1 Physiology Laboratory, Bradykinin (BK)-induced changes in intracellular calcium level
([Ca2+]i)
were studied on fura 2-loaded afferent (AA) and efferent glomerular arterioles (EA) microdissected from juxtamedullary renal cortex. A
distinction was made between thin and muscular EA. In AA and both types
of EA, BK increased
[Ca2+]i
through activation of B2 receptors
located only on the endothelium. The responses were not affected by
nifedipine (10
calcium; tyrosine kinase
THERE IS GENERAL AGREEMENT that intrarenal bradykinin
(BK) infusion induces a decrease in renal vascular resistance,
resulting in an increase in renal blood flow without a significant
change in glomerular filtration rate (3, 38), and that this renal vasodilation is mediated by BK B2
receptors (33). However, Lortie et al. (24) have suggested that
B1 receptors could also be
involved. The renal effects of exogenous BK have been much studied,
whereas only a few studies have examined the influence of endogenous BK on renal hemodynamics (29). Recent studies have detected the presence
of elements of the kallikrein-kinin system in the glomerular cells or
vessels, suggesting a local production of BK (2, 36). In particular,
mRNAs of kininogen and of B2
receptors have been colocalized in afferent arterioles (36), and
kallikrein gene expression has also been documented in juxtaglomerular
cells (8). There are, however, discrepancies in results with regard to
the glomerular arteriolar targets involved in the vasodilator effects of BK. Micropuncture experiments performed in Munich-Wistar rats have
shown that BK increases single-nephron plasma flow by reducing both
afferent and efferent arteriolar resistances with a greater afferent
decrease (3). A preferential vasodilation of afferent arterioles was
also observed in the dog after intra-arterial infusion of BK (38). On
the contrary, BK dilated only the efferent arteriole in experiments
carried out on isolated rabbit vessels (11). Some studies have
indicated that the vasodilation induced by BK implies the release of
endothelial factors, which requires a cytosolic Ca2+ increase (13, 15). In these
works, the intracellular Ca2+
concentration
([Ca2+]i)
increases were measured in cultured endothelial cells. Only a few
studies have reported
[Ca2+]i
variations in microdissected glomerular arterioles (7, 9, 10, 20, 28),
and they have been concerned mainly with the effects of ANG II. These
studies have clearly demonstrated a heterogeneity in afferent and
efferent arteriole responses to this agonist. To our knowledge,
[Ca2+]i
variations have never been examined in glomerular arterioles stimulated
by BK.
The aim of the present study was first to investigate the presence of
BK receptors in the juxtamedullary arterioles, because it has been
reported that intrarenal BK preferentially modulates the renal
medullary microcirculation (29, 34) and that the medullary blood flow
is supplied by efferent arterioles from the juxtamedullary cortex. The
second purpose of this study was to specify the subcellular signal
pathways activated by BK. In particular, we have investigated whether a
tyrosine kinase activity could mediate the effects of BK, as has been
suggested by some recent studies (21).
Therefore, we 1) examined the effect
of BK on
[Ca2+]i
in microdissected afferent arterioles and both types of efferent
arterioles and assessed the sensitivity and the maximal response to BK
of each type of arteriole, 2)
determined the subtype(s) of receptors involved in the
[Ca2+]i
response and its endothelial and/or muscular localization, 3) investigated the relative
contribution of intra- and extracellular Ca2+ pools to
[Ca2+]i
increases and tried to specify the
Ca2+ channels activated by BK, and
4) looked for a role of tyrosine kinase activity in intracellular
Ca2+ release and/or
Ca2+ influx.
Microdissection of renal microvessels.
