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Division of Nephrology, University of Maryland School of Medicine, Baltimore 21201-1595; and Department of Biology, Towson University, Towson, Maryland 21252
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The
intracellular calcium ([Ca2+]i)
response of outer medullary descending vasa recta (OMDVR) endothelia to
ANG II was examined in fura 2-loaded vessels. Abluminal
ANG II (10
8 M) caused
[Ca2+]i to fall in proportion to
the resting [Ca2+]i (r =
0.82) of the endothelium. ANG II (108 M)
also inhibited both phases of the
[Ca2+]i response generated by
bradykinin (BK, 107 M), 835 ± 201 versus 159 ± 30 nM (peak phase) and 169 ± 26 versus 103 ± 14 nM
(plateau phase) (means ± SE). Luminal ANG II reduced BK
(107 M)-stimulated plateau
[Ca2+]i from 180 ± 40 to 134 ± 22 nM without causing vasoconstriction. Abluminal ANG II added to the
bath after luminal application further reduced
[Ca2+]i to 113 ± 9 nM and
constricted the vessels. After thapsigargin (TG) pretreatment, ANG II
(108 M) caused
[Ca2+]i to fall from 352 ± 149 to
105 ± 37 nM. This effect occurred at a threshold ANG II concentration
of 1010 M and was maximal at
108 M. ANG II inhibited both the rate of
Ca2+ entry into
[Ca2+]i-depleted endothelia and the
rate of Mn2+ entry into
[Ca2+]i-replete endothelia. In
contrast, ANG II raised [Ca2+]i in
the medullary thick ascending limb and outer medullary collecting duct,
increasing [Ca2+]i from baselines
of 99 ± 33 and 53 ± 11 to peaks of 200 ± 47 and 65 ± 11 nM,
respectively. We conclude that OMDVR endothelia are unlikely to be the
source of ANG II-stimulated NO production in the medulla but that
interbundle nephrons might release Ca2+-dependent
vasodilators to modulate vasomotor tone in vascular bundles.
microperfusion; kidney; fura 2; bradykinin; thapsigargin
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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BLOOD IS SUPPLIED to the medulla of the kidney via the efferent flow of juxtamedullary glomeruli (16). Juxtamedullary efferent arterioles branch in the outer stripe of the outer medulla to form outer medullary descending vasa recta (OMDVR), which coalesce in the inner stripe to form vascular bundles. The orientation of OMDVR within vascular bundles is specific. Those on the bundle periphery give rise to the dense capillary plexus that supplies the outer medullary interbundle region and its nephrons. Those in the bundle center traverse the outer medulla to supply the inner medulla. Smooth muscle remnants known as pericytes surround the OMDVR wall, imparting contractile properties to these vessels. It is likely that OMDVR regulates regional perfusion of the outer versus inner medulla of the kidney. For example, preferential constriction of vessels in the center of vascular bundles or, equivalently, preferential dilation of OMDVR on the periphery, would tend to redirect blood flow away from the inner medulla toward the outer medullary interbundle capillary plexus. Such an effect would favor delivery of oxygen and nutrients to the metabolically active medullary thick ascending limb (MTAL) and outer medullary collecting duct (OMCD) (6).
