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(Hypertension. 1997;30:1223-1231.)
© 1997 American Heart Association, Inc.
Articles |
Calcium Signaling Mechanisms in Renal Vascular Responses to Vasopressin in Genetic Hypertension
From the Department of Physiology, University of North Carolina at Chapel Hill.
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Abstract |
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Abstract Previous blood flow studies demonstrated that arginine vasopressin (AVP) produces exaggerated renal vasoconstriction in young spontaneously hypertensive rats (SHR) compared with Wistar-Kyoto control rats (WKY). The purpose of the present study was to determine the role of postreceptor calcium signaling pathways in AVP-induced renal vasoconstriction in vivo. Renal blood flow (RBF) was measured by electromagnetic flowmetry in anesthetized, water-loaded, 8-week-old WKY and SHR pretreated with indomethacin to avoid interactions with prostaglandins. AVP was injected into the renal artery to produce a transient 25% to 30% decrease in RBF without affecting arterial pressure. To achieve similar control levels of vasoconstriction, SHR received a lower dose (2 versus 5 ng). Coadministration of nifedipine with AVP produced dose-dependent inhibition of the AVP-induced renal vasoconstriction. Nifedipine exerted maximum inhibition by blocking 30% to 35% of the peak AVP response, indicating the involvement of dihydropyridine-sensitive voltage-dependent calcium channels. To evaluate intracellular calcium mobilization, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) or heparin was coadministered with AVP. Each agent produced a dose-dependent inhibition of up to 65% of the maximum blood flow change produced by AVP. The degrees of inhibition produced by maximum effective doses of nifedipine and TMB-8 were additive; the combination blocked up to 85% of the response to AVP. These observations indicate that about one third of the AVP-induced constriction of renal resistance vessels is mediated by voltage-dependent L-type calcium channels responsive to the dihydropyridine nifedipine. Approximately two thirds of the change in vascular tone is due to inositol 1,4,5-trisphosphate–mediated calcium mobilization from intracellular sources sensitive to TMB-8 and heparin. The results suggest that the exaggerated renal vascular reactivity to AVP challenge in SHR is probably not due to a strain difference in postreceptor calcium signal transduction. After AVP receptor stimulation, calcium mobilization and calcium entry signaling pathways participate to similar degrees in WKY and SHR.
Key Words: renal circulation • arterioles • calcium channels • nifedipine • heparin • rats, inbred SHR
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Introduction |
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In essential hypertension, increased peripheral resistance and elevated arterial pressure are causally related to an increase in the tone and reactivity of vascular smooth muscle cells. Cross-transplantation studies provide strong evidence that the kidneys play a central role in the pathogenesis of genetic hypertension in the SHR.1 Abnormalities in renal hemodynamics are evident during the development of hypertension in young SHR. Renal vascular resistance is increased and glomerular filtration rate and RBF are reduced, compared with normotensive WKY.2 The reason for the elevated renal vascular resistance is not completely understood, although a combination of defects is probably involved. Vascular reactivity to constrictor agents such as Ang II, AVP, and thromboxane is increased, whereas the buffering effects of vasodilator prostanoids, such as PGE2 and PGI2, and dopamine are attenuated.3 4 The mechanisms responsible for the abnormalities may vary among vasoactive agents, receptor systems, and postreceptor second-messenger pathways.
Earlier studies by our laboratory have shown that young SHR exhibit almost twice the renal vascular response to Ang II as compared with WKY.3 This strain difference is primarily due to the counteracting effects of prostanoids in WKY. The vascular response to Ang II increases in WKY but is unchanged in SHR after inhibition of prostaglandin synthesis.3 Also, administration of PGE2 or PGI2 into the renal circulation effectively attenuates the vasoconstriction elicited by Ang II in WKY but is without effect in young SHR.4 Subsequent studies implicate weak coupling of the prostanoid receptors to stimulatory Gs proteins and the cAMP pathway in young SHR.5 On the other hand, the exaggerated reactivity to AVP observed in the renal microcirculation of young SHR appears to be related to a different defect, one that is predominantly independent of cyclooxygenase.6 The SHR responses to stimulation of the vascular AVP V1 receptors are exaggerated whether or not indomethacin is administered to inhibit cyclooxygenase and prostaglandin production. Thus, the abnormal effects of AVP on renal resistance vessels of SHR are probably mediated by a strain difference in V1 receptor function, or postreceptor second-messenger systems, or both.
A variety of postulates have been advanced to explain increased vascular reactivity in models of hypertension.7 Large changes in [Ca2+]i are most likely to mediate exaggerated responses of the most physiologically relevant resistance vessels to constrictor agents. Several intracellular signaling pathways can lead to changes in [Ca2+]i that are a major determinant of contractile tone and vascular reactivity. However, there is little definitive information about the functional role of the key mechanisms in peripheral resistance vessels in the various models of hypertension. After receptor activation, vasoconstrictors usually increase [Ca2+]i by one or more systems, including calcium influx from extracellular sources through calcium channels in the plasma membrane and/or release of intracellular calcium from sequestered pools such as the sarcoplasmic reticulum.
