Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7545
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ABSTRACT |
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This study provides
new information about the relative importance of calcium mobilization
and entry in the renal vascular response to adrenoceptor activation in
afferent arterioles isolated from 7- to 8-wk-old Wistar-Kyoto (WKY) and
spontaneously hypertensive rats (SHR). Intracellular free calcium
concentration ([Ca2+]i) was measured in
microdissected arterioles utilizing ratiometric photometry of fura 2 fluorescence. There was no significant strain difference in baseline
[Ca2+]i. Norepinephrine (NE;
106 and 10
7 M) elicited immediate,
sustained increases in [Ca2+]i. The general
temporal pattern of response to 10
6 M NE consisted of an
initial peak and a maintained plateau phase. The response to NE was
partially blocked by nifedipine (10
6 M) or
8-(N,N-diethylamino) octyl-3,4,5-trimetoxybenzoate (TMB-8; 10
5 M). A calcium-free external solution abolished the
sustained [Ca2+]i plateau response to NE,
with less influence on the peak response. In the absence of calcium
entry, TMB-8 (10
5 M) completely blocked the calcium
response to NE in WKY but not SHR, suggesting strain differences in
mobilization. A higher concentration of TMB-8 (10
4 M),
however, blocked all discernible mobilization in both strains. We
conclude that there are differences in Ca2+ handling in
renal resistance vessels between young WKY and SHR with respect to
mobilization stimulated by
-adrenoceptors. Afferent arterioles of
young SHR appear to have a larger
inositol-1,4,5-trisphosphate-sensitive pool or release from a site less
accessible to TMB-8.
spontaneously hypertensive rats; Wistar-Kyoto rats; norepinephrine; hypertension; 8-(N,N-diethylamino) octyl-3,4,5-trimetoxybenzoate; nifedipine; vascular smooth muscle; inositol-1,4,5-trisphosphate-mediated mobilization; L-type calcium channel; renal circulation
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INTRODUCTION |
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IT IS WELL KNOWN THAT HYPERTENSION is associated with increased peripheral vascular resistance. Less clear, however, is whether the increased vasomotor tone is a primary abnormality or whether it develops secondarily to the increased pressure. Cross-transplantation of kidneys between hypertensive and normotensive rat strains leads to development of hypertension in previously normotensive animals and to normalization of the elevated blood pressure in the hypertensive-prone strain. This indicates a crucial role of the kidneys and has been shown for various models of hypertension including very young spontaneously hypertensive rats (SHR) (39). The precise mechanism responsible for the renal defect is elusive, but the kidneys are known to be vasoconstricted during development of hypertension at the age of 6-8 wk in association with reduced glomerular filtration rate (GFR) and retention of salt and water (13, 20, 37). Furthermore, kidneys of young SHR exhibit increased reactivity to vasoconstrictors such as angiotensin II (ANG II), arginine vasopressin (AVP), and thromboxane A2 (8, 15). Increased extracellular fluid volume and autoregulatory- and paracrine-induced vasoconstriction in the peripheral circulation are likely to contribute to, if not cause, the elevated arterial pressure (24).
The renal circulation is richly endowed with sympathetic nerves, and the autonomic nervous system is poised to play an important role in the control of renal hemodynamics, GFR, extracellular fluid volume, and arterial blood pressure (3, 11). The critical function of renal nerves is indicated during the onset of hypertension in SHR. Renal denervation delays or attenuates the development of hypertension in genetic models (29) in association with exaggerated denervation natriuresis (40). With regard to renal vascular reactivity to adrenergic stimulation in genetic hypertension, studies to date have been less than conclusive. Some of the variation in results may result from the wide range of experimental conditions and age of the animals (4, 9, 12, 17, 45, 50).
Catecholamines released from nerve terminals and of hormonal origin
exert their effects by activation of cell surface adrenoceptors on
vascular smooth muscle cells (VSMC) to produce changes in cytosolic calcium concentration ([Ca2+]i) and the
resultant contractile response. Activation of -adrenoceptors induces
an increase in [Ca2+]i by recruitment of
calcium from one or two major sources, i.e., mobilization from
intracellular stores, and/or entry from the extracellular space, in
afferent and efferent arterioles (26, 42). Calcium entry
may occur through voltage-dependent and/or receptor-activated calcium
channels located in the plasma membrane; in some cell types,
store-operated calcium entry depends on depletion of intracellular
stores. VSMC exhibit two sites of internal calcium stores, the
sarcoplasmatic reticulum and mitochondria (33), although
the latter is thought to contribute little to the regulation of
[Ca2+]i in VSMC. Two channels for release of
calcium from sarcoplasmic reticulum are a ryanodine receptor and an
inositol-1,4,5-trisphosphate (IP3) receptor. The
IP3 receptor appears to be present in all VSMC, whereas the
contribution of the ryanodine receptor is poorly understood. Evidence
indicates that the relative contributions of entry vs. mobilization in
the response to stimulation with cathecholamines differ in VSMC
depending on location and function (7, 36).