A major role for production of nitric oxide (NO) in the control of medullary blood flow has been proposed based on the observation that nitric oxide synthase (NOS) inhibition leads to reduction of medullary blood flow and hypertension (20) and that a high-sodium diet results in upregulation of NOS isoforms (19). A reciprocal relationship between medullary ANG II and NO production is supported by the observation that interstitial infusion of ANG II leads to increased NO generation as measured by microdialysis with oxyhemoglobin trapping (37). Because OMDVR are the only microvessels with a continuous endothelium in the outer medulla, we tested the hypothesis that ANG II would elevate endothelial intracellular calcium concentration ([Ca2+]i) as required to stimulate NO production by the calcium-sensitive forms of NOS. In contrast with this, we found that ANG II lowers endothelial [Ca2+]i, blocks the elevation of [Ca2+]i induced by treatment with thapsigargin (TG), suppresses calcium signaling by bradykinin (BK), and inhibits divalent cation entry into the endothelia. Based on these observations, we hypothesize that in OMDVR ANG II inhibits [Ca2+]i for the purpose of decreasing production of Ca2+-dependent endothelial vasodilatory autacoids. This would tend restrict control of OMDVR vasomotor tone to vasodilators that diffuse to the vascular bundles from the adjacent surroundings. Given the radial arrangement of OMDVR within vascular bundles, dilation of OMDVR on the vascular bundle periphery preferentially favors distribution of juxtamedullary efferent blood flow to the interbundle region during times of increased metabolic demand.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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In vitro microperfusion. Tissue for microdissection was obtained from 67 Sprague-Dawley rats (80-150 g) (Harlan) anesthetized before nephrectomy by an intraperitoneal bolus of thiopental (50 mg/kg body wt). OMDVR, MTAL, OMCD, and the descending thin limb of long looped nephrons (LDLu) were dissected from the outer medulla as previously described (25). Vessels and nephron segments were mounted on glass pipettes and perfused using standard apparatus (ITM, San Antonio, TX) (22, 23, 25). For OMDVR, microperfusion pipettes were 4-6 µm (inner diameter), holding pipettes 13-15 µm (inner diameter) with constrictions of 6-9 µm, and collection pipettes ~10 µm inner diameter at the inlet. Pipette diameters were increased as appropriate to accommodate nephrons segments. Vessels and tubules were maintained at 37°C in custom-built microperfusion chambers during microperfusion. The following buffer was used for dissection, bath, and perfusate (in mM): 5 HEPES, 140 NaCl, 10 sodium acetate, 5 KCl, 1.2 MgCl2, 1.71 Na2HPO4, 0.29 NaH2PO4, 1 CaCl2, 5 alanine, 5 glucose, and 0.5 g/dl albumin, pH 7.4.
Videomicroscopy and measurement of vessel diameters. To quantify vasoconstriction, microperfusion experiments were captured on videotape using a video camera (Panasonic WV-BL90). Experiments were recorded on a Panasonic model AG 1960 videocasette recorder equipped with a microphone for voice recording and played back to enable diameter measurement calipers. As previously described, changes in vessel outer diameter are expressed as percent constriction (22).
Measurement of endothelial intracellular calcium. OMDVR were loaded with the Ca2+-sensitive fluorescent indicator fura 2 by exposure to bath containing 2 µM fura 2-AM (Molecular Probes, Eugene, OR). At the time the bath was exchanged to contain fura 2-AM, the feedback controller was turned on, gradually warming the vessel to 37°C over about 5 min. Total loading time was 20 min. We have previously shown that fura 2 preferentially loads into endothelial cells rather than pericytes (23). For measurement of [Ca2+]i, fura 2-loaded OMDVR were briefly excited at 340 and 380 nm or at 350 and 380 nm dual-wavelength combinations. The background-subtracted ratio of fluorescent emission (R340/380 or R350/380) was calculated for conversion to the equivalent [Ca2+]i assuming a dissociation constant for fura 2 at 37°C of 224 nM (10). Rmax and Rmin were measured as previously described by exposing vessels to buffer containing 5 mM CaCl2, or 0 CaCl2-0.5 mM EGTA along with 10 µM 4-Br-A-23187 (23).