With regard to the mechanism or mechanisms by which AVP produces vasoconstriction, most of our present knowledge is based on in vitro studies of cultured vascular smooth muscle cells derived from large-diameter vessels such as the aorta. These studies suggest that AVP may exert its vascular effects by activating V1 receptors that stimulate phospholipase C activity in a pertussis toxin–insensitive, GTP-dependent manner, which leads to hydrolysis of membrane phosphoinositides to increase IP3 and diacylglycerol production. Diacylglycerol can activate protein kinase C, and IP3 can activate a receptor on the sarcoplasmic reticulum to promote calcium release into the cytosolic compartment.8 9 Similar signaling pathways appear to be involved in the AVP effects on cultured glomerular mesangial cells.10 11 12 13 It is noteworthy that these in vitro studies on cultured cells suggest a strong reliance on calcium mobilization that is almost exclusively independent of calcium concentration in the external bathing medium. Furthermore, calcium signaling in these cultured cells is not affected by voltage-gated calcium entry channel blockers of the dihydropyridine class.12 14 15 16 17 18
The extent to which these cellular mechanisms can be extrapolated to in vivo conditions to apply to small-diameter resistance vessels awaits further investigation and clarification. The few animal studies conducted to date point to important functional differences. With regard to AVP effects on renal vascular responses in vivo, there is a prevalent dependence on calcium entry as revealed by significant inhibitory effects of calcium channel antagonists such as nifedipine and verapamil.19 20 21 One study also tested the importance of calcium mobilization in the dog renal circulation and found that AVP-induced renal vasoconstriction was mediated by a combination of calcium entry and mobilization.21 To the best of our knowledge, there is no information available about the relative contributions of calcium entry and intracellular calcium release in the renal vasoconstriction produced by AVP in vivo in normotensive or hypertensive animals.
The purposes of the present study were to investigate calcium signaling mechanisms in renal vascular responses to AVP in vivo and to gain insight into whether the enhanced vascular reactivity to AVP in SHR is associated with strain differences in postreceptor signaling pathways that involve calcium metabolism. A bolus of AVP was injected into the renal artery, and the relative contributions of calcium entry and mobilization pathways were evaluated in 8-week-old SHR and WKY. Nifedipine was used to determine the relative importance of extracellular calcium entry through dihydropyridine-sensitive, voltage-dependent calcium channels. Calcium mobilization was assessed by intrarenal administration of TMB-8 and heparin. Our results provide new evidence demonstrating that a calcium entry pathway inhibited by nifedipine accounts for about one third and a separate system of IP3-mediated mobilization sensitive to TMB-8 and heparin are responsible for two thirds of the AVP-induced renal vasoconstriction. The observation that these relative proportions of these major signal transduction systems do not differ between 8-week-old SHR and WKY suggests that similar postreceptor calcium signaling pathways operate in renal resistance vessels in vivo.
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Methods |
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Experiments were performed on 8-week-old anesthetized WKY and SHR obtained from our Chapel Hill breeding colony. The animals were maintained on a standard rat chow diet and tap water ad libitum. They were deprived of food but not water the night before an experiment. Anesthesia was induced by an intraperitoneal injection of sodium pentobarbital (65 mg/kg body wt), and the animals were placed on a servo-controlled heating table that maintained body temperature at 37°C. A tracheostomy was performed to facilitate free breathing. A carotid artery was cannulated to obtain blood samples and to monitor arterial pressure (Statham P23Db transducer). A jugular vein was cannulated for the administration of maintenance infusions, cyclooxygenase inhibitor, and supplemental doses of sodium pentobarbital. Isoncotic bovine serum albumin (47 g/L) was infused initially at a rate of 50 µL/min to replace losses associated with surgery (1.25 mL/100 g body wt) and then at 10 µL/min for the duration of an experiment to maintain hematocrit and plasma protein concentration at presurgical levels.2 3 To minimize time-dependent changes in AVP responses, 6% dextrose (100 µL/min) and 0.9% NaCl (50 µL/min) were infused intravenously throughout an experiment. These infusions effectively reduced urine osmolality to a level of 250 to 300 mOsm/kg H2O, which remained stable throughout an experiment.6 Midline and subcostal incisions were used to expose the abdominal aorta and left kidney. A noncannulating electromagnetic flow probe (1.5 mm circumference, Carolina Medical Electronics) was placed around the left renal artery to measure RBF. A tapered and slightly curved PE-10 catheter was introduced into the left femoral artery and advanced through the aorta until its tip was positioned approximately one mm into the left renal artery.3 4 5 6 Placement of the catheter in the renal artery did not affect RBF. The renal arterial catheter was used for the local administration of AVP and antagonists of calcium entry and mobilization. After completion of the surgical preparation, indomethacin (5 mg/kg body wt) was administered intravenously to inhibit cyclooxygenase activity and to minimize interactions between AVP and vasodilator prostaglandins.6 A previous study indicated that this dose of indomethacin produces a 60% to 80% decrease in the rate of urinary PGE2 excretion for at least 3 hours, as measured by radioimmunoassay.3 The animals were allowed to stabilize for 0.5 to 1 hour before starting the measurements.
Throughout an experiment, a continuous infusion (5 µL/min) of isotonic saline containing dilute heparin (30 U/mL) was administered via the renal artery catheter. One minute before the administration of drug, the rate of saline infusion was increased to 120 µL/min so that the entire bolus of drug was delivered to the kidney within 5 seconds. A Cheminert sample injection valve was used to introduce a 10-µL bolus into the infusion line. Previous studies have indicated the bolus reached the kidney 12 seconds after its introduction into the infusion line.3 4 5 6 After recovery of RBF to its baseline level (3 minutes), the infusion rate was returned to 5 µL/min.