Reports from work in several animals (e.g., rat, dog, cat, and monkey) have shown that an increase in renal sympathethic nerve activity decreases renal blood flow (RBF) and increases renal vascular resistance (11). Calcium entry blockers are known to block, at least in part, NE-induced renal vasoconstriction (25). Several studies have reported effects of norephinephrine (NE) on [Ca2+]i in isolated renal vessels (30, 42, 43). In this regard, we have previously shown in Sprague-Dawley rats that both the NE-induced [Ca2+]i response in isolated afferent arterioles and the renal vasoconstriction in vivo are attenuated ~50% by nifedipine (42). Moreover, the putative blocker of IP3-mediated calcium mobilization 8-(N,N-diethylamino) octyl-3,4,5-trimetoxybenzoate (TMB-8) inhibited NE-induced renal vasoconstriction or changes in [Ca2+]i by ~80% in both in vivo and in vitro preparations.
Differences in VSMC calcium handling may contribute to the increased vascular tone found in animals with genetic hypertension, during both developmental and established phases of hypertension. Evidence to date implicates strain differences in basal levels of [Ca2+]i (49), function of intracellular calcium stores (10, 35), and activity of calcium and potassium channels (41, 51). It should be recognized that most of these reports pertain to cultured VSMC obtained from large conduit vessels of relatively old SHR with established hypertension.
The present study was designed to identify mechanisms responsible for
mobilization and entry of [Ca2+]i in response
to stimulation of -adrenoceptors by NE in renal resistance vessels
microdissected from young normo- and hypertensive rats. The
contribution of calcium entry pathways was tested using a nominally
calcium-free bathing solution. The involvement of entry through
voltage-sensitive L-type calcium channels was assessed using the
pharmacological agent nifedipine. TMB-8 was used to inhibit
IP3-dependent mobilization of calcium from internal stores. [Ca2+]i was measured in vitro in isolated
afferent arterioles from 7- to 8-wk old SHR and WKY rats using the
ratiometric fluorescence of the indicator fura 2.
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METHODS |
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Glomeruli with an attached isolated afferent arteriole were microdissected from 7- to 8-wk-old WKY and SHR rats (weighing 208 ± 7 and 209 ± 5 g, respectively) from the Chapel Hill or Harlan colonies using methods described previously (42). Briefly, thin slices (thickness 0.5-1 mm) were cut from the midregion of the kidney. They were transferred to a dissection dish containing a chilled physiological salt solution (PSS) solution with BSA (Sigma) at a final concentration of 0.5 g/dl. The PSS solution had the following composition (mM): 135 NaCl, 5.0 KCl, 1.0 CaCl2, 1.0 MgCl2, 10 HEPES, and 5.0 D-glucose. Forceps were used for the isolation procedure under microscopic visualization (magnification ×12-100). The interlobular artery was localized at its origin from an arcuate artery, and a segment consisting of glomeruli, blood vessels, and tubular structures was removed. The tubular structures were carefully stripped away using forceps. A single afferent arteriole was cut at the bifurcation from an interlobular artery by using a knife blade. To obtain a homogenous population of superficial arterioles, we dissected arterioles from the outer one-third of the cortex. If no satisfactory preparation was obtained during the first 120 min of dissection, the kidney was discarded.