Experiments were conducted on glomerular arterioles isolated from rat
kidney by using a previously described protocol (10). Briefly, male
Sprague-Dawley rats (180-240 g) obtained from Iffa-Credo (L'Arbresle, France) were anesthetized with a single dose of
pentobarbital sodium (50 mg/kg ip). The left kidney was infused through
cannulation of the abdominal aorta with 3-5 ml of a standard
solution (4°C) containing (in mM) 127 NaCl, 5 KCl, 0.8 MgSO4, 0.33 Na2HPO4,
0.44 KH2PO4,
1 MgCl2, 4 NaHCO3, 2 CaCl2, 5 D-glucose, 10 CH3CO2Na, 20 HEPES, pH 7.4, and 0.1% BSA. The kidney was then
perfused with 5 ml of the same solution containing
collagenase A (8 mg/5 ml) and immediately removed, decapsulated, and
longitudinally sliced. Small pyramids were cut out and incubated in the
presence of collagenase (5 mg/5 ml) for 8 min at 30°C before they
were washed with the standard solution to eliminate collagenase. The
arterioles were microdissected from juxtamedullary cortex at 4°C
under a stereomicroscope (SZ3; Olympus, Tokyo, Japan). They were
isolated with their attachment to the glomerulus. Arterioles were then
identified as afferent (AA) or efferent arterioles (EA) according to
their morphology after observation under a Nikon microscope (×40
objective), and a distinction was made between thin and muscular EA
(10). AA were characterized by a thick and regular wall composed of
uniformly distributed smooth muscle cells. Both types of EA exhibited
marked morphological differences. Thin EA had a smaller diameter than AA and showed a bumpy wall due to the presence of irregularly shaped
and non-closely apposed smooth muscle cells. Muscular EA presented a
thick, muscular, and regular wall similar to that of AA. A common
feature of both types of EA was the presence of side branches, and only
the observation of branches made it possible to distinguish muscular EA
from AA (10). Before
[Ca2+]i
measurements, each sample was transferred on a thin glass slide in 1 µl of a standard solution containing 1% agarose (type IX). Agarose
was set by cooling the slide 1 min on ice. Arterioles were
then loaded by addition of 1 µl of 10 µM fura 2-AM (the
acetoxymethyl ester of fura 2) and incubated for 1 h at room
temperature in darkness.
Measurements of
[Ca2+]i.
In accordance with previous experiments (10),
[Ca2+]i
was evaluated by using a Photoscan II microfluorometer (Photon
Technology International, Kontron, France). The glass slide with the
sample was fixed at the bottom of a superfusion chamber and on the
stage of an inverted fluorescent microscope (Nikon). The sample was continuously superfused with the standard solution (0.6 ml/min, 37°C) carrying or not carrying the test substances. The microscope was fitted out with a quartz illumination system and a 40-fold magnification fluorescence immersion objective. The sample was alternately excited at 340 and 380 nm (4 s/cycle), and the fluorescence emitted at 510 nm from an area defined by an adjustable window (~25 × 30 µm) was measured.
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
6 M) and were
smaller in a Ca2+-free medium,
providing evidence that BK opens voltage-independent Ca2+ channels and mobilizes
intracellular Ca2+. Thin EA
differed from AA and muscular EA by a lower sensitivity to BK
(EC50 = 6.95 ± 3.81 vs. 0.21 ± 0.08 and 0.18 ± 0.13 nM, respectively;
P < 0.05), a higher maximal response
(89 ± 5 vs. 57 ± 5 and 44 ± 7 nM;
P < 0.001), and a spontaneous return
to basal Ca2+ level, even in the
presence of BK. Genistein
(10
4 M) and herbimycin A
(25 × 10
6
M), specific inhibitors of tyrosine kinases, inhibited the
[Ca2+]i
responses exclusively in AA. Genistein reduced the peak and plateau
phases of responses by 69 ± 9 and 82 ± 6%, respectively, in a
medium with Ca2+ and the peak by
48 ± 9% in a Ca2+-free
medium. Similar reductions were observed with herbimycin A. These results show that dissimilar signal transduction pathways are
involved in BK effects on juxtamedullary arterioles and that a tyrosine
kinase activity could participate in the regulation of BK effect on AA
but not on EA.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
(R
Rmin)/(Rmax
R), where
Kd (the
dissociation constant for the fura
2-Ca2+ complex) is 224 nM, R is
the ratio of fluorescence emitted for each wavelength (340/380 nm),
Rmax is the maximal ratio emitted in the presence of saturating free
Ca2+ (2 mM),
Rmin is the minimal ratio measured
in absence of free Ca2+ (0 mM),
and
is the ratio of fluorescence obtained at 380 nm in absence and
in presence of 2 mM Ca2+. The
values of Rmin,
Rmax, and
were periodically
determined by external calibration with the use of a buffer that
mimicked intracellular medium.