The following drugs were used: AVP (from Sigma), nifedipine (from Biomol), and TMB-8 (from Biomol), heparin sodium (from Elkins-Sinn), and indomethacin (from Sigma). We used nifedipine, a 1, 4-dihydropyridine calcium channel antagonist of high affinity, to reversibly inhibit calcium entry through dihydropyridine-sensitive L-type channels.22 Stock nifedipine was mixed in dimethyl sulfoxide and diluted in water immediately before use. TMB-8 was diluted in water and used to inhibit cellular mobilization by interfering with IP3-induced calcium release from the sarcoplasmic reticulum without affecting calcium influx from the extracellular compartment.23 24 Heparin also was used to antagonize IP3 binding to its receptor and IP3-induced calcium release from intracellular reservoirs.25 26
AVP was injected into the left renal artery at a dose of 2 or 5 ng. As shown in our previous report, a 5 ng AVP dose produces exaggerated renal vasoconstriction response in SHR compared with WKY.6 In most experiments, equally proportionate levels of renal vasoconstriction were achieved by injecting a lower dose of AVP into SHR (2 ng) than into WKY (5 ng). In the control period these amounts of AVP produced a reversible, transient 25% to 30% decrease in RBF in both strains. The effects of nifedipine, TMB-8, or heparin were evaluated in the experimental period. The role of voltage-dependent L-type calcium channels and IP3-mediated calcium mobilization from intracellular sources in AVP-induced renal vasoconstriction was evaluated by injecting AVP as a mixture with nifedipine or during intrarenal infusion of TMB-8 or heparin. The time interval between injections was 5 to 10 minutes. The order of inhibitor dose was randomly selected each day. None of the doses of inhibitor alone had an effect on baseline arterial pressure or RBF. Preliminary studies established that vehicle solutions had no effect on basal RBF or responses to AVP. To avoid treatment interactions, a single calcium pathway inhibitor was used in each rat except when the combined effects of two inhibitors were evaluated by design. To assess whether consecutive injections of an antagonist created an additive buffering effect, AVP was injected alone at the beginning, middle, and end of an experiment. In all cases, AVP produced a similar decrease in RBF.
The data acquisition system consisted of an IBM-compatible computer and a 12-bit analog/digital converter.3 The flow probe was interfaced to the data acquisition system using a Carolina Medical Electronics 500 electromagnetic flowmeter. A Hewlett-Packard 8805B carrier amplifier was used for the pressure sensor interface. The outputs of the transducers monitoring arterial pressure and RBF were sampled at a rate of 100 samples/sec for a period of 3 minutes, which usually was sufficient to allow flow to return to its baseline value after each injection of AVP. Each recording was started when AVP was introduced into the renal artery. Consecutive blocks of 100 data points were averaged to obtain second-by-second estimates of RBF and arterial pressure, and these averages were used to calculate second-by-second estimates of renal vascular resistance. The RBF, arterial pressure, and renal vascular resistance values were normalized and expressed as a percentage of baseline values. The baseline was calculated separately for each injection using the mean values of the corresponding variables observed during the time between the introduction of AVP into the infusion line and the onset of the renal vascular response. Plots of normalized arterial pressure, RBF, and renal vascular resistance as a function of time were prepared using the SigmaPlot scientific software package.
Nonlinear regression equation was given a best-fit to each blood flow recording as previously described.3 Once the best-fit curve had been estimated for each recording, it was used to calculate the maximum response, the time required to reach the maximum response, and the half-times for the constriction and recovery. These calculated data were used for the subsequent analysis of the results.
Statistical analyses were performed using ANOVA. This method permits simultaneous hypotheses to be tested regarding both categorical and quantitative variables, and represents the multivariate equivalent of an ANCOVA. Values of P<.05 were considered statistically significant. All values reported are mean±SE.
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Results |
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Basal renal hemodynamic data are summarized in Table 1
. The SHR were moderately
hypertensive compared with WKY control animals at 8 weeks of age. The
difference in mean arterial pressure was accompanied by
reduced RBF and elevated renal vascular resistance during the
water-loading state, in agreement with previous observations for
euvolemic conditions.3 4 5 6
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As has been observed previously, injection of AVP (5 ng) into the renal
artery produced exaggerated renal vasoconstriction in SHR as compared
with control responses in age-matched WKY.6 Because of the
strain differences in response to AVP, initial studies on SHR were
conducted by using not only the same dose of AVP (5 ng in SHR and WKY)
but also a lower dose of AVP (2 ng in SHR) so that the levels of renal
vasoconstriction were similar in the two strains of rats during control
conditions. As is shown in Fig 1, the
common AVP dose (5 ng) produced a greater maximum reduction in RBF in
SHR (42±8% versus 27±5%, P<.001). The absolute as well
as the percent changes in blood flow were larger in SHR (2.7±0.4
versus 2.1±0.3 mL · min-1 · g
kidney wt-1, P<.01). SHR receiving
the lower AVP dose (2 ng) responded with a transient, 25±8%, maximum
reduction in RBF, a level approximating that elicited in WKY by 5 ng
AVP (Fig 1).
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To determine the involvement of voltage-gated calcium channels, the
dihydropyridine nifedipine was injected
in combination with AVP in eight animals. Concurrent administration of
nifedipine (150, 750, and 1500 ng) with the AVP attenuated
the vasoconstrictor effect of AVP in a dose-dependent manner. As is
presented in Fig 1
, the two highest doses of
nifedipine produced similar degrees of blockade, suggesting
maximum inhibition of voltage-operated calcium entry channels during
these conditions. The highest nifedipine doses effectively
buffered the AVP-induced renal vasoconstriction, reducing the blood
flow changes produced by 5 ng AVP from 27±5% to 18±6% in WKY
(P<.01) and 42±8% to 27±4% in SHR (P<.001).
SHR responses to 2 ng AVP were attenuated by nifedipine,
from 25±8% to 16±4% (P<.001) (Figs 1 and 2, see left panel of Fig 2). The latter
results are similar to those recorded in WKY (Figs 1 and 2, see
left panel of Fig 2). In all three groups, the maximum inhibition
produced by nifedipine was about 35% of the maximum
AVP-induced renal vasoconstriction (Fig 2, right panel). It is
noteworthy that this maximum degree of blockade was similar even though
different doses of AVP were used in SHR. None of the
nifedipine doses used had an effect on basal RBF when
injected alone.
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In another group of eight animals, we evaluated the role of calcium
mobilization stimulated by the phospholipase C and IP3
pathway in the renal vascular response to AVP. Increasing doses of
TMB-8 were infused into the renal artery for 2 minutes to inhibit
AVP-induced IP3-dependent calcium release from
intracellular stores. The summarized results show that TMB-8
effectively reduced AVP-induced renal vasoconstriction in a
dose-dependent fashion. As is shown in the left panel of Fig 3, the two highest doses of TMB-8 blocked
a significant portion of the renal vasoconstriction produced by AVP.
RBF responses to 5 ng AVP were reduced from 26±5% to 9±2% in WKY
(P<.001) and from 43±8% to 18±4% in SHR
(P<.001). In SHR that received the lower dose of AVP (2
ng), decreases in RBF were attenuated by TMB-8 from 25±6% to 9±3%
(P<.001), a level almost identical to that recorded in
WKY. As noted earlier for nifedipine, the fractional effect
of TMB-8 was similar in the three groups of animals (Fig 3, right
panel). TMB-8 inhibited about 60% of the maximum response to AVP in
SHR receiving either AVP dose, as well as WKY. Thus, the degree of
maximum inhibition was similar in SHR and WKY even though different AVP
doses were used. These observations indicated that a majority of
AVP-induced renal vasoconstriction involves an intracellular calcium
mobilization mechanism.
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To test whether these two identifiable calcium signaling mechanisms
acted independently or shared common calcium steps in mediating the
renal vascular responses to AVP, the combined effect of
nifedipine and TMB-8 was evaluated. We selected a dose of
each inhibitor that exerted maximum inhibition in the
previous studies described above. For these studies we started with
similar control responses that were achieved by 5 ng AVP in WKY and 2
ng AVP in SHR. As shown in Fig 4, administration of the two inhibitors together reduced the
decrease in RBF elicited by AVP, from 27±5% to 6±3% in WKY
(P<.001) and 25±8% to 5±2% in SHR (P<.001).
No strain difference was noted. The combined inhibition averaged
21±9% in WKY and 18±8% in SHR (P>.05). The mixture of
nifedipine and TMB-8 produced significantly greater
inhibition than that observed when each inhibitor was given
alone (P<.01 for both). These results support the notion
that at least two different calcium pathways are involved in the
activation of renal resistance vessels in response to AVP
stimulation.
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To confirm further the role of calcium mobilization throughout the
IP3 pathway in the AVP-induced response, other experiments
were conducted using a chemically dissimilar agent, heparin, to inhibit
AVP-induced renal vasoconstriction. Heparin was infused into the renal
artery for 2 minutes in increasing doses to block
IP3-mediated calcium release from intracellular stores in
response to AVP. The summarized blood flow results for these
experiments are shown in Fig 5. Although
heparin by itself had no effect on baseline renal
hemodynamics or arterial pressure, it
effectively attenuated AVP-induced vasoconstriction in a dose-dependent
fashion. The highest doses of heparin attenuated the maximum decreases
in RBF, from 29±8% to 11±4% in WKY (P<.001) and from
24±7 to 10±3% in SHR (P<.001). As Figs 3 and 5 show, the
magnitude of the effects of heparin and TMB-8 on AVP-induced renal
vasoconstriction was similar. These results confirm our earlier
findings obtained with TMB-8 that indicated that an intracellular
calcium mobilization mechanism mediates about two thirds of the
AVP-induced renal vasoconstriction.
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Additional studies were performed to assess the specificity of action
of the putative inhibitors of calcium mobilization. Our
earlier results established a dose-response relationship between TMB-8
inhibition and AVP-induced renal vasoconstriction in some animals and
of heparin antagonism in others. High doses of these two potent
inhibitors of IP3-mediated calcium mobilization
were tested in a new group of animals to determine whether they were
acting by one or several mechanisms. The results in Fig 6 indicate that the apparent maximum
inhibitory effects of TMB-8 and heparin on AVP-induced
renal vasoconstriction were no different whether the agents were given
alone or in combination. Each agent antagonized 60% to 64% of the AVP
effect on blood flow as compared with 68% inhibition by the two
antagonists administered together in the same animals.