After the dissection procedure was completed, an afferent arteriole was
loaded with fura 2-acetoxymethyl ester (AM) for 45-60 min at room
temperature in the dark, as previously described (42). Fura 2-AM (Molecular Probes) was prepared as a stock solution in DMSO
(1 mM) and mixed with PSS to give a final concentration of 2 µM and
Pluronic F127 (Molecular Probes) (0.01%) immediately before use. After
the loading was completed, the vessel was transferred to a chamber
containing PSS on the stage of an inverted microscope (Olympus IX 70)
using an Eppendorf micropipette. The proximal end of the arteriole and
the glomerulus were then aspirated into concentric glass holding
pipettes to maintain mechanical stability. For measurements of
[Ca2+]i, the arteriole was centered in the
optical field of ×40 quartz oil immersion objective, and shutters were
adjusted to get an arteriole in a sampling window. The preparation was
continuously visualized by the use of a video camera (Sony) and
monitor. A dual-excitation wavelength DeltaScan equipped with dual
monochromators and a light pathway chopper (Photon Technology
International, NJ) was used for exciting with ultraviolet light of,
alternatively, 340- and 380-nm wavelength. The fluorescent emission was
detected by a photometer and was processed and stored by an
IBM-compatible Pentium computer and Felix software (Photon Technology
International). [Ca2+]i was calculated using
the Grynkiewicz equation (22):
[Ca2+]i = Kd · [(RRmin)/(Rmax
R)] · (Sf/Sb),
where Kd is the dissociation constant of fura 2 for calcium; Sf and Sb are 380-nm fluorescence at zero and saturating calcium concentrations, respectively; and Rmin and Rmax are values of R (fluorescence
ratio 340/380) at low and at saturating calcium concentration,
respectively. Values for Kd, Rmin,
Rmax, Sf, and Sb were determined in
vitro as previously described (27, 42).
During replacement of fluids, the volume in the experimental chamber
was maintained constant by the use of a vacuum suction system. All
experiments were performed at room temperature (26-28°C). The
experimental solutions were added by a volume large enough to allow
total exchange of the composition in the experimental chamber several
times. The nominally calcium-free solution was obtained by adding 1 mM
EGTA to PSS and replacing CaCl2 with NaCl. Pilot studies
established that 106 M of NE elicited one-half of maximal
response in [Ca2+]i; this concentration is
used throughout the present study unless stated otherwise. Nifedipine
was dissolved in DMSO and diluted in PSS to a final concentration of
10
6 M, a concentration known to inhibit L-type calcium
channels (18). TMB-8, an inhibitor of
IP3-mediated calcium release from sarcoplasmic reticulum,
was added to the same solutions to yield a final concentration of
10
5 or 10
4 M. The experiments were
conducted as follows. Initially, the viability of each vascular
preparation was determined by adding a short pulse (50 s) of NE. If
there was no immediate [Ca2+]i response, the
preparation was discarded. Inhibitory agents were added before or after
NE stimulation. Arterioles were exposed to nifedipine (50 s),
calcium-free medium (50 s), or TMB-8 (90 s) or combinations thereof
before and during NE stimulation. The mean prestimulation
[Ca2+]i values during the control period were
obtained between 10 and 15 s before the addition of NE. Similarly,
[Ca2+]i was measured as the immediate peak
response (maximal peak value during the first 15 s after the
addition of NE) and between 30 and 35 s (sustained plateau phase)
after the stimulation was initiated. To establish reversibility and to
exclude possible prolonged action and crossover effects of a particular
pretreatment, the experiments were performed in random order.
Statistical analyses. Data are presented as means ± SE. The SigmaStat (Jandel Scientific/SPSS) software was used for data analysis. Statistical significance was evaluated by ANOVA for repeated measurements and the post hoc Newman-Keuls test. Student's unpaired t-test was used for strain difference. A P value of < 0.05 is considered statistically significant.
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RESULTS |
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Measurements of basal afferent arteriolar
[Ca2+]i and
the response to NE.
Resting or prestimulation values of [Ca2+]i
did not differ between afferent arterioles from 7- to 8-wk-old WKY
(n = 22 arterioles from 18 rats) and age-matched SHR
(n = 21 arterioles from 19 rats): 97 ± 6 vs.
81 ± 10 nM (P > 0.1). Stimulation with NE
(106 M), which elicits a half-maximal response, caused an
abrupt, step increase in [Ca2+]i in afferent
arteriolar VSMC of WKY and SHR (Fig. 1).
The general response is characterized by a sharp rise with a transient
peak followed by a sustained plateau phase that remains 50-100 nM
above baseline. Overall, the basic response pattern was similar in
preparations from WKY and SHR. To more systematically analyze the
responses, we selected the time points of the initial peak
[Ca2+]i at 0-15 s and the average value
recorded during the sustained plateau phase between 30 and 35 s.
Preliminary studies established that the response at 30 s was
commonly maintained for several minutes. Contraction of the arteriole
observed on the video monitor correlated temporally with the increase
in [Ca2+]i.
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NE-induced calcium entry.