Either a digital fluorescent imaging system or a photon counting photomultiplier assembly (PMT) was employed to measure fluorescent emission at 510 nm from fura 2-loaded OMDVR endothelia. Light for excitation of fura 2 was provided from a 75-W xenon arc lamp directed through a computer-controlled shutter (Uniblitz, Rochester, NY). The excitation wavelengths were isolated using bandpass filters (Omega Optical, Brattleboro, VT) or with a computer-controlled monochrometer (PTI, South Brunswick, NJ). OMDVR were observed through a 1.3 NA Nikon CF fluor ×40 oil immersion objective. Fluorescent emission was isolated with a 510WB40 filter (Omega Optical) and directed to the intensified charge-coupled device (CCD) camera or PMT (23). Excitation (340 and 380 nm) was used with CCD detection, whereas later experiments employed 350- and 380-nm wavelengths with PMT detection. Output of the CCD camera was captured with a 4-MB GRB silicon video MUX framegrabber (Epix, Northbrook, IL) with a resolution of 8 bits/pixel. When the PMT was employed to measure the fluorescent emission, the output was monitored with a multichannel scaling card (ACE-MCS, EG&G Ortec, Oak Ridge, TN), using custom software or with commercial hardware and software (PTI).Measurement of Ca2+ influx
into OMDVR endothelia using fura 2.
To obtain a measurement of the rate of net Ca2+ influx into
OMDVR endothelia, [Ca2+]i was
measured at 6-s intervals as vessels were exposed to TG (107 M) and nominally
Ca2+-free buffer containing 100 µM EGTA to deplete
calcium stores. After the data acquisition software was
reset to increase the sampling rate,
[Ca2+]i was measured as frequently
as possible (approximately every 1.7 s) as the bath was exchanged from
zero-Ca2+ EGTA to 2.5 mM CaCl2 without EGTA.
Measurement of Mn2+ influx into OMDVR endothelia using fura 2. A method for examining the effect of agents on plasmalemmal influx of divalent cations is to exploit the effect of Mn2+ to quench the fluorescence of fura 2 (4). For these protocols, OMDVR were loaded with fura 2 and excited at the calcium-insensitive (isosbestic) wavelength, 360 nm, while fluorescent emission (F360) was monitored at 510 nm with a PMT. After MnCl2 (500 µM) was added to the bath, the rate of decline of F360 provides a measure of plasmalemmal Mn2+ influx through all available pathways.
Reagents.
BK, ANG II, and 4-bromo-A-23187 calcium ionophore (4-Br-A-23187) were
purchased from Sigma. These agents were dissolved in water at
103-105
M and stored frozen at 
20°C. Aliquots were thawed on the day of the experiment, and the excess was discarded at the end of each day.
4-Br-A-23187 was dissolved in ethanol at 10 mM. TG and fura 2-AM ester
was purchased from Molecular Probes (Eugene, OR) and stored frozen in
anhydrous DMSO. TG was always protected from light.
Statistical analysis. Experimental results are reported as means ± SE. Statistical comparisons employ paired t-test, unpaired t-test, or repeated-measures ANOVA as appropriate. For ANOVA, significance was determined by the Student-Newman-Keuls test. P < 0.05 was used for significance.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The effect of abluminal application of ANG II
(108 M) on endothelial
[Ca2+]i in shown in Fig.
1. As previously observed, baseline
endothelial [Ca2+]i in these
vessels generally lies between 50 and 150 nM. Baseline [Ca2+]i was measured for 1 min
after which the vessel was exposed to ANG II for an additional
5-10 min. The mean [Ca2+]i for
the endothelia of these vessels before ANG II exposure was 89 ± 10 nM. After application of ANG II,
[Ca2+]i fell by an average of 46 ± 9 nM (P < 0.05). An experimental record from the OMDVR
with the highest resting [Ca2+]i is
shown in Fig. 1A, and the results from 20 vessels are
summarized in Fig. 1B. On average, the change in
[Ca2+]i was proportional to the
preexisting [Ca2+]i level of the
endothelium (n = 20, r = 0.82, P < 0.05).
Equivalent results are obtained whether or not the vessel was perfused
(n = 13) or simply mounted on pipettes for immobilization
(n = 7). To increase the technical ease of obtaining large
numbers of observations, and to ensure equal luminal and abluminal
concentrations of the agents exchanged into the bath, subsequent
experiments were performed without perfusion except where otherwise
specified.