Clearly, the mean responses were similar and thus not additive,
supporting the view that TMB-8 and heparin act primarily via a common
site or mechanism of action to influence calcium mobilization.
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Further experiments were conducted to provide evidence that the
inhibitors of calcium mobilization we used had no
additional effect on signaling by acting on calcium entry through a
voltage-gated L-type channel. For this purpose, BAY K8644 was injected
into the renal artery to produce renal vasoconstriction by activation
of dihydropyridine-sensitive, calcium entry
channels. Responses to BAY K8644 were recorded before and during
intrarenal infusion of an inhibitor of calcium
mobilization. The results shown in Fig 7
clearly demonstrate that the 45% decrease in RBF elicited by BAY K8644
injection into the renal artery was unaffected by either TMB-8 or
heparin treatment. Thus, the agents used appear to be selective in
their action on calcium mobilization because they have no demonstrable
influence on calcium entry through
dihydropyridine-sensitive, calcium entry channels
in the rat renal vasculature in vivo. The kinetic
parameters describing the times to half-maximum
contraction, maximum contraction, and to half-recovery are summarized
in Table 2. There are no differences
between two strains. It is interesting to note that small but
statistically significant changes in recovery half-time were
recorded when antagonists of calcium were used with
AVP. Nifedipine, TMB-8, and heparin rendered the rate of
recovery more rapid.
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Discussion |
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Our previous studies demonstrated that AVP produces exaggerated renal vasoconstriction in young SHR relative to normotensive rats.6 The current study investigated mechanisms by which AVP receptor activation increases renal vasomotor tone in vivo. The purpose was to evaluate possible strain differences in postreceptor calcium signaling pathways activated by AVP in renal resistance vessels. RBF was measured in anesthetized 8-week-old SHR and WKY pretreated with indomethacin to avoid interactions with prostaglandins. To achieve similar levels of vasoconstriction, different doses of AVP were injected into the renal arteries of SHR and WKY to produce a transient 25% to 30% decrease in RBF without affecting arterial pressure. Coinjection of nifedipine with AVP buffered the AVP effect on renal hemodynamics in a dose-dependent manner, with maximum inhibition of 30% to 35% of the peak AVP response. Thus, AVP stimulation leads to activation of voltage-operated L-type calcium entry channels. To evaluate intracellular calcium mobilization, TMB-8 or heparin was infused intrarenally to antagonize the IP3 receptor of the sarcoplasmic reticulum. Each agent blocked AVP-induced renal vasoconstriction in a dose-dependent fashion, with maximum inhibition of 60% to 65% of AVP effect. These observations provide new information about the relative contributions of calcium entry and mobilization pathways in response to AVP stimulation of V1 vascular receptors in renal resistance vessels in the young SHR and WKY rat kidneys.
The predominant receptor type that mediates AVP effects in the renal vasculature is V1. A V1 receptor antagonist blocks virtually all of the AVP-induced renal vasoconstriction in vivo and in the isolated perfused kidney.6 27 A V2 receptor antagonist exerts from a very weak to no inhibitory influence on the vascular effects of AVP in the rat kidney.6
Analysis of the vascular reactivity is complicated by the fact that the same dose of AVP causes more profound renal vasoconstriction in SHR. Thus, our initial studies also included an assessment of calcium signaling when different amounts of AVP produce the same degree of renal vasoconstriction in SHR and WKY. We reasoned that a strain difference in receptor density is probably present with similar postreceptor second-messenger systems. On the other hand, a difference in the relative proportions of calcium signaling via calcium entry and mobilization would more likely reflect a strain difference in postreceptor events rather than in receptor number. We evaluated the relative contributions of calcium entry and mobilization when V1 receptors in SHR and WKY were stimulated by similar amounts of AVP to produce greater vasoconstriction in SHR. In addition, we analyzed the effects of smaller doses of AVP in SHR that were used to cause similar degrees of renal vasoconstriction in the two strains. Both sets of comparisons made using nifedipine demonstrate that approximately 35% of the AVP effects in both SHR and WKY are due to calcium entry through dihydropyridine-sensitive L-type calcium channels. The percentage of calcium mobilization inhibited by TMB-8 or heparin was also similar in SHR and WKY. Thus, the relative contributions of calcium entry and mobilization did not differ between rat strain, suggesting a similarity of postreceptor mechanisms and the proportion of the pathways activated by AVP receptor stimulation. Collectively, these results suggest that there is no major abnormality in postreceptor second-messenger systems associated with calcium signal transduction in renal resistance vessels of young SHR. Thus, it is reasonable to postulate that the exaggerated renal vascular reactivity to AVP is due primarily to an increased population of AVP V1 receptors in SHR at the age of 8 weeks.
The regulation of cytosolic calcium in vascular smooth muscle plays a central role in AVP-induced vasoconstriction. Previous studies have shown that [Ca2+]i is increased in the freshly isolated thoracic aorta of young SHR.28 Other studies reported that the aortic strips of SHR accumulate more 45Ca2+ than normal and that the calcium content of the aorta increases with an animal's age.29 In contrast, the basal level of [Ca2+]i in cultured aortic smooth muscle cells was found to be similar in WKY and SHR.30 Of particular interest, AVP but not Ang II caused a larger increase in [Ca2+]i in cultured aortic smooth muscle cells from SHR compared with WKY.16 31 The exaggerated calcium signal was reported to be due to a combination of increased calcium entry and mobilization of calcium from internal stores. Our observations extend this finding to the renal vasculature of young SHR.
Previous in vitro studies suggest that cultured aortic smooth muscle cells have a higher density or activity of voltage-dependent calcium channels than WKY.31 It also has been reported that AVP elicits more pronounced contraction of mesenteric arteries isolated from SHR than those from WKY.32 If the renal vasculature of SHR was to have a greater population of voltage-gated calcium channels, one would predict that a larger fraction of the AVP-induced renal vasoconstriction would be inhibited by the calcium entry antagonist in SHR. However, our results demonstrate similar inhibition in SHR and WKY, suggesting that V1 receptor activation leads to a similar degree of voltage-gated calcium entry in both strains of rat at the age of 8 weeks.
Earlier in vitro studies in which cultured vascular smooth muscle cells were used found that AVP induced the increase in [Ca2+]i in the presence and absence of extracellular calcium.14 15 18 33 Dihydropyridine-sensitive voltage-dependent calcium channel blockers such as nifedipine had no effect on an AVP-induced increase in [Ca2+]i.15 16 34 35 Responses to AVP can be completely abolished by an AVP V1 receptor–specific antagonist33 36 37 or agents that interfere with mobilization of intracellular calcium.15 18 Similar results have been presented for cultured rat glomerular mesangial cells.10 11 12 13 These in vitro studies indicate that the increase in calcium concentration induced by AVP is due in large part to V1 receptor stimulation of IP3-mediated release of calcium from intracellular stores.
In contrast to these in vitro preparations, results from in vivo studies demonstrate a larger role for calcium entry from extracellular sources. In one report, verapamil and nifedipine were found to block about one third of the changes in vascular resistance produced by AVP in the rat kidney.19 In the rabbit, nifedipine attenuates the AVP-induced RBF decrease by 40% to 50%.20 A calcium entry blocker and TMB-8 suppressed the RBF response to AVP by 80% and 40%, respectively, in dogs.21 These results suggest that vasoconstriction induced by AVP is mediated both by the influx of calcium through dihydropyridine-sensitive calcium channels and by the release of calcium from intracellular resources. Our observations extend this conclusion to include hypertensive rats of the Okamoto-Aoki strain in addition to Wistar-Kyoto rats studied under conditions of water loading. However, quantitative differences are noted. Our current in vivo observations demonstrate that nifedipine exerts maximum inhibition by blocking 30% to 35% of the renal vascular response to AVP. The present results also indicate that calcium mobilization plays an important role in the in vivo renal vasoconstriction elicited by AVP. Either TMB-8 or heparin antagonism of the IP3 receptor of the sarcoplasmic reticulum produced inhibition of up to 65% of the peak change in RBF.
The combined inhibition observed with nifedipine and TMB-8 indicates that AVP receptor activation triggers both calcium entry and mobilization. The results suggest that these pathways may mediate the renal vasoconstriction in an apparent independent manner because the maximum effects of nifedipine and TMB-8 were larger than either agent alone elicited. On the other hand, the combined treatment with nifedipine and TMB-8 at doses that produced near-maximum inhibitory effects only blocked about 85% of the AVP-induced vasoconstriction (rather than the almost 100% that was predicted) from the addition of two totally independent effects. It is not clear whether the residual response reflects an interaction of the pathways under investigation and/or the involvement of additional calcium signaling mechanisms. It is noteworthy that our results with the calcium channel agonist BAY K8644 are consistent with the presence of distinct pathways of calcium entry via a dihydropyridine-sensitive channel and of mobilization subject to specific antagonism by TMB-8 or heparin. Neither antagonist of the sarcoplasmic reticulum IP3 receptor, alone or together, affected renal vasoconstriction elicited by BAY K8644 stimulated calcium entry. Nevertheless, we cannot rule out the possibility that agonist-induced calcium release may stimulate a component of calcium entry. The present results with AVP are in general agreement with our earlier findings with Ang II. Previous hemodynamic studies in normotensive rats have indicated that calcium entry mediates about 50% of the renal vasoconstriction and that calcium mobilization is responsible for roughly 50% of the acute renal response induced by Ang II.38 As noted in the current study with AVP, the inhibitory effects of combined nifedipine and TMB-8 treatment were greater than either agent alone after stimulation by Ang II.
In summary, our observations provide new information about the vascular mechanisms mediating contraction of renal resistance vessels in response to AVP in SHR and WKY. The present in vivo hemodynamic data suggest that AVP produces constriction of renal resistance vessels by a combination of calcium entry and mobilization pathways. About one third of the vasoconstriction is mediated by voltage-gated L-type calcium channels that are antagonized by the dihydropyridine nifedipine. Two thirds of the vascular response to AVP results from calcium release from sarcoplasmic stores as revealed by inhibition of intracellular mobilization that is triggered by IP3 using either TMB-8 or heparin. Summation of these signal transduction pathways can account for almost all the AVP-induced constriction of renal resistance vessels. When AVP produces the same degree of vasoconstriction, calcium mobilization and calcium entry pathways participate to similar degrees in WKY and SHR. Thus, our studies demonstrate that there are no major differences between renal arterioles of SHR and WKY with regard to the relative importance to calcium entry and mobilization mechanisms in AVP-induced contraction. We conclude that the exaggerated renal vascular reactivity to AVP challenge in SHR is probably not due to a primary defect in postreceptor signaling. Our results are consistent with the view that the enhanced reactivity to AVP is mediated by a higher density of V1 receptors in the vasculature of SHR.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This research was supported by research grant HL-02334 from the Heart Blood Institute of the National Institutes of Health.
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Footnotes |
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Reprint requests to William J. Arendshorst, PhD, Department of Physiology, CB #7545, Room 152 Medical Sciences Research Bldg, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7545.
A preliminary report of portions of this work was published previously in abstract form (Calcium signaling pathways stimulated by vasopressin-induced renal vasoconstriction in normotensive and genetically hypertensive rats. Am Soc Nephrol. 1996;7:1580).
Received October 11, 1996; first decision November 8, 1996; accepted April 15, 1997.
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Rettig R, Schmitt B, Pelzl B, Speck T. The kidney and primary hypertension: contributions from renal transplantation studies in animals and humans. J Hypertens. 1993;11:883-891.[Medline] [Order article via Infotrieve]
2. Dilley JR, Stier CT Jr, Arendshorst WJ. Abnormalities in glomerular function in rats developing spontaneous hypertension. Am J Physiol. 1984;246:F12-F20.
3.
Chatziantoniou C, Daniels FH, Arendshorst WJ.
Exaggerated renal vascular reactivity to angiotensin and
thromboxane in young genetically hypertensive rats.
Am J Physiol. 1990;259:F372-F382.
4.
Chatziantoniou C, Arendshorst WJ. Impaired
ability of prostaglandins to buffer renal vasoconstriciton
in genetically hypertensive rats. Am J Physiol. 1992;263:F573-F580.
5.
Chatziantoniou C, Ruan X, Arendshorst WJ.
Defective G protein activation of the cAMP pathway in rat kidney during
genetic hypertension. Proc Natl Acad Sci U S A. 1995;92:2924-2928.
6.
Feng JJ, Arendshorst WJ. Enhanced renal
vasoconstriction induced by vasopressin in SHR is mediated by
V1 receptors. Am J Physiol. 1996;271:F304-F313.
7. Schiffrin EL. Intracellular signal transduction for vasoactive peptides in hypertension. Can J Physiol Pharmacol. 1994;72:954-962.[Medline] [Order article via Infotrieve]
8.
Nishizuka Y. Studies and perspectives of
protein kinase C. Science. 1986;233:305-312.
9. Berridge MJ, Irvine RF. Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature. 1984;312:315-321.[Medline] [Order article via Infotrieve]
10. Bonventre JV, Skorecki KL, Kreisberg JI, Cheung JY. Vasopressin increases cytosolic free calcium concentration in glomerular mesangial cells. Am J Physiol. 1986;251:F94-F102.
11. Hassid A, Pidikiti N, Gamero D. Effects of vasoactive peptides on cytosolic calcium in cultured mesangial cells. Am J Physiol. 1986;251:F1018-F1028.
12.
Takeda K, Meyer-Lehnert H, Kim JK, Schrier RW.
AVP-induced Ca fluxes and contraction of rat glomerular
mesangial cells. Am J Physiol. 1988;255:F142-F150.
13. Portilla D, Mordhorst M, Bertrand W, Irwin C, Morrison AR. Different guanosine triphosphate-binding proteins couple vasopressin receptor to phospholipase C and phospholipase A2 in glomerular mesangial cells. J Lab Clin Med. 1992;120:752-761.[Medline] [Order article via Infotrieve]
14. Vigne P, Breittmayer JP, Lazdunski M, Frelin C. The regulation of the cytoplasmic free Ca2+ concentration in aortic smooth muscle cells (A7r5 line) after stimulation by vasopressin and bombesin. Eur J Biochem. 1988;176:47-52.[Medline] [Order article via Infotrieve]
15. Hughes AD, Schachter M. Multiple pathways for entry of calcium and other divalent cations in a vascular smooth muscle cell line (A7r5). Cell Calcium. 1994;15:317-330.[Medline] [Order article via Infotrieve]
16.
Nabika T, Velletri PA, Lovenberg W, Beaven MA.
Increase in cytosolic calcium and phosphoinositide
metabolism induced by angiotensin II and
[Arg] vasopressin in vascular smooth muscle cells.
J Bio Chem. 1985;260:4661-4670.
17.
Wu SN, Yu HS, Seyama Y. Induction of
Ca2+ oscillations by vasopressin in the
presence of tetraethylammonium chloride in
cultured vascular smooth muscle cells. J
Biochem. 1995;117:309-314.
18.
Blatter LA, Wier WG. Agonist-induced
[Ca2+]i waves and Ca2+-induced
Ca2+ release in mammalian vascular smooth muscle
cells. Am J Physiol. 1992;263:H576-H586.
19. Goldberg JP, Schrier RW. Effect of calcium membrane blockers on in vivo vasoconstrictor properties of norepinephrine, angiotensin II and vasopressin. Miner Electro Metab. 1984;10:178-183.
20.
Seino M, Abe K, Tsunoda K, Yoshinaga K.
Interaction of vasopressin and prostaglandins through
calcium ion in the renal circulation. Hypertension. 1985;7:53-58.
21. Takahara A, Suzuki-Kusaba M, Hisa H, Satoh S. Effects of a novel Ca2+ entry blocker, CD-349, and TMB-8 on renal vasoconstriction induced by angiotensin II and vasopressin in dogs. J Cardiovasc Pharmacol. 1990;16:966-970.[Medline] [Order article via Infotrieve]
22. Xiong Z, Sperelakis N. Regulation of L-type calcium channels of vascular smooth muscle cells. J Mol Cell Cardiol. 1995;27:75-91.[Medline] [Order article via Infotrieve]
23. Ogawa N, Ono H. Effect of 8-(N,N-diethylaminl)octyl-3,4,5-trimetoxybenzoate (TMB-8), an inhibitor of intracellular Ca2+ release, on autoregulation of renal blood flow in the dog. Naunyn Schmiedeberg Arch Pharmacol. 1988;338:293-296.[Medline] [Order article via Infotrieve]
24. Chiou CY, Malagodi MH. Studies on the mechanisms of action of a new Ca2+ antagonist, 8-(N,N-diethylamino)octyl-3,4,5,-trimethoxybenzoate hydrochloride in smooth and skeletal muscles. Br J Pharmacol. 1978;53:279-285.[Medline] [Order article via Infotrieve]
25. Kobayashi S, Somlyo AV, Somlyo AP. Heparin inhibits the inositol 1,4,5-trisphosphate-dependent, but not the independent, calcium release induced by guanine nucleotide in vascular smooth muscle. Biochem Biophys Res Commun. 1988;153:625-631.[Medline] [Order article via Infotrieve]
26.
Ghosh TK, Eis PS, Mullaney JM, Ebert CL, Gill
DL. Competitive, reversible, and potent antagonism of inositol
1,4,5-trisphosphate-activated calcium release by
heparin. J Biol Chem. 1988;263:11075-11079.
27.
Cooper CL, Malik KU. Mechanism of action of
vasopressin on prostaglandin synthesis and vascular
function in the isolated rat kidney: effect of calcium
antagonists and calmodulin
inhibitors. J Pharmacol Exp Ther. 1984;229:139-147.
28.
Jelicks LA, Gupta RK. NMR measurement of
cytosolic free calcium, free magnesium, and intracellular sodium in the
aorta of the normal and spontaneously hypertensive rat.
J Biol Chem. 1990;265:1394-1400.
29.
Bhalla RC, Webb RC, Singh D, Ashley T, Brock T.
Calcium fluxes, calcium binding, and adenosine cyclic
3',5'-monophosphate-dependent protein kinase activity in the aorta of
spontaneously hypertensive and Kyoto Wistar normotensive rats.
Mol Pharmacol. 1978;14:468-477.
30. Nabika T, Velletri PA, Beaven MA, Endo J, Lovenberg W. Vasopressin-induced calcium increases in smooth muscle cells from spontaneously hypertensive rats. Life Sci. 1985;37:579-584.[Medline] [Order article via Infotrieve]
31. Yamada K, Goto A, Matsuoka H, Sugimoto T. Alterations of calcium channels in vascular smooth muscle cells from spontaneously hypertensive rats. Jpn Heart J. 1992;33:727-734.[Medline] [Order article via Infotrieve]
32. Bund SJ, Heagerty AM, Aalkjaer C, Swales JD. Vasopressin-mediated contractions in mesenteric arteries from spontaneously hypertensive rats: differences in response compared with Wistar-Kyoto animals. Clin Sci. 1988;75:449-453.[Medline] [Order article via Infotrieve]
33. Vallotton MB, Wuthrich RP, Lew PD, Capponi AM. Effects of vasopressin and its analogs on rat aortic smooth muscle and renal medullary tubular cells: characterization of receptor subtypes. J Cardiovasc Pharmacol. 1986;8(suppl):S5-11.
34. Kondo K, Kozawa O, Takatsuki K, Oiso Y. Ca2+ influx stimulated by vasopressin is mediated by phosphoinositide hydrolysis in rat smooth muscle cells. Biochem Biophys Res Commun. 1989;161:677-682.[Medline] [Order article via Infotrieve]
35.
Capponi AM, Lew PD, Vallotton MB. Cytosolic free
calcium levels in monolayers of cultured rat aortic smooth muscle
cells: effects of angiotensin II and vasopressin.
J Biol Chem. 1985;260:7836-7842.
36. Doyle VM, Ruegg UT. Vasopressin induced production of inositol trisphosphate and calcium efflux in a smooth muscle cell line. Biochem Biophys Res Commun. 1985;131:469-476.[Medline] [Order article via Infotrieve]
37. Aiyar N, Nambi P, Stassen FL, Crooke ST. Vascular vasopressin receptors mediate phosphatidylinositol turnover and calcium efflux in an established smooth muscle cell line. Life Sci. 1986;39:37-45.[Medline] [Order article via Infotrieve]
38.
Ruan X, Arendshorst WJ. Calcium entry and
mobilization signaling pathways in ANG II-induced renal
vasoconstriction in vivo. Am J Physiol. 1996;270:F398-F405.
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