Arteriolar deendothelialization.
The technique reported by Beierwaltes (4) was used to deendothelialize
renal microvessels without altering the arteriolar smooth muscle layer.
Kidneys were infused in situ with standard buffer to clear them from
blood; they were then perfused with a solution of
102 M
H2O2
for 2 min. After elimination of residual
H2O2
by the infusion of 20 ml of standard buffer, kidneys were either
infused with the collagenase solution and prepared for microdissection
or excised and fixed for histological observation. Deendothelialization
was confirmed by the absence of a
[Ca2+]i
response to 10
6 M ACh, a
well-established endothelial stimulator (14). The integrity of the
smooth muscular layer was evaluated by comparing the arteriolar
[Ca2+]i
response to ANG II of deendothelialized with control arterioles. Thus,
in each experiment, the
[Ca2+]i
response to 10
8 M BK was
followed by 10
6 M ACh and
10
7 M ANG II tests.
Experimental protocols. The same experimental protocol was applied to the different types of arterioles. BK was superfused only once, for 5 min on each arteriole. The tested substances were superfused for 5 min before and during agonist stimulation except for herbimycin A, which required preincubation for 45-60 min before the experiment.
The Ca2+-free medium contained the same components as the standard solution except that Ca2+ was omitted and 0.1 mM EGTA was added (pH 7.4). When BK was tested in the absence of external Ca2+, the Ca2+-free medium was superfused for only 2 min before BK. In some experiments, the inhibition of [Ca2+]i increase was investigated by preincubating arterioles with a membrane-permeable Ca2+-chelating compound: 10 µM acetoxymethyl ester of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM) for 45 min. To investigate the effects of genistein on [Ca2+]i responses, 10Chemicals. Collagenase A (Clostridium Histolyticum, 1.1 PZ U/mg) was purchased from Boehringer Mannheim; fura 2-AM was from Molecular Probes (Leiden, The Netherlands); HOE 140 (D-Arg0-[Hyp3,Thi5,D-Tic7,Oic8]-BK) was from Hoechst; BAPTA-AM, EGTA, DMSO, agarose, H2O2, BK, des-Arg9,[Leu8]-BK, ANG II, ACh, nifedipine, genistein, and herbimycin A were from Sigma-Aldrich. Genistein and herbimycin A were dissolved in DMSO. The final concentrations of DMSO were 0.1 and 1.4%, respectively. We checked to ensure that these concentrations did not alter the [Ca2+]i responses to BK.
Statistics. Results are reported as means ± SE. When each arteriole served as its own control, significance was obtained by paired Student's t-test. Differences between two groups were analyzed with the use of unpaired Student's t-test. Multiple comparisons in similar protocol were evaluated by ANOVA, followed by Scheffé's test. Values were considered significantly different at P < 0.05. The commercially available Kaleidagraph software was used to fit dose-response curves and to estimate EC50 and mean maximal response.
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RESULTS |
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Characterization of
[Ca2+]i
responses induced by BK in AA and muscular and thin EA.