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Most vessels have a low baseline
[Ca2+]i (77 ± 33 nM, means ± SD
for all vessels in this report), and the extent of the ANG II induced
fall in [Ca2+]i is small
(Fig. 1). We next performed a series of experiments to
determine whether stimulation by ANG II modulates the increase in
[Ca2+]i generated by the
endothelium-dependent vasodilator BK. Baseline [Ca2+]i was recorded for 2 min
after which BK or BK + ANG II was added to the bath for 10 min. BK ± ANG II was then removed from the bath for 3 min. To quantify the
baseline, plateau, and recovery phases of the response,
[Ca2+]i concentrations were
averaged for five data points before bath exchange or termination of
the experiment. A Ca2+ response typical of that observed on
abluminal application of BK (107 M) by
exchange into the bath is shown in Fig.
2A, and the smaller response
typical of BK application in the presence of ANG II
(108 M) are shown in Fig. 2B.
The means ± SE of a group comparison (n = 9 each) is shown in
Fig. 2C. In a manner similar to that previously described (23),
BK increased [Ca2+]i from ~70 nM
to a peak of 835 ± 201 nM. The peak phase is followed by a sustained
and reversible plateau of 169 ± 26 nM in control vessels. ANG II
significantly inhibited both the peak (159 ± 30 nM) and plateau (103 ± 14 nM) phases of the BK-induced Ca2+ response. In a
separate series of experiments,
[Ca2+]i was first raised by BK
(107 M, 5 min) after which abluminal ANG
II (108 M, 4 min) was applied and then
removed (5 min). With this protocol, ANG II again consistently and
reversibly reduced the plateau phase of the
[Ca2+]i response (n = 10, Fig. 3). Time controls (n = 8) showed no effect.
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We next studied the effect of luminal versus abluminal ANG II on
Ca2+ signaling and vasomotion (Fig.
4). To raise
[Ca2+]i, vessels were perfused and
bathed with BK (107 M) throughout the
experiment. Baseline diameter was recorded, after which
[Ca2+]i was measured by fluorescent
microscopy with fura 2 as ANG II (108 M)
was exchanged into the lumen (5 min). Subsequently, white light images
were again recorded by videomicroscopy to determine the effect of
luminal ANG II on vessel outer diameter.
[Ca2+]i measurements were restarted
as ANG II (n = 8) or vehicle (n = 6) was exchanged into
the bath (5 min). After this, vessel diameters were recorded a third
time to measure the effect of concomitant abluminal ANG II on
vasomotion. In these experiments, to perform diameter measurements,
vessel images were captured on videotape for 2 min. The total time
required to suspend fluorescent data acquisition, redirect white light
to the camera, and then restart data acquisition was ~4-5 min.
Luminal ANG II reduced [Ca2+]i, an
effect that was enhanced by subsequent, simultaneous addition of ANG II
to the bath (Fig. 4A). In contrast, luminal ANG II alone failed
to alter vessel diameter but concomitant abluminal ANG II produced a
typical vasoconstrictor response (Fig. 4B). White light images
of OMDVR perfused and bathed in 108 M
ANG II are shown in Fig. 5. Time controls
with sham exchange of the bath in the second period produced no
additional effect on [Ca2+]i
(n = 7, data not shown) or vessel diameters (Fig. 4B).
This selective effect of abluminal ANG II to induce vasoconstriction is
consistent with the location of pericytes on the vessel periphery. The
ability of luminal ANG II to modulate endothelial
[Ca2+]i suggests the presence of
luminal ANG II receptors on OMDVR endothelia, activation of which
modulates Ca2+ signaling.
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TG is a sesquiterpene lactone that blocks active uptake of
Ca2+ into internal stores by irreversibly binding to the
sarcoplasmic-endoplasmic Ca2+ ATPase (34). It is generally
accepted that depletion of the stores activates a plasmalemmal pathway
for "capacitative" Ca2+ entry, which augments a
secondary increase in cytoplasmic
[Ca2+]i (26). In a manner
consistent with this, an increase in
[Ca2+]i of OMDVR endothelia was
generally observed on addition of TG to the bath (Fig.
