. In these and many other experiments performed by us with microvascular cells, the greatest variability in secretory rates between experiments occurred not in the treated samples but in those representing the control or basal conditions. In virtually every case, moreover, the basal secretory rates were inversely related to the ability of a stimulatory agent like 1,25(OH)2D3 to increase secretion of PA activity over the baseline levels. The highest basal secretory rates observed were those associated with an established endothelial cell line whose PA secretory rates were not increased by 1,25(OH)2D3, isoproterenol, or thapsigargin. The reason(s) for the variability in basal secretory rates and degrees of its induction by stimulatory agents are not yet known. We speculate that unintended induction of tPA secretion may have occurred when the experimental period was initiated, or perhaps before. One possible variable was the amount of shearing stress that cells were subjected to during the media changes associated with initiation of the experiment. It has been reported, for example, that shear stress of endothelial cells causes Ca2+ entry into the cells by a mechanism involving myosin light-chain kinase (49). Variable degrees of shear stress between experiments could have caused variable degrees of Ca2+ entry that, in turn, may have induced release of PA activity from control and experimental groups of cells. If the results of Ca2+ entry and other experimental treatments were not additive (as seems the case), then the results would be observed as a reduced effect of test agents on the rates of release of PA activity into media. In the case of endothelial cells in particular, shear stress during media changes should probably be minimized as much as possible.
Effects of 1,25(OH)2D3 and 1,25-Dihydroxy-16-ene-24-oxo-vitamin D3 (Ro 25-8272) on Secretion of PA Activity
Both 1,25(OH)2D3 and a less calcemic analog strongly stimulated PA release; 1,25(OH)2D3 was more potent than the analog. Most notable about their induction of PA secretion is the range of concentrations over which they occurred. In the experiment shown in Fig. 3, significant increases in PA secretion were observed at 10One basis for the high potency of 1,25(OH)2D3
and its analogs in our experimental culture model rests on the
treatment of the cells with a medium containing 2% of a lipid-depleted
serum substitute before experimentation. We observed in early
experiments that, without this treatment, the effects of
1,25(OH)2D3 on PA secretion were variable and
sometimes insignificant; the maximal effects of the hormone were
observed at concentrations of 1010 to
10
11 M. The reason that the treatment
with lipid-depleted serum was initiated, however, was not to deplete
the cells of 1,25(OH)2D3 but to eliminate the
high concentrations of 25-OH-D3 normally present in serum.
It has been reported that vascular endothelial cells can convert
25-OH-D3 to 1,25(OH)2D3 (32). The
lipid-depleted serum replacement presumably contains little
25-OH-D3 or 1,25(OH)2D3, and so its
inclusion in media for the final 72 h of culture may render the cells
vitamin D deficient in vitro. In addition to the absence of
vitamin D metabolites in the serum substitute, moreover, the treatment
may additionally deplete the cells of bound
1,25(OH)2D3 because of its content (~100 nM)
of lipid-stripped vitamin D-binding protein(s) that might extract
receptor-bound hormone from the cells. Unfortunately, the
concentrations of vitamin D metabolites in the serum replacement were
not available.
In cells rendered vitamin D deficient, biochemical characteristics of
the cells might change in ways that would increase the sensitivity of
the cells to 1,25(OH)2D3. For example,
1,25(OH)2D3 reduces the levels of mRNA for the
-1C subunit of voltage-sensitive calcium channels in ROS 17/2.8
osteosarcoma cells (33). Depletion of
1,25(OH)2D3 in the rat heart microvascular
cells might therefore increase the synthesis and thus the number per
cell of such calcium channels. If part of the activity of
1,25(OH)2D3 in these cells is based on
regulation of cytosolic [Ca2+], exposure to the
hormone after a period of depletion would maximize the initial rate at
which Ca2+ could enter the cells and might by that induce
physiological responses at lower hormone concentrations.