As shown in Fig. 1, a 5-min superfusion of
108 M BK elicited an
increase in
[Ca2+]i
in AA and both types of EA. In AA and muscular EA, the response was
biphasic. It was characterized by a peak followed by a plateau phase
maintained as long as BK was applied. The
[Ca2+]i
peaks were reached within a similar delay in AA (1.73 ± 0.21 min)
and muscular EA (1.78 ± 0.32 min), and their magnitudes were not
significantly different (AA: 57 ± 5 nM,
n = 14; muscular EA: 44 ± 7 nM;
n = 11). The level of the plateau
phase was ~75% of the peak response. After BK removal,
[Ca2+]i
decreased and returned progressively to the basal level. The whole
[Ca2+]i
response lasted 9.3 ± 1.3 min in AA and 9.0 ± 0.9 min in
muscular EA (Table 1). In thin EA, the time
course of
[Ca2+]i
response to 10
8 M BK was
different, since it did not display a plateau phase. After reaching a
peak value
(
[Ca2+]i = 50 ± 5 nM, n = 11) at 1.03 ± 0.2 min,
[Ca2+]i
declined slowly and returned to the basal level, even in the presence
of the agonist. The duration of the
[Ca2+]i
response was significantly shorter (5.6 ± 0.5 min,
P < 0.05) than in AA and muscular
EA.
|
|
Identification and characterization of the
receptors involved.
The nature of BK receptors involved was investigated with the use of
specific B2 (HOE 140) and
B1
(des-Arg9,[Leu8]-BK)
receptor antagonists. For the three types of arterioles, 106 M HOE 140 but not
10
6 M
des-Arg9,[Leu8]-BK
prevented the responses to BK (Fig. 2). In
the presence of 10
6 M
des-Arg9,[Leu8]-BK,
the
[Ca2+]i
peak values were as follows: 60 ± 8 nM for AA, 40 ± 9 nM for muscular EA, and 92 ± 10 nM for thin EA. They did not differ from control values reported in Table 1. These results indicate that only
B2 receptors were involved.
|
|
Role of endothelial layer in
[Ca2+]i
responses to BK.
As indicated in METHODS, ACh was used
to test the effectiveness of
H2O2
deendothelialization. When arterioles were not treated by
H2O2,
106 M ACh increased
[Ca2+]i
by 50 ± 6, 71 ± 11, and 135 ± 20 nM in AA
(n = 5), muscular EA
(n = 6), and thin EA
(n = 5), respectively. After
H2O2
infusion, 40-60% of the tested arterioles (5/14 AA, 8/13 muscular
EA, and 6/14 thin EA) did not respond to ACh, nor did these arterioles respond to BK, substantiating the claim that only the endothelial layer
is involved in the
[Ca2+]i
response (Fig. 4). In deendothelialized
arterioles, ANG II-induced [Ca2+]i
increases were similar to those in control arterioles [AA, 97 ± 11 (n = 5) vs. 110 ± 6 nM
(n = 9); muscular EA, 76 ± 6 (n = 8) vs. 87 ± 12 nM
(n = 5); and thin EA, 62 ± 9 (n = 6) vs. 70 ± 10 nM
(n = 8)], indicating the
integrity of the muscular layer. Histological analysis also showed that
the endothelial layer was differently altered in glomerular arterioles.
Some arterioles displayed a significant disruption of the endothelium
integrity, whereas others presented only slight changes (data not
shown).
|
[Ca2+]i
responses in the absence of external
Ca2+.
Contribution of intracellular Ca2+
stores to the rise in
[Ca2+]i
elicited by BK represents only a small part of the
[Ca2+]i
response (Table 1). It is only 8 ± 1, 14 ± 4, and 20 ± 2% for AA, muscular EA, and thin EA, respectively. BAPTA-AM (10 µM) suppressed the BK-induced increase in
[Ca2+]i
regardless of arteriole type (results not shown). As
shown in Table 1, the magnitude of the
[Ca2+]i
peak did not change in AA and muscular EA but was significantly decreased in thin EA. In contrast, the three types of arterioles exhibited a large and significant shortening of the time course and a
decrease of the integral of the
[Ca2+]i
responses (Table 1, Fig. 5), indicating
that the sustained phase of the
[Ca2+]i
responses observed with 2 mM Ca2+
was due to Ca2+ influx.
|
[Ca2+]i
responses in the presence of the
[Ca2+]i
entry blocker, nifedipine.
