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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Experiments were conducted to gain insight into mechanisms responsible for exaggerated renal vascular reactivity to ANG II and vasopressin (AVP) in spontaneously hypertensive rats (SHR) during the development of hypertension. Cytosolic calcium concentration ([Ca2+]i) was measured by ratiometric fura 2 fluorescence and a microscope-based photometer. Vascular smooth muscle cells (SMC) from preglomerular arterioles were isolated and dispersed using an iron oxide-sieving method plus collagenase treatment. ANG II and AVP produced rapid and sustained increases in [Ca2+]i. ANG II elicited similar dose-dependent increases in [Ca2+]i in SMC from SHR and Wistar-Kyoto rats (WKY). In contrast, AVP caused almost twofold larger responses in afferent arteriolar SMC from SHR. ANG II effects were inhibited by the AT1 receptor antagonist losartan. AVP action was blocked by the V1 receptor antagonist [d(CH2)5,Tyr(NH2)9]AVP. In SMC pretreated with nifedipine, neither ANG II nor AVP elicited [Ca2+]i responses. Poststimulation nifedipine reversed elevated [Ca2+]i to basal levels. Short-term reductions in external [Ca2+]i (EGTA) mimicked the nifedipine effects. Our study shows that AT1 and V1 receptors stimulate [Ca2+]i by a common mechanism characterized by preferential action on voltage-gated L-type channels sensitive to dihydropyridines. Calcium signaling elicited by AT1 receptors does not differ between SHR and WKY; thus the in vivo exaggerated reactivity may be dependent on interactions with other cell types, e.g., endothelium. In contrast, AVP produced larger changes in [Ca2+]i in arteriolar SMC from SHR, and such direct effects can account for the exaggerated renal blood flow responses.
smooth muscle cells; calcium channel; voltage-gated channel; dihydropyridine; nifedipine; spontaneously hypertensive rat; inbred rats; renal circulation; glomerular filtration rate; segmental vascular resistance; angiotensin AT1 receptor; vasopressin V1 receptor
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INTRODUCTION |
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IN MOST MODELS of essential hypertension, the kidney plays a critical role in the development of hypertension. Crosstransplantation studies show that replacing a kidney from a hypertensive-prone rat leads to the development of elevated arterial pressure in a previously normotensive rat. Conversely, transplanting a kidney from a normal rat into a hypertensive-prone rat prevents the development of hypertension in young animals and lowers arterial pressure in animals with established hypertension. These results were obtained in various models of genetic hypertension including spontaneously hypertensive rats (SHR) of Okamoto-Aoki and Milan strains and also the Dahl salt-sensitive rat (5, 17, 42). The mechanisms responsible for the kidney determining arterial pressure have been an important area of investigation.
Evidence indicates young rats of hypertensive strains retain salt and water and that the kidneys of these animals are vasoconstricted with increased renal vascular resistance and reduced glomerular filtration rate. The preglomerular afferent arteriole, the main site of resistance control in the kidney, plays a major role in the regulation of renal blood flow, glomerular filtration rate, and arterial pressure. The afferent arteriole is constricted in early as well as middle stages of hypertension in the SHR model, although relative vasodilation has been noted in old animals during hyperfiltration and adaptation to nephron loss (18, 22, 30). The observed abnormalities in renal hemodynamics and function are associated frequently with increased vascular reactivity. For example, the renal vasculature is more responsive to vasoconstrictor agents such as ANG II and arginine vasopressin (AVP) (11, 13, 15, 19, 20). Also contributing are attenuated buffering actions of vasodilators such as prostaglandins (PG) and dopamine (11, 14, 15, 31). The precise mechanisms underlying these functional abnormalities remain to be determined. Possibilities include strain differences in excitation-contraction coupling related to receptor types and relative densities and their functional interactions with intracellular second messenger systems. Hormones and paracrine agents elicit changes in cytosolic calcium concentration ([Ca2+]i) to produce contraction of vascular smooth muscle cells (SMC) and regulate vascular resistance.
The purpose of the present study was to gain insight into mechanisms that are responsible for increased renal vascular reactivity in young SHR during the developmental phase of hypertension. Calcium signaling was evaluated in individual SMC from afferent arterioles isolated using an iron oxide-sieving technique combined with collagenase digestion. Changes in [Ca2+]i produced by ANG II and AVP were determined by measuring fura 2 fluorescence using a microscope-based photometer. Both agonists produced an immediate step increase in [Ca2+]i that was maintained throughout the 200-s stimulation period. AVP elicited a larger [Ca2+]i increase in SMC from SHR than from Wistar-Kyoto rats (WKY); there was, however, no strain difference in [Ca2+]i stimulation elicited by ANG II. Losartan antagonized ANG II-induced responses via the angiotensin AT1 receptor(s); AVP effects were inhibited by a selective V1 receptor antagonist. The presence and function of dihydropyridine-sensitive L-type calcium channels in freshly isolated preglomerular arteriolar SMC were indicated by the inhibitory effects of nifedipine and of a nominally calcium-free medium. Nifedipine completely inhibited [Ca2+]i responses to ANG II and AVP; pretreatment prevented a response, and application during stimulation reversed the hormone effect. Thus AT1 and V1 receptor activation leads to [Ca2+]i changes that are mediated predominantly, if not exclusively, by calcium entry through L-type channels. These channels are critical for the initiation as well as the maintenance phases. This signaling pathway participates in the exaggerated response of SHR arteriolar SMC to AVP and may be responsible for the exaggerated renal reactivity observed in vivo. On the other hand, AT1 receptor coupling to calcium entry does not appear to be abnormal in renal arteriolar SMC; thus other factors (e.g., endothelium-derived vasoactive agents) may account for the exaggerated renal vascular reactivity to ANG II in vivo.
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METHODS |
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Isolation of preglomerular vessels. All experiments were performed on male SHR (Chapel Hill colony) averaging 6 wk of age with a body weight of 160-200 g. Age-matched WKY served as controls. Data about cardiovascular and renal function in these animals have been published previously (1, 11-15, 19, 20). Preglomerular afferent arterioles were isolated using an iron oxide-sieving technique previously described by Chatziantoniou and Arendshorst (12). Rats were anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg/kg body wt), and the abdominal aorta was cannulated below the renal arteries through a midline abdominal incision. For each experiment, the kidneys of two to three rats were perfused with ice-cold isotonic PBS (in mM: 125 NaCl, 17 K2HPO4, 3 NaH2PO4, and 5 MgCl2 at 4°C and pH 7.3) after ligation of the aorta above the renal arteries. Thereafter, the left renal vein was cut and the kidneys were perfused with ice-cold iron oxide suspension (1% Fe2O4). The kidneys were excised, decapsulated, and placed in ice-cold PBS.
Cortical tissue was minced with a razor blade and transferred to a tube containing 5 ml cold PBS. The tissue was homogenized in a Polytron homogenizer at a low speed (2 times for ~5 s each). Renal vessels, glomeruli, and surrounding tissue were removed from the crude homogenate with the aid of a magnet. The tissue was resuspended in PBS and injected three times through needles of decreasing size (22-23 gauge). The suspension was then filtered through a 100-µm sieve. The microvessels were recovered from the top of the sieve and added to PBS. The suspension contained microvessels and some attached tubular elements; glomeruli were rare. A magnet was used to remove microvessels and separate them from tubular fragments and cells. More than 95% of the vessels were free of glomeruli and tubular segments, primarily consisting of afferent arterioles with some interlobular arteries with diameters <50 µm. The microvessels were incubated with collagenase (1 mg/ml PBS, type 1A; Sigma, St. Louis, MO) for 30 min with constant shaking at 37°C. After collagenase digestion, the vessel-cell mixture was shaken vigorously to disburse iron oxide from the vessels and to disrupt the vessels. The free iron oxide was removed using a magnet, and the SMC were stored on ice.Measurement of [Ca2+]i. Measurements of [Ca2+]i in SMC were performed at room temperature using the calcium-sensitive fura 2-acetoxymethyl ester (AM) fluorescent dye as previously described (24, 28, 50). Cells were incubated with fura 2-AM (2 µM) mixed with Pluronic acid (0.02 µM; Molecular Probes, Eugene, OR) in PBS for 60 min at room temperature in the dark. The solution was centrifuged for 1-2 min, and the fura 2 solution was removed. The remaining pellet of cells was placed on ice, and, after 10-15 min, 300 µl of HEPES-buffered physiological salt solution (PSS, in mM: 135 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 5 D-glucose, and 10 HEPES at pH 7.3) was slowly added to the SMC over 2-3 min. The cells were stored on ice until measurements were made.
Measurements were performed on a 10-µl droplet of cell solution placed on a glass cover slip and centered in the optical field of a ×40 oil-immersion fluorescence objective of an inverted Olympus IX-70 microscope. The cells were excited alternatively with lights of 340 and 380 nm wavelength from a dual-excitation wavelength DeltaScan system equipped with dual monochronometers and a chopper (Photon Technologies). After signals were passed through a barrier filter (510 nm), fluorescence was detected by a photomultiplier tube; signal intensity was stored and processed by an IBM-compatible Pentium computer running PTI Felix software. Calibration of free calcium concentration was based on the ratio of 340/380 nm as originally described by Grynkiewicz et al. (24). Internal calibration of [Ca2+]i in our SMC was determined using ionomycin (2.5 × 10Statistical analyses. The data are presented as means ± SE. Sets of data were tested by ANOVA. A P value of <0.05 was considered statistically significant.
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RESULTS |
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The acute effects of ANG II and AVP were tested by the ability of these
agents to increase
[Ca2+]i
in SMC isolated from preglomerular arterioles of 6- to 8-wk-old WKY and
SHR during the developmental phase of hypertension. Figure 1 shows group averages for temporal
responses to stimulation of [Ca2+]i
by ANG II (106 M;
left) and AVP
(10
6 M;
right). The baseline
[Ca2+]i
during control conditions averaged 183 ± 5 nM in WKY
(n = 82) and 189 ± 7 nM in SHR
(n = 84)
(P > 0.6). After a 50-s control period, ANG II was added to the bathing medium to stimulate
[Ca2+]i.
ANG II receptor activation increased
[Ca2+]i
in a steplike fashion, reaching maximum values within 10 s. Thereafter,
[Ca2+]i
remained elevated at a plateau level that was sustained close to the
level of the initial response. This general response pattern was
observed in three of the four sets of experiments or protocols shown in
Fig. 1. The same general shape of the response pattern was observed
with ANG II in SMC from SHR and WKY (Fig. 1,
left) and with AVP in SMC from WKY
(Fig. 1, top
right). The one exception appeared
to be with AVP stimulation in SHR arteriolar SMC (Fig. 1,
bottom
right), as evidenced by a tendency
for calcium levels to rise through the stimulation period. However, as
seen in subsequent experiments, this tendency was not universal,
because AVP frequently produced a stable sustained increase in
[Ca2+]i
in SMC from SHR.
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Figure 2 shows the summarized data for the
effects of different concentrations of ANG II on
[Ca2+]i
in renal arteriolar SMC from WKY and SHR. Concentration-response relations to stimulation by ANG II
(1010-10
6
M) are evidenced by progressive stimulation of steady-state
[Ca2+]i
values. All tested ANG II concentrations produced significant increases
in
[Ca2+]i.
Stimulation by the two highest ANG II concentrations did not differ
from each other, suggesting maximum effects or saturation. The
responses to ANG II tended to be slightly greater in SHR than in WKY,
but the 21% strain difference was not statistically significant (P > 0.2).
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Similar experiments were performed using AVP as a receptor agonist. The
results presented in Fig. 3 demonstrate
that increasing amounts of AVP produced
[Ca2+]i
changes that were dose dependent. All three concentrations produced
significant increases in
[Ca2+]i
in SMC from both rat strains. A major finding was that the [Ca2+]i
increases were statistically greater in SHR than in WKY. This was the
case for all three concentrations evaluated. On the average, the SHR
responses to AVP were ~2-2.5 times greater for AVP
concentrations between 1010
and 10
6 M.
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Comparisons of SMC responsiveness in Fig. 4
revealed that ANG II and AVP produced similar increases in
[Ca2+]i
in WKY. For a given concentration, the agonist-induced
[Ca2+]i
changes did not differ between hormones tested. In marked contrast, SMC
of SHR exhibited roughly twofold larger responses to a given concentration of AVP compared with responses to the same amount of ANG
II. ANOVA of all of the SHR data revealed that the effects of AVP are
statistically greater than those of ANG II
(P < 0.01).
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Additional studies were conducted to identify the major receptor type
initiating ANG II and AVP effects in our freshly isolated renal
arterioles. The mediation of ANG II effects by the
AT1 receptors was evaluated using
the nonpeptide losartan. As Fig. 5 shows, this AT1 receptor antagonist
markedly attenuated the calcium response to ANG II when the inhibitor
was added concurrently with agonist at equimolar concentrations
(106 M). Losartan blocked
~55% of the ANG II-induced change in
[Ca2+]i
in SHR. In SMC from WKY, losartan reduced the calcium response by 70%.
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Figure 6 shows that almost all of the AVP
effect was mediated by the vascular
V1 receptor. The
V1 receptor antagonist used reduced the
[Ca2+]i
change elicited by AVP to a value not different from the prestimulation level (P > 0.4). This was true for
renal arteriolar SMC from SHR and WKY.
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Recent studies on preglomerular arteriolar SMC from Sprague-Dawley rats
revealed that a major pathway mediating
[Ca2+]i
changes is calcium entry through L-type calcium channels (28). To
evaluate the functional role of these channels during receptor stimulation, we tested responses to ANG II and AVP when calcium entry
was inhibited by nifedipine or a nominally calcium-free medium. The
results in Fig. 7 show that nifedipine had
a small nonsignificant effect on basal
[Ca2+]i.
On the average, nifedipine reduced baseline by 22 ± 6 nM
(P > 0.4) in the two WKY groups and
by 36 ± 8 nM (P > 0.4) in SHR. The data also demonstrate that during inhibition of calcium entry through dihydropyridine-sensitive channels, ANG II and AVP had no
discernible effect on
[Ca2+]i.
While WKY preparations were exposed to nifedipine,
[Ca2+]i was stable, with a slight rise of 10 ± 6 or 10 ± 7 nM, respectively (P > 0.5) in response to ANG II or
AVP. In SMC from SHR, ANG II and AVP produced small,
insignificant increases in
[Ca2+]i
of 32 ± 8 and 22 ± 7 nM, respectively
(P > 0.4) during nifedipine treatment. Likewise, no changes in
[Ca2+]i
were triggered by either hormone after the external solution was made
nominally calcium free by addition of EGTA just before (15 s) addition
of agonist (data not shown). As a negative control, neither ANG II nor
AVP had any effect on
[Ca2+]i
when the combination of nifedipine plus EGTA calcium-free medium was
tested.
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Other nifedipine studies were conducted to further evaluate the
involvement of dihydropyridine-sensitive calcium entry channels during
receptor activation. Basal
[Ca2+]i
was recorded for 50 s, and then SMC were stimulated with either AVP
(108 M) or ANG II
(10
8 M). Figure
8 shows that addition of nifedipine at 150 s during continued stimulation by receptor agonist completely reversed the response. The elevated
[Ca2+]i
was restored to basal or resting levels in SMC from SHR and WKY. Thus
there was no marked strain difference in calcium signaling mechanisms
or in the near-complete dependence on calcium entry through L-type
channels. Similar poststimulation reversal was observed when the
external solution was made nominally calcium free by addition of EGTA
at 150 s (data not shown).
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DISCUSSION |
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The present study provides new information about calcium signaling in SMC freshly isolated from preglomerular resistance arterioles of SHR of the Okamoto-Aoki strain. A major finding is the enhanced [Ca2+]i response in SMC of SHR during AVP stimulation. All concentrations of this vasoconstrictor hormone tested produced twofold greater [Ca2+]i increases in 7-wk-old SHR than in age-matched WKY. Interestingly, the exaggerated response is specific to AVP, because there is no strain difference in the ANG II-induced calcium responses recorded in identical preparations of dispersed arteriolar SMC. Both vasoconstrictor agents produced dose-dependent increases in [Ca2+]i that are maintained close to the initial peak level throughout the period of continued stimulation. This is the case for renal arteriolar SMC from young WKY and SHR. These findings confirm and extend our earlier observations on ANG II and AVP actions on renal arteriolar SMC of Sprague-Dawley rats isolated using the same methodology (28). The use of specific receptor antagonists indicates that ANG II and AVP primarily cause changes in [Ca2+]i after activation of AT1 and V1 receptors, respectively. Essentially all of the calcium responses are mediated through L-type voltage-gated calcium channels. The dihydropyridine calcium channel antagonist nifedipine, introduced before or during receptor activation, completely inhibited [Ca2+]i changes elicited by either hormone. A strong dependence on a calcium entry mechanism is also supported by the observations that the short-term use of EGTA to render the medium nominally calcium free completely inhibited the [Ca2+]i response to receptor activation. Like nifedipine, this was the case when EGTA was introduced either just before or during agonist stimulation. It is noteworthy that responses in renal arteriolar SMC from WKY are qualitatively and quantitatively similar to those observed in renal SMC from normotensive Sprague-Dawley rats (28). Thus the abnormality identified in the present study pertains to SMC from preglomerular resistance vessels of animals with genetic hypertension compared with the same preparation of SMC from two strains of normotensive rats. Our AVP results implicate a primary abnormality in V1 receptor function and/or postreceptor signal transduction in afferent arteriolar SMC.
The work of other laboratories supports the view that increased renal
vascular resistance and reactivity to vasoconstrictors such as ANG II,
AVP, and sympathetic nerve stimulation are major abnormalities in
isolated perfused kidneys of 4- to 7-wk-old stroke-prone SHR compared
with WKY (3, 16). The exaggerated vascular reactivity becomes greater
with age and the duration of hypertension. We previously found that
both AVP and ANG II produced exaggerated renal vasoconstriction in vivo
in 7-wk-old SHR relative to age-matched normotensive rats of WKY and
Sprague-Dawley strains (11, 13-15, 19, 20). There is evidence
suggesting that this strain difference in vascular reactivity to ANG II
is more readily evident in the renal than in the mesenteric vascular
bed at the age of 4 wk (30). Studies on isolated rabbit vessels
demonstrate that AVP contracts afferent arterioles in a dose-dependent
manner, with 108 M
producing a half-maximal response (49). Relatively small amounts of AVP
constrict the afferent arterioles of the rat juxtamedullary nephron
preparation and reduce blood flow in descending vasa recta in the outer
medulla (25). In addition, higher concentrations of AVP are required to
constrict interlobular arteries and efferent arterioles feeding
juxtamedullary nephrons. With regard to ANG II effects on the renal
vasculature, recent studies document the presence of ANG II receptors
in preglomerular resistance vessels (12). Functional studies establish
that ANG II can contract the afferent as well as the efferent
arterioles in vivo and in vitro (see Ref. 38). The extent to which ANG
II increases renal vascular resistance depends in part on local
production of paracrine agents such as PG and endothelial factors (11,
14, 15, 39). It is noteworthy that different calcium signaling
mechanisms are thought to operate in pre- and postglomerular
arterioles. The ANG II-induced contraction of afferent arterioles
displays a very strong dependence on calcium entry through
voltage-gated calcium channels, whereas efferent arteriolar responses
to ANG II are largely independent of L-type channels (9, 35, 39).
Chloride channels are thought to mediate calcium changes in the
preglomerular arterioles during ANG II-induced contraction, whereas
they do not appear to be involved in postglomerular arterioles (8, 32,
39).
In isolated mesenteric arteries, AVP causes a greater contractile response in SHR than in WKY between 8 and 16 wk of age (7). Other investigators report normal mesenteric arterial reactivity to AVP of 4-wk-old SHR, with increased reactivity after 8 wk of age (33). In another perfusion study of mesenteric arteries, 10-wk-old SHR exhibited an enhanced vasoconstriction during AVP infusion compared with WKY responses. The strain-dependent increase in reactivity appeared to be specific for AVP, because no pressor response was observed during ANG II infusion (43). In this vessel type, postreceptor events are thought to mediate enhanced inositol 1,4,5-trisphosphate (IP3) production, [Ca2+]i responses, and contraction of isolated mesenteric vessels from SHR at the ages of 8, 12, and 16 wk (33). It is interesting to note that the response to AVP in mesenteric arteries is abolished in a calcium-free medium (7). Increased vasoconstriction is also elicited by AVP infusion into the mesenteric vascular bed of Goldblatt hypertensive rats (34); however, the effect in this model of hypertension is thought to be independent of AVP receptor density. AVP is reported to have equal effects on cultured aortic SMC, mesenteric arterial SMC, and mesangial cells from 6- to 7-wk-old SHR and WKY in terms of increasing [Ca2+]i or intermediates such as IP3 and diacylglycerol (38, 41). These results in prehypertensive animals or young animals with relatively mild hypertension suggest that primary changes in the SMC responsible for pressure regulation are more related to functional abnormalities than to structural adaptations or remodeling secondary to high intravascular pressure. Structural changes of the vessel wall appear to be more prevalent and may play a larger role in vascular reactivity in older animals with established hypertension (21, 37).
Our earlier in vivo blood flow studies demonstrated that ANG II and AVP produced exaggerated renal vasoconstriction in 6- to 8-wk-old SHR compared with normotensive controls (11, 13-15, 19, 20). The reasons for the exaggerated responses appear to differ between these peptides. With regard to ANG II, indomethacin administration amplifies the ANG II-induced renal vasoconstriction in WKY while having virtually no effect in SHR, indicating weaker anticonstrictor buffering action of endogenous vasodilatory cyclooxygenase metabolites in kidneys of young hypertensive animals (13). This view is reinforced by the finding that intrarenal infusion of endothelium-derived PG such as PGI2 and PGE2 more effectively attenuates ANG II-induced renal vasoconstriction in WKY (11, 14). Subsequent studies conducted in Chapel Hill reveal that AVP injection into the renal artery produced twofold larger reductions in renal blood flow in 7-wk-old SHR versus age-matched WKY (19, 20). In contrast to the stimulation with ANG II, however, endogenous cyclooxygenase products had relatively small effects on AVP-induced renal vasoconstriction and there was a significant strain difference in the vascular response to AVP during as well as before cyclooxygenase inhibition (19). To explain the exaggerated AVP effect in SHR, we proposed a difference in AVP vascular V1 receptor density and/or in a postreceptor signal transduction step. Follow-up blood flow studies were conducted to evaluate the contributions of calcium signaling pathways to AVP-induced renal vasoconstriction in vivo (20). The results of this study show that although a given dose of AVP elicited more pronounced renal vasoconstriction in SHR, the relative contributions of calcium entry and mobilization were similar on a fractional basis. The results clearly demonstrate similar degrees of participation of calcium entry antagonized by the calcium channel antagonist nifedipine and of calcium mobilization inhibited by 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester interference with an IP3-mediated step. The relative contributions of calcium entry and mobilization to AVP effects on total renal vascular resistance do not differ between 7-wk-old SHR and WKY when the initial responses to AVP are normalized (20). These observations suggest that the strain difference in renal vascular reactivity to AVP observed earlier in vivo and in the present in vitro study on arteriolar SMC can be explained by an increased receptor density of V1 receptors in young SHR (2). There is no need to postulate an abnormality in postreceptor signaling pathways coupled to the vascular V1 receptor.
In the present study of renal arteriolar SMC, a calcium entry mechanism(s) plays a major role in the response to either ANG II or AVP receptor activation. Calcium entry through L-type channels is the predominant pathway, as evidenced by marked inhibition of agonist effects by pretreatment with nifedipine or by poststimulation reversal by adding nifedipine to the responsive cells. It is noteworthy that the concentration of nifedipine used exhibited no autofluorescence and that the resting calcium values are not affected by nifedipine. In marked contrast, nifedipine abolished the stimulatory effect of the agonists ANG II and AVP. Similar results are obtained when a different calcium channel blocker, verapamil, is added either before or after agonist-induced stimulation (S. K. Fellner and W. J. Arendshorst, unpublished observations). In addition, a nominally calcium-free medium prevents a calcium response to either ANG II or AVP. Collectively, our results suggest that in the fresh cell preparation, calcium signaling pathways differ appreciably from the mechanisms thought to be operating in cultured renal arteriolar cells, in which there is a combination of calcium entry and mobilization (51). Other investigators have presented evidence for calcium entry as a major mechanism in freshly isolated preglomerular arteriolar SMC of normotensive rats (27). The precise explanation for the difference in the relative roles of calcium entry versus calcium mobilization in freshly isolated versus cultured SMC is not clear. One possibility is that the SMC undergo phenotypic changes while proliferating in culture conditions such that release from sarcoplasmic reserves becomes a dominant mechanism. Another possibility is that response of SMC from small-diameter terminal arteriolar resistance vessels differs from those of SMC from the aorta, a conduit vessel. In this regard, the AVP-induced vasoconstriction of mesenteric arteries is reported to be strongly dependent on calcium entry and is abolished when the medium is calcium free (7). The reason(s) for the differences between preparations is worthy of future investigation.
Most of our background knowledge of intracellular signaling mechanisms in SMC derives from studies of cultured SMC from the aorta or other conduit vessels or cultured mesangial cells (23, 36, 44, 45, 48). Most of these reports indicate that AVP and ANG II elicit contraction of cultured SMC by means of mobilization of calcium from an intracellular sequestration pool. The transient increase in [Ca2+]i can be inhibited by maneuvers designed to deplete internal calcium stores or to inhibit IP3-induced release of calcium from intracellular stores (49). Calcium influx plays a relatively minor role in cultured cells, because the responses elicited by receptor agonists are largely independent of calcium channel antagonists or removal of external calcium (4, 10, 26, 36). A small calcium entry phase may be associated with a plateau phase during which [Ca2+]i is maintained slightly above basal values; the sustained increase disappears after blockade of calcium channels with nifedipine or verapamil treatment.
With regard to the actions of AVP, the fact that a V1 receptor antagonist produces complete inhibition of the calcium response in the SMC from afferent arterioles from WKY and SHR extends our earlier observations for freshly isolated renal SMC from Sprague-Dawley rats (28). This observation is consistent with a relatively pure vascular preparation devoid of tubular epithelial cells with V2 receptors. There is convincing evidence establishing V1 receptor as the dominant receptor type in fresh preparations of rat afferent arteriolar (2, 19) and cultured human and rat aortic SMC and in cultured rat mesangial cells (4, 26, 36, 44, 45, 48). Our present studies localize the major defect in vascular reactivity to AVP to individual vascular SMC of preglomerular arterioles studied ex vivo. Thus the primary action of AVP is on arteriolar SMC, and there is no need to propose more complex interactions with different cell types to account for the enhanced renal vascular reactivity in young SHR. The isolation and purification of SMC minimizes possible interactions with the vascular endothelium and glomerular mesangial cells as well as tubular cells associated with the juxtaglomerular apparatus. The calcium responses we observed in individual and groups of dispersed renal SMC make a strong case for a primary functional defect distinct from vessel wall thickness or structure in young SHR (21, 37). Our present results of enhanced reactivity to AVP in hypertensive animals can be explained by our recent finding that AVP receptor density in preglomerular resistance vessels is two to three times higher in 7-wk-old SHR than in WKY (2). Cultured aortic SMC obtained from 12- to 13-wk-old SHR have two times the number of AVP receptors relative to SMC from WKY (40).
Concerning ANG II effects, the proposed mechanism focuses on reactivity to PG intermediates produced by endothelial cells and their buffering action mediated by receptor-Gs protein interaction (15). The lack of strain differences in the ANG II effect on [Ca2+]i in our freshly isolated preglomerular arteriolar SMC increases the probability that the defect in vascular reactivity to ANG II resides in other cell types or interactions present in vivo but not in vitro. Our present findings argue against a major strain difference in receptor-induced calcium signaling in preglomerular SMC. Such conclusions are consistent with and complementary to our previous radioligand binding studies of ANG II receptors demonstrating that neither ANG II receptor density nor affinity differs between preglomerular arteriolar SMC freshly isolated from 7-wk-old SHR and WKY (12). In addition, previous studies from our laboratory indicate up to 80% of ANG II binding in vitro to freshly isolated preglomerular SMC and ANG II effects on renal vascular resistance in vivo are mediated primarily by AT1 receptors, with a small percentage of the binding and effects resulting from a non-AT1 receptor that is sensitive to PD-123319 but not CGP-42112 (12). It is noteworthy that there were no differences between preglomerular microvessels isolated from 7-wk-old SHR and WKY. In a recent study, we observed that the AT1 receptor blocker candesartan inhibits 88% of the calcium response to ANG II in preglomerular SMC from young SHR (29). On the other hand, other investigators have reported an enhanced calcium response to ANG II in cultured aortic and mesenteric arterial SMC from SHR compared with WKY at 7, 9, and 17 wk of age (6, 46, 47). The larger responses were noted in SMC of primary culture and up to the fifth subpassage. Greater calcium turnover is suggested in SHR.
In conclusion, our study provides new information about freshly isolated SMC from afferent arterioles, demonstrating that AVP produces a significantly larger [Ca2+]i increase in SHR developing genetic hypertension compared with renal SMC of normotensive WKY. In contrast, ANG II had similar stimulatory effects in renal arteriolar SMC from 7-wk-old SHR and WKY. The greater response to AVP was unique to SHR. There was no strain difference in the calcium responses to AVP and ANG II in WKY. Also, WKY responses were similar to those observed previously for preglomerular arteriolar SMC from normotensive Sprague-Dawley rats. Almost all of the [Ca2+]i increase was mediated by calcium entry mechanism through voltage-gated L-type channels, with little evidence of appreciable calcium mobilization during the experimental conditions. The effects of ANG II and AVP on [Ca2+]i can be explained by a similarity of ANG II AT1 receptor density and efficiency of signal transduction in renal SMC from SHR and WKY. The action of AVP may reflect an increased number of V1 receptors or increased postreceptor signaling in arteriolar SMC of young SHR. Our current observations of exaggerated [Ca2+]i responses to AVP in vitro in isolated SMC from renal resistance vessels extend our earlier finding of enhanced renal vascular reactivity to AVP in rats during the developmental phase of genetic hypertension.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Research Grant HL-02334.
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FOOTNOTES |
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A preliminary report of portions of this material was made in abstract form (27a) and in presentations at the 30th Annual Meeting of the American Society of Nephrology, San Antonio, TX, November 1997.
The sabbatical leave of B. M. Iversen was supported by the Research Council of Norway.
Current address of B. M. Iversen: Renal Research Group, Medical Department A, University of Bergen, Haukeland University Hospital, Bergen N-5021, Norway.
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: W. J. Arendshorst, Department of Cell and Molecular Physiology, CB no. 7545, Rm. 152 Medical Sciences Research Building, University of North Carolina Chapel Hill, Chapel Hill, NC 27599-7545 (E-mail: arends{at}med.unc.edu).
Received 11 February 1998; accepted in final form 22 October 1998.
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