In addition to the treatment with a lipid-depleted serum substitute,
the high potency of 1,25(OH)2D3 in rat heart
microvascular cells may be associated with cooperative effects on
ligand binding to the receptors or with postreceptor binding effects
such as increased metabolic stability of the receptor-ligand complex or high affinity for the DNA binding site (23). These possibilities have
been discussed in connection with the biological activities of
structural analogs of 1,25(OH)2D3, such as KH
1060, a 20-epi-analog of 1,25(OH)2D3 that does
not bind to vitamin D-binding protein (51). KH 1060 has been shown to
induce differentiation of U 937 cells at
1014 M and to inhibit
interleukin-1-induced proliferation of thymocytes at an
IC50 of 3 × 10
15 M,
while displaying a Kd for receptor binding of
10
11 M (4). These concentrations are
more in concert with the doses of 1,25(OH)2D3
shown here to increase the secretion of PA. This effect, therefore, may
have resulted from the ability of low doses of
1,25(OH)2D3 to maintain or reinstate
differentiated characteristics of rat heart microvascular cells, rather
than a direct receptor-mediated effect of the hormone on transcription
of the mRNA for tPA.
A third possibility to explain the high sensitivity to 1,25(OH)2D3 is that, in rat microvascular cells, 1,25(OH)2D3 may be acting by more than one signal transduction pathway and that the pathways used may interact synergistically to produce the high sensitivity to the hormone observed in our experiments. This hypothesis is based on recent demonstrations that 1,25(OH)2D3 is able to demonstrate effects by "nongenomic" as well as "genomic" pathways (37). For example, 1,25(OH)2D3 activates plasma membrane Ca2+ channels in target cells (3, 7, 34), thereby increasing the rate of Ca2+ entry and the cytosolic [Ca2+]. To examine the genomic and nongenomic pathways more deeply, numerous structural analogs of 1,25(OH)2D3 have been synthesized and tested for their abilities to bind to the vitamin D receptor and to influence the activity of Ca2+ channels (5, 25).
Two analogs of 1,25(OH)2D3 with different profiles of interaction with the vitamin D receptor or with plasma membrane Ca2+ channels were chosen to examine the hypothesis that the high sensitivity of rat vascular endothelial cells to 1,25(OH)2D3 may result from synergy between two interacting mechanisms. As described in the previous section, a genomic analog, 1,25-dihydroxy-16-ene-23-yne-26,27-hexafluoro-vitamin D3, binds to the vitamin D receptor like 1,25(OH)2D3 and is a more powerful initiator of transcription (15), but it interacts only weakly with Ca2+ channels (15). A nongenomic analog, 25-hydroxy-16,23E-diene-vitamin D3, on the other hand, binds poorly to the vitamin D receptor but is more powerful than 1,25(OH)2D3 at activating Ca2+ channels (25).
The results of the study with the analogs showed that they were both
individually active at inducing PA secretion from rat microvascular
cells. The genomic analog was the more powerful inducer and increased
PA secretion consistently at 1014 M. The
nongenomic analog required 10
13 M or
greater concentration to induce PA secretion. When added together at
10
15 M or even
10
16 M each, however, the combination
was more potent than either compound alone, suggesting that synergistic
effects may occur between the mechanisms used by each analog.
To determine whether the hypothesis was tenable that 1,25(OH)2D3 could increase PA secretion in part through increased cytosolic [Ca2+] resulting from increased Ca2+ channel activity, two drugs were tested that increase cytosolic [Ca2+]. BAY K 8644 is a Ca2+ (or cation) channel agonist (17), and thapsigargin is an inhibitor of the Ca2+ pump that transports Ca2+ from the cytosol into the lumen of the endoplasmic reticulum (24). Both agents stimulated PA secretion; thapsigargin did so very strongly. The results are in overall agreement with those of Tranquille and Emeis (42), who concluded that Ca2+ influx was essential for the acute release of tPA. The effects of altering cytosolic [Ca2+] on steady-state rates of tPA secretion have not yet been systematically investigated, however, and the effects of altered cytosolic [Ca2+] under physiological conditions remain to be shown. The results obtained after thapsigargin treatment may be complicated by its demonstrated induction of apoptosis in several cell types (50, 52).