6, vehicle). Rarely, vessels failed to
respond to TG and were excluded from further experimentation and
analysis. In the protocol shown in Fig. 6, endothelial
[Ca2+]i was measured for a total of
11 min. After a 1-min baseline observation period, TG
(107 M) was exchanged into the bath.
Five minutes later ANG II (n = 8) or vehicle (n = 7)
was also added to the bath and
[Ca2+]i measured for another 5 min.
In the presence of TG and vehicle, [Ca2+]i continued to rise in all
vessels. In contrast, ANG II reversed the effect of TG by nearly
returning endothelial [Ca2+]i to
baseline levels. The concentration dependence of ANG II to modulate
endothelial [Ca2+]i was also
examined by measuring its effects on TG-treated vessels. For this
protocol, OMDVR were only tested if the endothelial
[Ca2+]i increased and stabilized
within 10 min of application of abluminal TG. If
[Ca2+]i was stable after 10 min of TG, the experiment was continued by increasing ANG II
concentration in the bath by log molar increments from
1010 to
107 M at 3-min intervals. A small
reduction of [Ca2+]i was observed
at 1010 M, and the effect to suppress
[Ca2+]i was maximal at
108 M (Fig.
7).
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The remarkable ability of ANG II to lower
[Ca2+]i in TG-treated vessels
suggests the existence of mechanism(s) that inhibit conductance
pathways for plasmalemmal Ca2+ influx. To test this we
determined whether the rate of Ca2+ influx into
Ca2+-depleted OMDVR was blocked by ANG II.
[Ca2+]i was monitored for 2 min in
vessels previously exposed to TG (107 M)
with or without ANG II (108 M). The bath
was changed to nominally Ca2+-free EGTA after which
[Ca2+]i fell dramatically (Fig.
8A). After the rate of data
acquisition was increased, CaCl2 was replaced in the bath
as [Ca2+]i was monitored. The rate
of rise of [Ca2+]i was much slower
in ANG II-treated OMDVR, suggesting inhibition of Ca2+
influx into the endothelia (Fig. 8B).
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Another method for examining the effect of agents on plasmalemmal
influx of divalent cations is to exploit the effect of Mn2+
to quench the fluorescence of fura 2 (4). As fluorescence was measured,
vehicle (n = 12), MnCl2 (500 µM, n = 12),
ANG II (108 M, n = 7), or ANG II + MnCl2 (n = 7) was exchanged into the bath. An
equal rate of fall of fura 2 fluorescence was observed in control and
ANG II-treated OMDVR, likely due to leak of fura 2 from the endothelial
cells. In the presence of Mn2+, the rate of decline of
fluorescence was markedly enhanced due to quench of fura 2 by
Mn2+ (Fig. 9A).
Consistent with inhibition of plasmalemmal Mn2+ influx, the
quench rate by Mn2+ was markedly reduced by ANG II
(Fig. 9B).