To evaluate the importance of extracellular calcium in the NE response,
the effects of NE were assessed with and without a calcium-free bath solution containing EGTA (1 mM). WKY arterioles in
the normal calcium-containing solution responded to NE
(106 M) with increases in
[Ca2+]i from 134 ± 27 to a peak value
of 237 ± 38 nM (~78% increase above baseline) between 0 and 15 s and to 200 ± 31 nM (~50% increase above baseline) at
30-35 s (n = 5, P < 0.001). After
short-term (60 s) exposure to the calcium-free solution, the small
decrease in baseline [Ca2+]i (from 128 ± 25 to 106 ± 27 nM) was not statistically significant (P = 0.16). NE challenge in the calcium-free solution
caused a transient peak in [Ca2+]i to ~54%
above baseline (to 163 ± 36 nM, P < 0.01, n = 5). The calcium values during continued NE
stimulation for 30-35 s declined to control levels (110 ± 25 vs. 106 ± 27 nM) (Fig. 2). Thus NE stimulation was significantly attenuated when calcium entry was limited
by calcium-free extracellular fluid, especially during the sustained
plateau phase (P < 0.001). SHR afferent arterioles in
the presence of normal extracellular calcium responded to NE (10
6 M) with increases in
[Ca2+]i from 107 ± 18 to a peak value
of 208 ± 27 nM (~95% increase above baseline) and to a
sustained plateau level of 168 ± 20 nM at 30-35 s (~57%
increase, n = 5, P < 0.001). After
pretreatment of SHR vessels with Ca2+-free solution for
60 s, resting [Ca2+]i was relatively
stable, falling slightly (from 96 ± 21 to 72 ± 15 nM)
(P = 0.6), and NE elicited an increase of ~138% (to
171 ± 45 nM). At 30-35 s, [Ca2+]i
had declined to prestimulation levels (80 ± 14 nM). Thus in contrast to WKY, the mean absolute peak response of roughly 100 nM in
SHR was not significantly attenuated by pretreatment with a
calcium-free solution.
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Calcium release from intracellular stores.
To further assess the involvement of calcium mobilization, the response
to NE was assessed before and during treatment with TMB-8, an inhibitor
of IP3-induced calcium release (38). Before drug application, NE elicited a peak increase in
[Ca2+]i (108 ± 13 to 256 ± 28 nM)
at 0-15 s and a sustained response at 210 ± 22 nM at
30-35 s (P < 0.001) in 7 WKY preparations. Ninety seconds of exposure to TMB-8 did not affect baseline
[Ca2+]i but did attenuate NE effects on
[Ca2+]i. TMB-8 inhibited both the peak and
plateau responses (P < 0.01). As Fig.
3 shows,
[Ca2+]i rose from 101 ± 12 to 178 ± 24 nM between 0 and 15 s and to 135 ± 13 nM at 30-35 s
(P < 0.05, n = 7). Likewise, the
NE-induced response was attenuated by TMB-8 in afferent arterioles of
SHR; baseline values were unaffected. In SHR control experiments, NE caused an immediate rise in [Ca2+]i from
102 ± 12 to 221 ± 28 nM, with a sustained elevation
(167 ± 15 nM at 30-35 s, P < 0.001, n = 8). After TMB-8 pretreatment, NE produced an
initial rise of 82% above baseline (90 ± 12 to 164 ± 20 nM) that remained elevated above control (113 ± 12 nM) at
30-35 s (P < 0.05, n = 8).
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TMB-8 sensitivity of calcium release.
To examine sensitivity of calcium release to TMB-8, afferent arterioles
were pretreated for 90 s with a combination of TMB-8 (105 M or 10
4 M) and a calcium-free
solution + 1 mM EGTA. Representative tracings are shown in Fig. 1.