At maximally effective concentrations of either 1,25(OH)2D3 or isoproterenol, no additive effects were observed when maximally effective doses of thapsigargin or BAY K 8644 were also present. Similar results were obtained when the effects of 1,25-dihydroxy-16-ene-23-yne-25,26-hexafluoro-vitamin D3 combined with thapsigargin were examined. The results suggested that cytosolic [Ca2+] may be an important component of the 1,25(OH)2D3-mediated mechanism for increasing PA secretion, but they do not infer that it comprises a separate pathway from those already in use by the hormone to increase PA secretion.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported by a grant from the American Heart Association, Kansas Affiliate (KS-97-GS-60).
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: R. R. MacGregor, Dept. of Anatomy and Cell Biology, Univ. of Kansas Medical Center, 39th St. and Rainbow Blvd., Kansas City, KS 66160-7400 (E-mail: rmacgreg{at}kumc.edu).
Received 14 May 1999; accepted in final form 15 September 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Allen, E. H.,
and
T. J. Martin.
Prostaglandin E2 regulates production of plasminogen activator isozymes, urokinase receptor, and plasminogen activator inhibitor-1 in primary cultures of rat calvarial osteoblasts.
J. Cell. Physiol.
165:
521-529,
1995[ISI][Medline].
2.
Bansal, D. D.,
and
R. R. MacGregor.
Secretion of plasminogen activator from bovine parathyroid cells.
Endocrinology
126:
2245-2251,
1990[Abstract].
3.
Baran, D. T.,
A. M. Sorenson,
V. Shalhoub,
T. Owen,
A. Oberdorf,
S. Stein,
and
J. Lian.
1,25-Dihydroxyvitamin D3 rapidly increases cytosolic calcium in clonal rat osteosarcoma cells lacking the vitamin D receptor.
J. Bone Miner. Res.
6:
1269-1275,
1991[ISI][Medline].
4.
Binderup, L.,
S. Latini,
E. Binderup,
C. Bretting,
M. Calverley,
and
K. Hansen.
20-epi-Vitamin D3 analogs: a novel class of potent regulators of cell growth and immune responses.
Biochem. Pharmacol.
42:
1569-1575,
1991[ISI][Medline].
5.
Bouillon, R.,
W. H. Okamura,
and
A. W. Norman.
Structure-function relationships in the vitamin D endocrine system.
Endocrine Rev.
16:
200-257,
1995[ISI][Medline].
6.
Burkitt, H. G.,
B. Young,
and
J. W. Heath.
Circulatory system.
In: Wheater's Functional Histology (3rd ed.). Edinburgh: Churchill Livingstone, 1993, chapt. 8, p. 140-152.
7.
Caffrey, J. M.,
and
M. C. Farach-Carson.
Vitamin D3 metabolites modulate dihydropyridine-sensitive calcium currents in clonal rat osteosarcoma cells.
J. Biol. Chem.
264:
20265-20274,
1989
8.
Campbell, E. E.,
M. A. Shitman,
J. G. Lewis,
J. J. Pasqua,
and
S. V. Pizzo.
A colorimetric assay for releasable plasminogen activator.
Clin. Chem.
28:
1125-1128,
1982
9.
Chen, T. C.,
K. Persons,
M. R. Uskokovic,
R. L. Horst,
and
M. F. Holick.
An evaluation of 1,25-dihydroxyvitamin D3 analogues on the proliferation and differentiation of cultured human keratinocytes, calcium metabolism and the differentiation of human HL-60 cells.
J. Nutr. Biochem.
4:
49-57,
1993[ISI].
10.
Chertow, B. S.,
G. R. Baker,
H. L. Henry,
and
A. W. Norman.
Effects of vitamin D metabolites on bovine parathyroid hormone release in vitro.
Am. J. Physiol. Endocrinol. Metab.
238:
E384-E388,
1980
11.
Collen, D.
On the regulation and control of fibrinolysis.
Thromb. Haemost.
43:
77-89,
1980[ISI][Medline].
12.
De Boland, A. R.,
and
A. W. Norman.
Evidence for involvement of protein kinase C and cyclic adenosine 3',5'-monophosphate dependent protein kinase in the 1,25-dihydroxyvitamin D3-mediated rapid stimulation of intestinal calcium transport (transcaltachia).