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Because ANG II does not raise endothelial
[Ca2+]i of isolated OMDVR but is
known to enhance generation of NO in the renal medulla, we reasoned
that it might elevate [Ca2+]i in
outer medullary nephrons which express calcium-sensitive isoforms of
NOS. With this in mind, we tested the ability of ANG II applied from
the lumen or bath to increase
[Ca2+]i in MTAL, OMCD, and LDLu
(Fig. 10). In response to ANG II,
[Ca2+]i increased in the MTAL and
OMCD from baseline of 99 ± 33 and 53 ± 11 to peaks of 200 ± 47 and 65 ± 11, respectively. The increase in
[Ca2+]i achieved significance
(P < 0.05) during the time period from 3.25 through 6.58 min
in the MTAL and 3.17 through 7.08 min in the OMCD. We recognize that
both principal and intercalated cells are present in the OMCD so that
the magnitude of the OMCD [Ca2+]i
response might be underestimated if fura 2 loads into both cell types
and ANG II affects only one of them. ANG II had no effect on the
LDLu. The effect on MTAL and OMCD
[Ca2+]i was observed when ANG II
was applied from the bath but not the lumen. The effect of ANG II on
short-looped thin descending limbs could not be reliably examined due
to the difficulty of dissection and identification of that structure.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Measurement of endothelial [Ca2+]i in isolated OMDVR. The OMDVR wall is composed of two cell types, pericytes and endothelial cells. We have found that fura 2 loading occurs largely, if not exclusively, into OMDVR endothelia. We have previously provided high-resolution white light and fluorescent images of fura 2-loaded vessels that show no fluorescent emission from pericytes (23). We also found that vasopressin constricts OMDVR (36) but fails to raise [Ca2+]i in fura 2-loaded vessels (23). Vasoconstriction via the vasopressin V1 receptor is mediated by the inositol trisphosphate (IP3) Ca2+ signaling pathway, reinforcing the notion that fura 2 is not present to detect [Ca2+]i changes in pericyte cytoplasm. Initially, motivated by the desire to localize the fura 2 signal by digital imaging, we quantified [Ca2+]i by measuring ratio images with an intensified CCD camera (23). After determining that the fluorescent signal is endothelial in origin, we abandoned this approach in favor of photon-counting PMT detection. The latter is more accurate because image analysis in our system is limited to 8-bit resolution (0-28 gray scale), whereas photon counting PMT detection is a 16-bit operation. The greater sensitivity of PMT detection also enables reduction of excitation intensity, limiting the risk of photobleaching. Our investigations with either detection method have yielded similar results (Fig. 1).
Suppression of Ca2+
signaling in OMDVR endothelia.
We have shown that treatment with ANG II reduces basal
[Ca2+]i in OMDVR endothelium (Fig.
1), peak and plateau [Ca2+]i
responses in BK-stimulated vessels (Figs. 2-4), and endothelial [Ca2+]i in TG-treated OMDVR (Figs.
6 and 7). If [Ca2+]i in the
cytoplasm is at a steady level, the rate of plasmalemmal influx must
equal the rate of Ca2+ extrusion. ANG II could exert its
actions on OMDVR endothelia by influencing the set point of either or
both of these transport pathways. As part of this study we tested the
hypothesis that plasmalemmal influx might be inhibited by ANG II. Under
conditions that favor intracellular Ca2+ store depletion
(TG, zero bath CaCl2, and external EGTA), ANG II reduced
the rate of increase of [Ca2+]i
when CaCl2 was replaced in the bath (Fig. 8).
The precise route involved in this process is unknown, but the
conditions of the experiment favor activation of capacitative
Ca2+ entry pathway(s) resulting from intracellular store
depletion (26). Under Ca2+-replete conditions, the rate of
influx of Mn2+ was also reduced by ANG II (Fig. 9). This
divalent cation can traverse nonselective cation channels in
some endothelia, suggesting modulation of some similar pathway(s)
that conduct divalent cations in OMDVR. Another possibility is that ANG
II lowers the electrochemical driving forces for Ca2+
influx by depolarizing the endothelia. In this case, modulation of
K+ or Cl channels might account for the
actions of ANG II (1).
) is lacking in many
cell types in which ANG II increases IP3. Instead, ANG II
stimulation activates the Ca2+-insensitive PLC-
1 isoform
by tyrosine phosphorylation. Interestingly, given the results of this
study, PLC-1 generation of IP3 can sometimes be blocked
through tyrosine kinase inhibition (2, 7, 8, 18). cGMP-dependent
protein kinase (cGK) activation has been shown to block
[Ca2+]i responses of various
endothelia. The cGK1 isoform is known to phosphorylate and inactivate
the IP3 receptor, inhibiting Ca2+ release, and
to phosphorylate phospholamban, leading to enhancement of
Ca2+ compartmentalization (5, 17).