In control experiments in WKY arterioles (n = 7), NE
caused an initial increase from 102 ± 14 to 236 ± 26 nM,
followed by a sustained plateau of 189 ± 16 nM at 30-35 s
(P < 0.01). After pretreatment with the lower
concentration of TMB-8 (10
5 M) in the calcium-free
solution, the response to NE was completely abolished (Fig. 1
A; Fig. 4). Basal
[Ca2+]i declined to 72 ± 10 nM on
average, and no significant changes were noted during NE stimulation at
0-15 s (89 ± 15 nM) and at 30 to 35 s (76 ± 11 nM, P > 0.2). In contrast to the complete inhibition
in WKY, responses were clearly evident in SHR arterioles. Here we found
that NE still elicited an immediate peak response in the presence of
TMB-8 and the calcium-free solution. In the SHR control experiments, NE
caused a peak increase in [Ca2+]i of 169 nM
above baseline (100 ± 14 to 269 ± 28 nM) and a sustained plateau level (179 ± 22 nM, n = 6, P < 0.01). Combined TMB-8 treatment with the
calcium-free solution for 90 s did not significantly reduce
baseline [Ca2+]i (from 97 ± 14 to
63 ± 12 nM, P = 0.07). Subsequent stimulation with NE caused a significant peak increase of 59 nM (to 122 ± 22 nM, P < 0.001) with a subsequent return to baseline at
30 s (69 ± 12 nM). A higher concentration of TMB-8
(10
4 M) totally abolished the intracellular release in
both strains. In WKY, basal [Ca2+]i in the
presence of TMB-8 and the EGTA-containing, calcium-free solution was
82 ± 10 nM (n = 4). Subsequent stimulation with
NE did not affect [Ca2+]i, which remained at
80 ± 8 nM. In SHR, the corresponding values were 60 ± 7 and
60 ± 8 nM (n = 7). Thus the results from this series of experiments suggest that afferent arterioles of young SHR
exhibit a lower sensitivity to TMB-8 (Fig.
5).
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Involvement of voltage-operated calcium channels.
To investigate the involvement of voltage-gated L-type calcium
channels the dihydropyridine nifedipine was used to antagonize the
NE-induced [Ca2+]i response. In WKY before
nifedipine treatment, NE caused the usual peak and plateau responses of
145 and 87 nM (85 ± 10 to 230 ± 24 nM and then 172 ± 26 nM; P < 0.01; n = 6) (Fig. 6).
Pretreatment with nifedipine (106 M) for 60 s did
not influence basal [Ca2+]i
(P > 0.4); however, it attenuated both the peak and
plateau response to NE by ~50% (P < 0.05). As Fig.
6 shows, for WKY vessels treated with the
calcium channel blocker, the peak [Ca2+]i
response to NE was attenuated to 80 nM and the pleateau phase to 33 nM;
the absolute [Ca2+]i values rose from 72 ± 12 to 152 ± 11 nM and then declined to 105 ± 6 nM
(P < 0.05 vs. nifedipine). In afferent arterioles from SHR (n = 6), NE in the control period increased
[Ca2+]i from 71 ± 7 nM to an initial
peak (204 ± 23 nM) and a plateau (144 ± 9 nM) that were 187 and 102% above baseline, respectively (P < 0.01). In
the experimental period, nifedipine pretreatment did not significantly
decrease basal [Ca2+]i (68 ± 7 to
57 ± 5 nM, P > 0.05); however, it attenuated the responses to NE, reducing the immediate peak response to 152% above
baseline (144 ± 24 nM) and the sustained plateau to 53% above
control (87 ± 6 nM) (P < 0.05). Thus nifedipine
attenuated the NE-induced [Ca2+]i response to
the same extent in WKY and SHR.
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DISCUSSION |
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The present study presents new information about the cellular mechanisms mediating adrenoceptor-induced activation of cytosolic calcium in VSMC of resistance vessels in the renal microcirculation. One objective was to determine the relative importance of entry of extracellular calcium and mobilization of calcium from intracellular stores in microdissected afferent arterioles. A related aim was to investigate possible strain differences in these calcium signaling pathways between young WKY and SHR. The present studies extend to afferent arterioles of young WKY and SHR our previous findings on calcium signaling in the control of renal vascular resistance in vivo and cytosolic calcium in afferent arterioles of Sprague-Dawley rats (42). Our earlier work established that the vasoconstrictor actions of NE involve a combination of calcium entry that is antagonized by conventional calcium channel blockers with calcium mobilization sensitive to an antagonist of the IP3-mediated pathway. NE-induced renal vasoconstriction was attenuated in a dose-dependent manner by dihydropyridine nifedipine, with maximal ~50% inhibition of both the NE-induced renal vasoconstriction and the increase in arteriolar [Ca2+]i. TMB-8 blocked ~80% of the NE response in vivo and in vitro. In vivo blood flow experiments showed that there was no additive effect of combined treatment with nifedipine and TMB-8, indicating a continuous and complex interplay between calcium mobilization and entry.
In the present study, the [Ca2+]i response to
NE (106 M) was similar in afferent arterioles isolated
from 7- to 8-wk-old WKY and SHR. However, the overall response pattern,
characterized by an initial peak followed by a sustained plateau phase,
differed somewhat from what we previously found in Sprague-Dawley rats (42). In our earlier work, the majority of the
[Ca2+]i responses to NE were square shaped
with the initial peak and sustained plateau of similar magnitudes. Both
WKY and SHR exhibited a slightly more pronounced initial transient peak
relative to the maintained plateau phase. It is not clear whether the
response patterns reflect strain differences or the fact that younger
animals were used in the present study. The methods utilized for
microdissection of afferent arterioles and the measurement of fura 2 fluorescence were identical.
It is noteworthy that a lower concentration of NE (107 M)
also elicited equivalent [Ca2+]i changes in
arterioles from WKY and SHR. In both strains there was a small but
clearly significant increase in response to this concentration of NE.
However, only in SHR was there a statistically significant difference
between the peak and plateau recordings of
[Ca2+]i using this lower dose of NE. We
cannot, however, convincingly conclude that this reveals a more
pronounced susceptibility of the SHR to stimulation with NE in the form
of an initial transient [Ca2+]i response.
There are conflicting reports in the literature regarding the magnitude of the constrictor response to adrenergic stimulation of VSMC from WKY and SHR. For example, in one study utilizing the isolated perfused kidney preparation, administration of NE or nerve stimulation caused more pronounced renal vasoconstriction in young (4-5 wk) SHR than in WKY (45). However, the contractile sensitivity to NE was reduced in SHR. In other isolated kidney studies, increased reactivity to NE was found in both young (4 wk) and adult (2 and 4 mo) stroke-prone SHR compared with age- and sex-matched WKY (4). On the other hand, reduced renal vascular reactivity of ex vivo blood-perfused kidneys to NE has been reported for 12- to 16-wk-old SHR compared with WKY (17), whereas other investigators found no strain difference for this age group in vivo (12).
It has been proposed that VSMC from hypertensive rats have abnormalities in calcium metabolism (44). Dysfunction may involve intracellular overload of calcium (19), handling of calcium by sarcoplasmic reticulum (5, 10, 31), ion channels (32), or an Na+/Ca2+ exchanger (34). In the present study, we used young rats at 7 to 8 wk of age to characterize SHR during the developmental phase of hypertension, characterized by rapid increases in arterial pressure and relative retention of salt and water (13). In contrast to what other investigators have found in VSMC from other vascular beds (47), baseline [Ca2+]i did not differ statistically from normal in SHR afferent arterioles, although it tended to be slightly lower than WKY values. Our observations agree with the lack of a strain difference in baseline [Ca2+]i found in small muscular arteries (15-35 µm outer diameter) (46) and cultured aortic VSMC (35). Furthermore, no differences in basal [Ca2+]i levels were found between cultured VSMC from mesenteric resistance arteries during primary and first passages (6). Interestingly, basal [Ca2+]i rose in VSMC from SHR during subculture to beyond passage 3 (6). Clearly, reports of possible strain differences in basal or stimulated [Ca2+]i need to be qualified by detailed experimental conditions and interpreted with caution. Furthermore, because the morphology of the resistance vessels may differ between preparations from WKY and SHR, one cannot completely exclude structural modifications and their potential impact on optical properties and measurements of dye fluorescent emission (37).
We have previously shown in Sprague-Dawley rats that the NE-induced [Ca2+]i response in isolated afferent arterioles exhibited a similar ~50% attenuation by nifedipine (42). These results indicated the importance of L-type voltage-gated calcium channels in the rat renal vascular response to activation of adrenoceptors. The existence of voltage-gated calcium channels was further supported by the fact that potassium-induced depolarization of the isolated arterioles caused calcium entry exclusively via a nifedipine-sensitive pathway. However, we did not find any significant alterations of the baseline [Ca2+]i by administration of nifedipine in Sprague-Dawley rats.
Alterations of calcium channels may at least partially account for variability seen in baseline [Ca2+]i, and the [Ca2+]i response to agonist activation between hypertensive and normotensive animals. Support for that notion has been provided by several laboratories (32, 48). In the present study on vessels from young rats, pretreatment with nifedipine attenuated the response to NE to the same extent in WKY and SHR. Nifedipine attenuated the sustained plateau response to NE by roughly 50% when extracellular calcium concentration was normal, as previously demonstrated for renal arterioles of Sprague-Dawley rats (42). Thus our findings indicate that dihydropyridine-sensitive calcium channels contribute to the NE-induced [Ca2+]i response to the same extent in SHR and WKY. The fact that low extracellular calcium inhibited the NE-induced sustained increase in [Ca2+]i more strongly than nifedipine implicates a second calcium entry pathway, present in SHR and WKY, as previously found in renal arterioles of Sprague-Dawley rats (42). By definition, calcium entry via this pathway is insensitive to the dihydropyridine class of agents and thus independent of L-type calcium channels. Previous studies provide support for the participation of more than one calcium entry in smooth muscle preparations of different vascular origin, e.g., store-operated capacitative entry (14, 21). Such mechanisms may be responsible for distinction between the effects of nifedipine and the calcium-free medium. It is possible that exposure to a calcium-free medium could influence calcium release from internal stores. Our preliminary studies reveal, however, that a [Ca2+]i response to NE exists after several min of pretreatment with a calcium-free solution.
With regard to the relative importance of calcium mobilization from
intracellular stores, we tested TMB-8, an inhibitor of IP3-mediated calcium release from intracellular stores, on
the NE-induced [Ca2+]i response
(38). We found that pretreatment with TMB-8 attenuated both the peak and the plateau increases in
[Ca2+]i produced by NE. Both were inhibited
to the same extent in WKY and SHR, in accord with previous findings for
Sprague-Dawley rats (42). A generally accepted model for
VSMC predicts that calcium entry is solely responsible for the
sustained phase of [Ca2+]i increase and that
inhibition of calcium mobilization, an initial event, thus should not
interfere with the sustained stimulation. In our present studies, it is
clear that the plateau phase is totally dependent on extracellular
calcium because this phase is absent in a calcium-free environment. The
fact that TMB-8 attenuates both the immediate peak and the sustained
[Ca2+]i plateau in the presence of
extracellular calcium indicates a continuous interaction between
IP3-induced mobilization and entry. This finding holds for
afferent arterioles from WKY, SHR, and Sprague-Dawley rats. The fact
that the peak is substantially higher in the presence than in the
absence of extracellular calcium, at least in WKY vessels, indicates
that the initial peak depends on entry as well as intracellular
release. Calcium entry via L-type channels during the peak and plateau
is indicated by nifedipine inhibition of both phases of calcium
responses. Also, several reports differ from the "noninteracting"
time-dependent model and support the notion that calcium recruitment
pathways may vary between large-diameter conduit vessels and
small-diameter resistance vessels (7, 36). It is difficult
to draw any conclusions from the shape of the response and as to
whether it consists of a peak and a plateau or a square-shaped
response. A square-shaped response may have an initial response that
consists of the same components as in the "peak-plateau type"; it
just does not surpass the level of the sustained plateau. The
specificity of TMB-8 to block the IP3 pathway has been
questioned (1). Earlier studies from our laboratory have
shown that the lower concentration of TMB-8 (105 M) used
in this study has no demonstrable effect on the calcium entry through
voltage-sensitive calcium entry channels in isolated arterioles
(42). Furthermore, it has no effect on renal
vasoconstriction elicited by stimulation of L-type calcium entry
channels with BAY K 8644 in vivo (16).
If one assumes that the mobilization of calcium from intracellular stores relies solely on release from IP3-sensitive pathways and that this release is totally blocked by the employed low dose of TMB-8, no agonist-induced [Ca2+]i response should be expected after pretreatment with a combination of TMB-8 and a calcium-free solution. In the present study, these assumptions hold true for afferent arterioles of WKY. After pretreatment with both concentrations of TMB-8 in a calcium-free solution, there was no NE-induced response in afferent arterioles from WKY. In contrast, in SHR there was a clear transient response to NE after treatment with the lower concentration of TMB-8. On the other hand, the higher concentration of TMB-8 in combination with the calcium-free solution totally abolished the response to NE. For both strains and both concentrations used, the combination of TMB-8 and the calcium-free medium totally abolished the sustained plateau phase in both strains.
Thus the present results strongly suggest a difference in calcium handling of the intracellular calcium stores between SHR vs. WKY afferent arteriolar VSMC at the age of 7 to 8 wk. This might reflect either different properties of the IP3-sensitive pool or the contribution of a non-IP3-sensitive pool. In this regard, a 1.6-fold higher IP3 receptor activity has been found in aortic microsomes of SHR compared with WKY (5). Other investigators found an increased responsiveness to IP3 in cardiac myocytes from SHR relative to WKY (28). In cultured aortic myocytes from 9- to 10-wk-old rats, it is reported that thapsigargin causes a larger calcium release that might indicate a larger intracellular calcium pool (10). This study also reported an enhanced angiotensin II-induced release of calcium in calcium-free conditions in SHR and that this augmented mobilization could be blocked by ryanodine. This might indicate a larger contribution of a ryanodine-sensitive pool in SHR. Furthermore, the presence of additional thapsigargin- and nonthapsigargin-sensitive intracellular calcium pools in SHR vessels (35) has been suggested. A third possibility is that IP3 is accumulated to a higher degree by NE stimulation in SHR. This has indeed been suggested for VSMC from SHR, presumably due to a decreased rate of dephosphorylation of IP3, rather than an increased production (23).
It is therefore evident that there are several levels in the chain of
events leading to agonist-induced release of calcium from
intracellullar stores that may differ between VSMC of SHR and WKY. From
our results, it is not possible to determine whether the intracellular
calcium pool consists of one or two compartments. One interpretation is
that one single mechanism accounts for the total release and that this
mechanism is simply more sensitive to attenuation by TMB-8 in WKY. This
notion is supported by the fact that the higher concentration of TMB-8
(104 M) totally abolished the effect of NE under
calcium-free conditions. Another explanation is that there are two
distinct release mechanisms, one of which is more pronounced in
afferent arteriolar VSMC of SHR than in those of WKY and less sensitive
to TMB-8. The sensitivity to the higher concentration of TMB-8 could
then be due to a nonspecific effect, as previously suggested
(1). On the other hand, we found in preliminary
experiments, both in WKY and SHR, that the transient increase in
[Ca2+]i stimulated by response to caffeine,
an agent commonly used to activate a ryanodine receptor channel in a
calcium-free solution, is essentially unaffected by the highest
concentration of TMB-8 employed in the present study, which supports
the notion that TMB-8 is selective, without general inhibition of all
mobilization pathways. Other investigators have found that the effect
of caffeine is not affected by TMB-8 in single VSMC from rat tail
artery (2). If our preliminary finding holds true, this
indicates that caffeine-sensitive receptors are not involved in the
relatively TMB-8-sensitive response to NE in SHR. From the results
obtained in the present study we cannot, however, totally discriminate
between a single mechanism that is less TMB-8 sensitive in SHR or two
distinct intracellular calcium pools. We were not able to detect any
strain differences when blocking the NE-induced response with TMB-8 in
the presence of extracellular calcium. This could be explained by the
possibility that the calcium release mechanism(s) might be suppressed
by an elevation of [Ca2+]i. It is possible
that during TMB-8 treatment alone the NE-induced calcium influx via
dihydropyridine-sensitive channels and/or the alternative entry pathway
might account for the attenuation of the mobilization pathway in SHR.
Further studies are required to provide more in-depth information about
conditions leading to the existence of and the functional relative
importance of the less TMB-8-sensitive, or additional calcium
mobilization pathway, in VSCM of resistance arterioles.
In summary, our study presents novel findings regarding calcium signaling mechanisms during stimulation of adrenergic receptors in renal resistance vessels from 7- to 8-wk-old WKY and SHR. In both strains, NE-induced increases in [Ca2+]i were dependent on both calcium entry via dihydropyridine-sensitive calcium channels and on TMB-8-sensitive intracellular mobilization. Exposure of isolated vessels to a calcium-free solution results in marked attenuation of the NE-induced sustained increase in [Ca2+]i in arterioles from both strains; the fact that inhibition was more marked than the nifedipine effect suggests the operation of calcium entry pathways in addition to L-type channels. The fact that, in SHR, a [Ca2+]i response was induced after pretreatment with a combination of calcium-free solution and TMB-8 (low dose) might indicate a larger intracellular calcium pool, a single release mechanism that is less TMB-8 sensitive in SHR or an alternative pathway for calcium mobilization distinct from IP3. The observed strain differences in calcium signaling may represent a mechanism responsible for enhanced renal vascular reactivity in young SHR during the development of genetic hypertension.
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ACKNOWLEDGEMENTS |
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These studies were supported by National Heart, Lung, and Blood Institute Grant HL-02334. M. Salomonsson's visit was sponsored in part by the Swedish Medical Research Council, The Royal Physiographic Society in Lund, Medical Faculty Lund University, Maggie Stephens Foundation, and the Berth von Kantzow Foundation.
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FOOTNOTES |
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Present address of M. Salomonsson: Dept. of Medical Physiology, Univ. of Copenhagen, Blegdamsvej 3, 2200 Copenhagen N, Denmark.
Address for reprint requests and other correspondence: W. J. Arendshorst, Dept. of Cell and Molecular Physiology, CB #7545, School of Medicine, Rm. 152, Medical Sciences Research Bldg., Univ. of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7545 (E-mail: arends{at}med.unc.edu).
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. Section 1734 solely to indicate this fact.
Received 20 June 2000; accepted in final form 20 April 2001.
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