Endocrinology
127:
39-45,
1990[Abstract].
13.
DeLuca, H. F.,
and
C. Zierold.
Mechanisms and functions of vitamin D.
Nutr. Rev.
56:
S4-S10,
1998[ISI][Medline].
14.
Evans, R. M.
The steroid and thyroid hormone receptor superfamily.
Science
240:
889-895,
1988[ISI][Medline].
15.
Farach-Carson, M. C.,
I. Sergeev,
and
A. W. Norman.
Nongenomic actions of 1,25-dihydroxyvitamin D3 in rat osteosarcoma cells: structure-function studies using ligand analogs.
Endocrinology
129:
1876-1884,
1991[Abstract].
16.
Ferrara, J.,
K. McCuaig,
G. N. Hendy,
M. Uskokovic,
and
J. H. White.
Highly potent transcriptional activation by 16-ene derivatives of 1,25-dihydroxyvitamin D3.
J. Biol. Chem.
269:
2971-2981,
1994
17.
Franckowiak, G.,
M. Bechem,
M. Schramm,
and
G. Thomas.
The optical isomers of 1,4-dihydropyridine BAY K 8644 show opposite effects on Ca channels.
Eur. J. Pharmacol.
114:
223-226,
1985[ISI][Medline].
18.
Fukumoto, S.,
E. H. Allan,
and
T. J. Martin.
Regulation of plasminogen activator inhibitor-1 (PAI-1) expression by 1,25-dihydroxyvitamin D3 in normal and malignant rat osteoblasts.
Biochim. Biophys. Acta
1201:
223-228,
1994[ISI][Medline].
19.
Gils, A., and P. J. Declerck. Modulation of
plasminogen activator inhibitor 1 by Triton X-100identification of
two consecutive conformational transitions. Thromb. Haemost.
286-291, 1998.
20.
Grulich-Henn, J.,
and
G. Müller-Berghaus.
Regulation of endothelial tissue plasminogen activator and plasminogen activator inhibitor type 1 synthesis by diacylglycerol, phorbol ester, and thrombin.
Blut
61:
38-44,
1990[ISI][Medline].
21.
Hanss, M.,
and
D. Collen.
Secretion of tissue-type plasminogen activator and plasminogen activator inhibitor by cultured human endothelial cells: modulation by thrombin, endotoxin, and histamine.
J. Lab. Clin. Med.
109:
97-104,
1987[ISI][Medline].
22.
Heussen, C.,
and
E. B. Dowdle.
Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing SDS and copolymerized substrates.
Anal. Biochem.
102:
196-202,
1980[ISI][Medline].
23.
Inaba, M.,
S. Okuno,
A. Inoue,
Y. Nishizawa,
H. Morii,
and
H. F. DeLuca.
DNA binding property of vitamin D3 receptors associated with 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3.
Arch. Biochem. Biophys.
268:
35-39,
1989[ISI][Medline].
24.
Jackson, T. R.,
S. I. Petterson,
O. Thastrup,
and
M. R. Hanley.
A novel tumor promoter, thapsigargin, transiently increases cytoplasmic free Ca2+ without generation of inositol phosphates in NG 115-401L neuronal cells.
Biochem. J.
253:
81-86,
1988[ISI][Medline].
25.
Kabat, M. M.,
W. Burger,
S. Guggino,
B. Hannessy,
J. A. Iacobelli,
K. Takeuchi,
and
M. R. Uskokovi.
Total synthesis of 25-hydroxy-16,23E-diene vitamin D3 and 14,25-dihydroxy-16,23E-diene vitamin D3: separation of genomic and nongenomic vitamin D activities.
Bioorg. Med. Chem.
6:
2052-2059,
1998.
26.
Kooistra, T.,
J. Opdenberg,
K. Toet,
H. F. Hendriks,
R. M. Van den Hoogen,
and
J. J. Emeis.
Stimulation of tissue type plasminogen activator synthesis by retinoids in cultured human endothelial cells and rat tissue in vivo.
Thromb. Haemost.
65:
565-572,
1991[ISI][Medline].
27.