To determine whether L-type voltage-sensitive
Ca2+ channels are involved in the
influx phase, 10 µM nifedipine was applied before (5 min)
and during superfusion of maximal doses of BK
(108 M for AA and muscular
EA, and 10
7 M for thin EA).
Nifedipine did not change the magnitude of the [Ca2+]i
responses in AA [44 ± 4 nM
(n = 8) vs. 58 ± 7 nM
(n = 5)], muscular EA [55 ± 7 nM (n = 5) vs. 49 ± 9 nM
(n = 9)], or thin EA [74 ± 14 nM (n = 6) vs. 77 ± 5 nM
(n = 9)]. Similarly, the integral of
[Ca2+]i
responses,
(
[Ca2+]i) · dt,
did not significantly differ when nifedipine was present or absent in
either AA (15,453 ± 1,176 vs. 18,258 ± 4,358 nM · s), muscular EA (12,994 ± 1,209 vs. 14,482 ± 3,821 nM · s), or thin EA (9,748 ± 621 vs.
10,238 ± 1,050 nM · s).
[Ca2+]i
responses in the presence of tyrosine kinase inhibitors.
As indicated in Fig. 6,
104 M genistein
significantly reduced the
[Ca2+]i
response of AA (n = 9). Compared with
control arterioles (n = 14), the
Ca2+ peak was inhibited by 69 ± 9% (20 ± 6 vs. 66 ± 7 nM;
P < 0.001), and the
plateau phase was inhibited by 82 ± 6% (9 ± 3 vs. 49 ± 6 nM; P < 0.001). The duration
of the response was not altered, but whole
Ca2+ mobilization, evaluated by
the integral of response, was decreased by approximately five times
(Table 2). The same treatment had no effect
on muscular (n = 8) and thin
(n = 9) EA. Note that
10
4 M genistein caused a
small but significant decrease in basal [Ca2+]i
in the three types of arterioles (Table 2).
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DISCUSSION |
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The present study provides the following information: 1) BK receptors are present in AA and in both types of EA from juxtamedullary glomeruli, 2) these receptors are B2 receptors and they are located on the endothelial layer, 3) BK induces a release of Ca2+ from intracellular stores and a Ca2+ influx through voltage-independent channels in all arterioles, 4) the BK-induced [Ca2+]i increase in thin EA differs from that in AA and muscular EA, and 5) tyrosine kinase activity may be involved in the [Ca2+]i response of AA to BK.
We observed that both afferent and efferent juxtamedullary glomerular arterioles respond to BK by an increase in the cell [Ca2+]i level only via an activation of B2 receptors. The analysis of the [Ca2+]i responses allowed us to detect differences in the BK regulation of juxtamedullary glomerular arterioles, as previously reported for ANG II (10). The EC50 value for BK, as well as the maximal magnitude of the responses, was similar in AA and muscular EA. However, thin EA had a lower sensitivity and exhibited a higher maximal response to BK. If a direct relationship exists between BK-induced vasodilation and BK-stimulated endothelial [Ca2+]i elevation, our results are not consistent with some data in the literature. Indeed, Baylis et al. (3) and Thomas et al. (38) have shown that BK induces a greater decrease in the resistances of preglomerular rat and dog vessels. Our results also disagree with the data obtained by Edwards (11) showing no effect of BK on rabbit isolated AA. Note that in these studies the thin or muscular aspect of EA was not taken into consideration. In the present work, the muscular EA dividing into vasa recta have a higher sensitivity to BK. Because the medullary blood flow is derived from these arterioles, this observation suggests a greater vasodilatory effect of BK on this part of the vasculature.
To demonstrate the cells (endothelial or muscular) in which a [Ca2+]i increase was induced by BK, we used a technique of deendothelialization of renal microvasculature by H2O2 intrarenal infusion, according to the method reported by Beierwaltes (4). The criterion for correct deendothelialization was the suppression of the response to ACh, which increases [Ca2+]i potently in endothelial cells but has no effect on muscular cells (14). Our results indicate that only some of the microdissected arterioles from the H2O2-treated kidneys lost their ability to respond to ACh, regardless of the morphological type. Thus renal H2O2 infusion does not ensure a complete deendothelialization of all glomerular arterioles. However, all arterioles not responding to ACh did not respond to BK, and all arterioles responding to ACh responded to BK. In addition, arterioles responding to neither ACh nor BK still responded to ANG II, indicating that BK does not increase [Ca2+]i in muscular cells. Therefore, it is possible to conclude that BK causes an increase in [Ca2+]i exclusively in endothelial cells of the three types of glomerular arterioles. Our results are consistent with most data in the literature, which report the presence of BK receptors on endothelial cells (27, 31). Our data disagree with immunochemical studies by Figueroa et al. (12), who detected the presence of B2 receptors on the smooth muscle cells of AA, an observation that was not confirmed by autoradiography.
As previously reported (25, 30, 35), a second stimulation with BK induced a homologous desensitization process in the three types of arterioles, which remained less responsive to BK for at least 20 min after agonist removal. Such a rapid homologous desensitization of the BK-induced increase in [Ca2+]i has been described in bovine endothelial cells (25). We previously observed that a sustained intrarenal infusion of BK induces only transient effects on glomerular hemodynamics and that the brief in vivo BK effects are associated with a negative autoregulation of BK B2 receptors in isolated glomeruli (33).
Thin EA displayed a 30-times-lower sensitivity than AA or muscular EA, associated with a greater maximal response. Difference in sensitivity may be related to dissimilar affinities for BK of the receptors, as reported in the literature (18, 26). An alternative explanation is that BK was bound to B2 receptors in a negative cooperative manner (32). This characteristic of BK binding has recently been described in Chinese hamster ovary (CHO) cells, resulting in a decreased receptor affinity (32). Indeed, receptor-receptor interaction, which is responsible for the phenomenon of negative cooperativity, would be more pronounced in thin EA than in AA or muscular EA if one assumes a higher receptor density in thin EA, which exhibited a greater [Ca2+]i response to BK.
BK released Ca2+ from intracellular stores and induced a Ca2+ influx from external medium in all arterioles. Ca2+ release from intracellular pools accounts for almost all of the initial phase of the [Ca2+]i response in AA and muscular EA. Conversely, the peak response of thin EA was higher in the presence of 2 mM Ca2+, suggesting that a Ca2+ influx also participates in the initial phase of the [Ca2+]i response. The [Ca2+]i responses of thin EA also reveal a dissimilar time course. Unlike other arterioles, a sustained plateau phase was absent in thin EA, and [Ca2+]i returned to the basal level even in the presence of an agonist, suggesting a more rapid desensitization of B2 receptors. The inconstant presence of a sustained plateau phase in the response to BK has also been described in fibroblasts (6). Lack of a nifedipine effect indicates that the Ca2+ channels involved are not voltage-operated Ca2+ channels. Most studies, except for a few (15), have failed to detect classical voltage-operated Ca2+ channels in endothelial cells.
Over the last years, ample evidence has indicated that tyrosine kinases are involved in signal transduction from receptors devoid of intrinsic tyrosine kinase activity (5). To investigate a possible role of protein tyrosine phosphorylation in the regulation of Ca2+ entry and/or Ca2+ release from intracellular stores, we used two specific tyrosine kinase inhibitors that act via two distinct mechanisms: genistein and herbimycin. Whereas genistein inhibits tyrosine kinase by competing with an ATP binding site (1), herbimycin interacts with essential sulfhydryl groups (39). Unlike the widely used tyrosine kinase inhibitor tyrphostin, genistein and herbimycin A do not interfere with fluorometric [Ca2+]i measurements made with fura-2. For the same reasons, we could not use daidzen, an analog of genistein devoid of inhibitor effect. Our results show that genistein and herbimycin A decrease the [Ca2+]i responses to BK of AA only, both in the presence and absence of external Ca2+.
The role of tyrosine kinase activity in
Ca2+ release from intracellular
pools agrees with the observation by Leeb-Lundberg and Song (22) that
genistein inhibits both inositol phosphate (IP) formation in Swiss 3T3
fibroblasts and BK-stimulated tyrosine phosphorylation of proteins.
These observations suggest that these two events may be linked and that
tyrosine phosphorylation might play a role in some step of BK-promoted
IP formation. More recently, Fleming et al. (13) reported
that BK induces a transient tyrosine phosphorylation of phospholipase
C (PLC-
) and stimulates inositol trisphosphate
(IP3) production in endothelial
cells. In these experiments, genistein partially inhibited
(40-50%) IP3 formation. Similarly, in the absence of external
Ca2+, we found a partial
inhibition of the peak of
[Ca2+]i
in AA by genistein. These results suggest that BK stimulation of IP
metabolism involves other forms of PLC besides PLC-
(PLC-
and
PLC-
) that, unlike PLC-
, are not activated by tyrosine
phosphorylation (5).
With regard to Ca2+ influx, data in the literature also indicate that a tyrosine kinase activity may be involved in an agonist-induced increase in transmembraneous Ca2+ entry. Lee et al. (21) were the first to propose a role for tyrosine kinases in BK-stimulated Ca2+ influx on the basis of three effects of two tyrosine kinase inhibitors (genistein and/or tyrphostin) on BK-promoted Ca2+ influx in fibroblasts: 1) genistein inhibited the plateau phase of the [Ca2+]i response to BK, 2) genistein and tyrphostin blocked BK-stimulated 45Ca uptake, and 3) both tyrosine kinase inhibitors attenuated BK-induced protein phosphorylation. By using endothelial cells, Fleming et al. (13) showed that genistein decreases the plateau phase of the [Ca2+]i response to BK by 62% and detected BK-phosphorylated tyrosine residues of two low-molecular-weight proteins that were identified as isoforms of mitogen-activated protein kinases. Note that the reported Ca2+ influx inhibition caused by tyrosine kinase inhibitors was only partial, as it is in the present study, indicating that other mechanisms are also involved in the regulation of extracellular Ca2+ entry. One of these mechanisms could imply a "myosin light-chain kinase," as suggested by Takahashi et al. (37). It is noteworthy that genistein caused a small but significant decrease in the basal [Ca2+]i in the three types of arterioles, suggesting a tonic effect of tyrosine kinase on the basal [Ca2+]i level. A similar observation by Liu et al. (23) pointed out that L-type Ca2+ channels could be tonically phosphorylated by tyrosine kinase in rat portal vein.
The functional significance of the role of tyrosine kinase in the BK-induced [Ca2+]i response of AA remains to be established. It could be involved in BK vasodilation mediated by NO (19, 40). Moreover, Ito et al. (17) showed that NO attenuates ANG II-induced constriction of AA but has no effect on EA. Taken together, these observations and our results suggest a role of protein tyrosine phosphorylation in NO release by BK in AA.
In conclusion, this study clearly demonstrates that BK induces [Ca2+]i responses in glomerular arterioles of the juxtamedullary cortex, i.e., AA and both types of EA. The differences in sensitivity observed between thin EA and muscular EA, which divide to form vasa recta, suggest that BK regulates local microcirculation in different ways. In addition, BK-stimulated signal-transduction pathways are not similar in AA and EA. Tyrosine kinase activity is involved in the regulation of the BK effect in AA but not in EA. The consequences of dissimilar BK effects according to the type of juxtamedullary glomerular arterioles on the regulation of medullary hemodynamics remain to be investigated.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Rabary Rajerison for helpful discussions and suggestions throughout the course of this study. We acknowledge the skillful technical assistance of Marie-Thérèse Garelli and Catherine Chollet.
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FOOTNOTES |
---|
This work was supported by grants from the Institut National de la Santé et de la Recherche Médicale and by a grant from the Bristol Myer-Squibb Institute for Medical Research (Princeton, NJ).
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: F. Praddaude, Physiology Laboratory, School of Medicine, 133, route de Narbonne, 31062 Toulouse Cedex 4, France.
Received 13 October 1998; accepted in final form 30 June 1999.
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REFERENCES |
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