Effect of ANG II on renal blood flow and NO production. The effect of ANG II on perfusion of the renal medulla has been the subject of many investigations. The majority favor tonic constriction of the juxtamedullary microcirculation (24, 28). A reciprocal relationship between ANG II stimulation and intrarenal NO production has been observed by several investigators. Ito and colleagues (12) showed that NOS inhibition enhances the effects of ANG II in the afferent but not the efferent arteriole of the rabbit. Similarly, studies with NO trapping by oxyhemoglobin microdialysis have shown that infusion of ANG II into the medullary interstitium leads to an increase in global NO generation implying a possible role for NO to abrogate ANG II vasoconstriction of juxtamedullary resistance vessels (37). Direct evidence for stimulation of NO production was provided by Thorup et al. (35) who showed that renal cortical microvessels generate NO when stimulated with ANG II.
It is known that perfusion of the renal medulla is under the influence of kinins and is highly sensitive to NO. Chronic inhibition of NO production reduces medullary blood flow and induces hypertension in rats (20). Enhancement of NO production in the spontaneously hypertensive rat by L-arginine infusion normalized renal medullary hemodynamics (15). ANG AT1 receptors and the Ca2+-sensitive forms of NOS (NOS I and NOS III) have been localized to thick ascending limb, vasa recta, and collecting ducts (11, 13). Because the major endothelial isoform of NOS (eNOS or NOS III) is stimulated by elevation of [Ca2+]i, we reasoned that NO generation might self-limit ANG II-induced OMDVR constriction by raising endothelial [Ca2+]i to activate NOS III. In contrast to this, we found that ANG II reduces OMDVR endothelial [Ca2+]i and inhibits Ca2+ signaling by the endothelium-dependent vasodilator bradykinin. ANG II does, however, raise [Ca2+]i in OMCD and MTAL (Fig. 10). In the absence of specific hormonal stimulation, [Ca2+]i levels found in isolated OMDVR are in the range of 50 to 100 nM, concentrations which are probably too low to activate Ca2+/calmodulin-sensitive forms of NOS. In contrast, steep dependence of NOS is observed as [Ca2+]i is increased between 100 and 1,000 nM (30). BK elevates OMDVR endothelial [Ca2+]i to plateau values between 150 and 400 nM (Figs. 2-4), levels that are expected to stimulate NO generation. Siragy et al. (33) found that blockade of ANG II receptors led to reduction of renal interstitial BK concentrations in dogs. The ability of ANG II to modulate BK-stimulated OMDVR endothelial [Ca2+]i levels provides additional insight into possible mechanisms by which these hormones interact to influence intrarenal hemodynamics.ANG II concentrations and possible effects on medullary blood flow.
We previously observed that ANG II constricts OMDVR with threshold and
one-half maximal effects at ~1011 M
and 5 × 1010 M, respectively.
Interestingly, the threshold and one-half maximal effects of ANG II on
OMDVR endothelial [Ca2+]i occur at
~10-fold higher concentration (Fig. 7). Circulating ANG II
concentrations are much lower than this, typically
~1011 M (21, 31). OMDVR originate from
the branching of the efferent arterioles of juxtamedullary glomeruli
and local ANG II concentrations within the kidney can be quite high.
ANG II concentrations in proximal tubule fluid and star vessel plasma
downstream of superficial glomeruli can exceed circulating levels by as
much as 1,000-fold (31). Juxtamedullary nephrons are inaccessible to
micropuncture; however, renin content of juxtamedullary arterioles is
probably high (24). Taken together, it would seem that concentrations of ANG II within vascular bundles in vivo as high as
109-108
M cannot be ruled out.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institutes of Health Grants DK-42495 and HL-62220.
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FOOTNOTES |
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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: T. L. Pallone, Division of Nephrology N3W143, Univ. of Maryland at Baltimore, 22 S. Greene St., Baltimore, MD 21201-1595.
Received 28 June 1999; accepted in final form 1 November 1999.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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, Microperfused OMDVR
in which measurements were obtained with an intensified charge-coupled
device (CCD) camera; 
, unperfused vessels mounted on collecting
style pipettes for immobilization in which fluorescent emission is
monitored with a photon-counting photomultiplier detection
assembly.