Kooistra, T.,
J. Van den Berg,
A. Tons,
G. Platenburg,
D. C. Rijken,
and
E. Van den Berg.
Butyrate stimulates tissue-type plasminogen activator synthesis in cultured human endothelial cells.
Biochem. J.
247:
605-612,
1987[ISI][Medline].
28.
Lemire, J. M.,
D. C. Archer,
and
G. S. Reddy.
1,25-Dihydroxy-24-oxo-16-ene-vitamin D3, a renal metabolite of the vitamin D analog 1,25-dihydroxy-16-ene-vitamin D3 exerts immunosuppressive activity equal to its parent without causing hypercalcemia in vivo.
Endocrinology
135:
2818-2821,
1994[Abstract].
29.
Martin, D. L.,
and
H. F. DeLuca.
Calcium transport and the role of vitamin D.
Arch. Biochem. Biophys.
134:
139-148,
1969[ISI][Medline].
30.
Mathiasen, I. S,
K. W. Colston,
and
L. Binderup.
EB 1089, a novel vitamin D analogue, has strong antiproliferative and differentiation-inducing effects on cancer cells.
J. Steroid Biochem. Molec. Biol.
46:
365-371,
1993[ISI][Medline].
31.
Merke, J.,
W. Hofmann,
D. Goldschmidt,
and
E. Ritz.
Demonstration of 1,25(OH)2 vitamin D3 receptors and actions in vascular smooth muscle cells in vitro.
Calcif. Tissue Int.
41:
112-114,
1987[ISI][Medline].
32.
Merke, J.,
P. Milde,
S. Lewicka,
U. Hügel,
G. Klaus,
D. J. Mangelsdorf,
M. R. Haussler,
E. W. Rauterberg,
and
E. Ritz.
Identification and regulation of 1,25-dihydroxyvitamin D3 receptor activity and biosynthesis of 1,25-dihydroxyvitamin D3. Studies in cultured bovine aortic endothelial cells and human dermal capillaries.
J. Clin. Invest.
83:
1903-1915,
1989[ISI][Medline].
33.
Meszaros, J. G.,
N. J. Karin,
K. Akanby,
and
M. C. Farach-Carson.
Down-regulation of the L-type Ca2+ channel transcript levels by 1,25-dihydroxyvitamin D3. Osteoblastic cells express L-type alpha1C Ca2+ channel isoforms.
J. Biol. Chem.
271:
32981-32985,
1996
34.
Nemere, I.,
Y. Yoshimoto,
and
A. W. Norman.
Studies on the mode of action of calciferol. LIV. Calcium transport in perfused duodena from normal chicks: enhancement within 14 min of exposure to 1,25-dihydroxyvitamin D3.
Endocrinology
115:
1476-1483,
1984[Abstract].
35.
Nishida, M.,
W. W. Carley,
M. E. Gerritsen,
Ø. Ellingsen,
R. A. Kelly,
and
T. W. Smith.
Isolation and characterization of human and rat cardiac microvascular endothelial cells.
Am. J. Physiol. Heart Circ. Physiol.
264:
H639-H652,
1993
36.
Norman, A. W.,
and
S. Hurwitz.
The role of the vitamin D endocrine system in avian bone biology.
J. Nutr.
123:
310-316,
1993[ISI][Medline].
37.
Norman, A. W.,
I. Nemere,
L. Zhou,
J. E. Bishop,
K. E. Lowe,
A. C. Maiyar,
E. D. Collins,
T. Taoka,
I. Sergeev,
and
M. C. Farach-Carson.
1,25(OH)2-vitamin D3, a steroid hormone that produces biological effects via both genomic and nongenomic pathways.
J. Steroid Biochem. Molec. Biol.
41:
231-240,
1992[ISI][Medline].
38.
Ohlsson, M.,
G. Leonardsson,
X.-C. Jia,
P. Feng,
and
T. Ny.
Transcriptional regulation of the rat tissue type plasminogen activator gene: localization of DNA elements and nuclear factors mediating constitutive and cyclic AMP-induced expression.
Mol. Cell. Biol.
13:
266-275,
1993[Abstract].
39.
Radcliffe, R.,
and
T. Heinze.
Stimulation of tissue plasminogen activator by denatured proteins and fibrin clots: a possible additional role for plasminogen activator.
Arch. Biochem. Biophys.
211:
750-761,
1981[ISI][Medline].
40.
Ross, T. K.,
J. M. Prahl,
and
H. F. DeLuca.
Overproduction of rat 1,25-dihydroxyvitamin D3 receptor in insect cells using the baculovirus expression system.
Proc. Natl. Acad. Sci.
88:
6555-6559,
1991[Abstract].
41.
Selles, J.,
and
R. L. Boland.
Evidence on the participation of the 3',5'-cyclic AMP pathway in the nongenomic action of 1,25-dihydroxyvitamin D3 in cardiac muscle.
Mol. Cell. Endocrinol.
82:
229-235,
1991[ISI][Medline].
42.
Tranquille, N.,
and
J. J. Emeis.
On the role of calcium in the acute release of tissue-type plasminogen activator and von Willebrand factor from rat perfused hindleg region.
Thromb. Haemost.
66:
479-483,
1991[ISI][Medline].
43.
Van den Eijnden-Schrauwen, Y.,
T. Kooistra,
R. E. M. De Vries,
and
J. J. Emeis.
Studies on the acute release of tissue-type plasminogen activator from human endothelial cells in vitro and in rats in vivo: evidence for a dynamic storage pool.
Blood
85:
3510-3517,
1995
44.
Van Hinsbergh, V. W.,
T. Kooistra,
J. J. Emeis,
and
P. Koolwijk.
Regulation of plasminogen activator production by endothelial cells: role in fibrinolysis and local proteolysis.
Int. J. Radiat. Biol.
60:
261-272,
1991[ISI][Medline].
45.
Vieth, R.
Simple method for determining specific binding capacity of vitamin D-binding protein and its use to calculate the concentration of "free" 1,25-dihydroxyvitamin D.
Clin. Chem.
40:
435-441,
1994
46.
Wali, R. K.,
C. L. Baum,
M. J. G. Bolt,
T. A. Brasitus,
and
M. D. Sitrin.
1,25-Dihydroxy-vitamin D3 inhibits Na+-H+ exchange by stimulating membrane phosphoinositide turnover and increasing cytosolic calcium in CaCo-2 cells.
Endocrinology
131:
1125-1133,
1992[Abstract].
47.
Waller, E. K.,
and
W.-D. Schleuning.
Induction of fibrinolytic activity in HeLa cells by phorbol myristate acetate.
J. Biol. Chem.
260:
6354-6360,
1985
48.
Walters, M. R.
Newly identified actions of vitamin D endocrine system.
Endocrine Rev.
13:
719-764,
1992[ISI][Medline].
49.
Watanabe, H.,
R. Takahashi,
X. Zhang,
Y. Goto,
H. Hayashi,
J. Ando,
M. Isshiki,
M. Seto,
H. Hidaka,
I. Niki,
and
R. Ohno.
An essential role of myosin light-chain kinase in the regulation of agonist- and fluid flow-stimulated Ca2+ influx in endothelial cells.
FASEB J.
12:
341-348,
1998
50.
Wei, H.,
W. Wei,
D. E. Bredesen,
and
D. C. Perry.
Bcl-2 protects against apoptosis in neuronal cell line caused by thapsigargin-induced depletion of intracellular calcium stores.
J. Neurochem.
70:
2305-2314,
1998[ISI][Medline].
51.
Wiberg, K.,
S. Ljunghall,
L. Binderup,
and
Ö. Ljunggren.
Studies on two new vitamin D analogs, EB 1089 and KH 1060: effects on bone resorption and osteoclast recruitment in vitro.
Bone
17:
391-395,
1995[ISI][Medline].
52.
Zhou, Y. P.,
D. Teng,
F. Dralyuk,
D. Ostrega,
M. W. Roe,
L. Philipson,
and
K. S. Polonsky.
Apoptosis in insulin-secreting cells. Evidence for the role of intracellular Ca2+ stores and arachidonic acid metabolism.
J. Clin. Invest.
101:
1623-1632,
1998
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |