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作者：Joen Steendahl, Niels-Henrik Holstein-Rathlou, Charlotte Mehlin Sorensen, and Max Salomonsson作者单位：Division of Renal and Cardiovascular Research, Department of Medical Physiology, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen N, Denmark
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0 q* J0 P! t g W 【摘要】
4 x; G7 G) q' ^, {' H4 @% ~ The aim of the present study was to investigate the role of Ca 2 -activated Cl - channels in the renal vasoconstriction elicited by angiotensin II (ANG II) and norepinephrine (NE). Renal blood flow (RBF) was measured in vivo using electromagnetic flowmetry. Ratiometric photometry of fura 2 fluorescence was used to estimate intracellular free Ca 2 concentration ([Ca 2 ] i ) in isolated preglomerular vessels from rat kidneys. Renal arterial injection of ANG II (2-4 ng) and NE (20-40 ng) produced a transient decrease in RBF. Administration of ANG II (10 -7 M) and NE (5 x 10 -6 M) to the isolated preglomerular vessels caused a prompt increase in [Ca 2 ] i. Renal preinfusion of DIDS (0.6 and 1.25 µmol/min) attenuated the ANG II-induced vasoconstriction to 35% of the control response, whereas the effects of NE were unaltered. Niflumic acid (0.14 and 0.28 µmol/min) and 2-[(2-cyclopentenyl-6,7-dichloro-2,3-dihydro-2-methyl-1-oxo-1 H -inden-5-yl)oxy]acetic acid (IAA-94; 0.045 and 0.09 µmol/min) did not affect the vasoconstrictive responses of these compounds. Pretreatment with niflumic acid (50 µM) or IAA-94 (30 µM) for 2 min decreased baseline [Ca 2 ] i but did not change the magnitude of the [Ca 2 ] i response to ANG II and NE in the isolated vessels. The present results do not support the hypothesis that Ca 2 -activated Cl - channels play a crucial role in the hemodynamic effects of ANG II and NE in rat renal vasculature.
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microcirculation; vascular smooth muscle; 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; niflumic acid; calcium channels ) J. h F% z9 X. q9 |( J
【关键词】 chloride blockers vascular responses angiotensin norepinephrine
; t% z) `! m3 m7 y' c CALCIUM - ACTIVATED Cl - (Cl Ca ) channels have been suggested as a link in the chain of events leading to the initiation of vascular smooth muscle contraction ( 17 ). The intracellular cytosolic free Cl - concentration is believed to be substantially above equilibrium in resting vascular smooth muscle cells of different origin ( 1, 4 ). Stimulation and opening of Cl Ca channels will thus lead to Cl - efflux and depolarization of the cell membrane. The depolarization causes the opening of voltage-sensitive Ca 2 channels, preferentially of the L-type, subsequently giving rise to vasoconstriction. Stimulation of cell-surface receptors with vasoconstrictors [e.g., angiotensin II (ANG II), norepinephrine (NE), and endothelin] activates the inositol trisphosphate (IP 3 ) pathway, causing release of Ca 2 from intracellular stores. This initial rise in cytosolic free Ca 2 concentration ([Ca 2 ] i ) activates the Cl Ca channels. A complicating circumstance is that an increase in [Ca 2 ] i also activates Ca 2 -sensitive K channels, an event that will lead to hyperpolarization, closure of L-type Ca 2 channels, and vasorelaxation ( 10 ).5 C& h0 ~( R* l- Q% h$ l5 u+ c
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Because of the lack of antagonists specific for Cl Ca channels, it is difficult to fully depict the constrictive pathways possibly involving these channels. Some studies have dealt with the role of Cl Ca channels in the control of the renal vasculature or mesangial cell function ( 3, 11, 20, 28, 29, 32 ). Several of these studies suggest a role for Cl Ca channels in the ANG II-mediated vasoconstriction ( 3, 11, 29, 32 ). A role for Cl Ca channels has also been proposed in the afferent arteriolar vasoconstriction elicited by NE ( 29 ). In this study, the hypothesis that activation of Cl Ca channels is of crucial importance in agonist-induced vasoconstriction in the renal vasculature was challenged. We tested whether three different antagonists of Cl Ca channels, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), 2-[(2-cyclopentenyl-6,7-dichloro-2,3-dihydro-2-methyl-1-oxo-1 H -inden-5-yl)oxy]acetic acid (IAA-94), and niflumic acid, affected the renal vascular response to ANG II and NE. All the agents were tested in vivo in rats with the use of electromagnetic flowmetry to measure renal blood flow (RBF). Ratiometric fluorescence of the indicator fura 2 was used to assess the effect of niflumic acid and IAA-94 in vitro by measuring [Ca 2 ] i in isolated intact rat preglomerular vessels.# u& h+ @/ l& z" F. z
* C6 O4 ?1 Q f6 k# dMETHODS
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# Q, ]! K; u8 G5 p+ t. e0 h% l/ jThe experimental protocols were approved by the Danish National Research Animal Committee.1 f' ~+ R; c. x9 s3 q' k
! z1 z$ u6 a: W" O6 u/ x2 O+ dRBF Measurements
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Male Sprague-Dawley rats (270-330 g body wt) were obtained from Mollegard (Lille Skensved, Denmark). They were fed standard rat laboratory chow and tap water ad libitum.0 N4 l- q$ E2 [
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Anesthesia was induced with 5% halothane delivered in 35% N 2 -65% O 2. Polyethylene catheters were placed in the right jugular vein (PP-10) for infusion and in the left carotid artery (PP-50) for continuous measurement of systemic blood pressure by a pressure transducer (Statham P23-dB, Gould, Oxnard, CA). A tracheostomy was performed, and the rat was placed on a servo-controlled heating table to maintain body temperature at 37°C. The rat was connected to and ventilated by a small animal ventilator at tidal volume of 1.7-2.5 ml, depending on the body weight, and frequency of 60 breaths/min. The final halothane concentration needed to maintain sufficient anesthesia was 1%. A continuous intravenous injection of 4 mg/2 ml pancuronium bromide (Pavulon, N. V. Organon Oss) in 4.7 ml of 0.9% saline (0.6 mg/ml) was given at a rate of 20 µl/min. Additional isotonic saline was given continuously at the same rate.
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# ]* f0 c/ v; }& LMidline and subcostal incisions were made to expose the abdominal aorta and left kidney. A noncannulating precalibrated electromagnetic flow probe (model 101, Scalar Medical) was placed around the accessed left renal artery to measure RBF. A tapered and curved polyethylene catheter was introduced into the left iliac artery and advanced through the abdominal aorta and 1 mm into the left renal artery. This catheter was used to administer test agents directly into the renal artery. The preparation was discarded if the NE or ANG II bolus produced a substantial rise in arterial pressure. Mean arterial pressure sometimes increased 0-5 mmHg 4 s after the bolus infusion, indicating an effect of increased renal resistance on arterial pressure or, possibly, minor shunting of the agonist to the systemic circulation.
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An Upchurch six-port injection valve was used to introduce a 10-µl bolus of NE or ANG II into the renal artery infusion line. At 1 min before administration of the agonist, the rate of renal artery infusion was increased from 10 to 144 µl/min. (In the experiment involving niflumic acid, an infusion rate of 288 µl/min was used to double the plasma concentration.) This rate of infusion allowed administration of the bolus in + z) a) o: G# S+ }, U" F
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The RBF values were normalized and expressed as a percentage of baseline values, which were calculated separately for each injection using the mean value observed during the 15-s time interval between introduction of the agonist (NE or ANG II) and onset of the renal vascular response. The RBF values were averaged for 5 s, during which the blood flow response to the agonist was maximal. The recordings obtained using the Cl - channel blocker and the agonists were analyzed in the same fashion.8 g. h# ?# G" f4 c5 l
/ I: _ D7 ]! V7 hMeasurements of [Ca 2 ] i |$ u5 N7 |; g% N& @; v
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Isolation and fura 2 loading of the preparation. Preglomerular resistance vessels were microdissected from Sprague-Dawley rats (250-300 g body wt; Møllegard) as described previously ( 24 ). Briefly, thin slices (0.5-1 mm thick) of the kidney cortex were cut from the midregion and transferred to a dissection dish containing a physiological salt solution [PSS (in mM): 135 NaCl, 5.0 KCl, 1.0 CaCl 2, 1.0 MgCl 2, 10 HEPES, and 5.0 D -glucose (pH 7.38-7.42)] with BSA (0.5 g/dl; Sigma). All solutions were equilibrated with atmospheric air. The isolation procedure was performed using sharpened forceps under a dissection microscope ( x 9-120 magnification). An interlobular artery was localized at its origin from an arcuate artery, and sharpened forceps and knives were used to remove the tubular structures. Only the most-distal part of the interlobular artery and cortical afferent arterioles were used. The kidney was discarded if no preparation was obtained during the first 120 min of dissection. After completion of dissection, the vessel was loaded with 5 µM fura 2-AM for 45-60 min in the dark at room temperature. Fura 2 loading was facilitated with 0.01% Pluronic F-127 (Sigma). A 5-µl micropipette was used to transfer the vessel to a PSS-containing chamber on the stage of an inverted microscope (Olympus). The ends of the vessel were aspirated into glass holding pipettes to obtain mechanical stability.% ^9 d) {+ k' K$ C" R
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Measurements of [Ca 2 ] i. A x 40 quartz oil immersion objective was used for measurements of [Ca 2 ] i. A digital video camera (PCO, Kelheim, Germany) and Image Workbench software (Axon Instruments, Union City, CA) were used. The vessel was visualized on a computer screen using the video camera and the software. An area for measurement of [Ca 2 ] i was then encircled using a software-based routine. UV light of alternatively 340- and 380-nm wavelengths was provided from a monochromator controlled by the software. The fluorescent emission was detected by the digital video camera, and the ratio of fluorescence obtained with 340-nm excitation to that obtained with 380-nm excitation (R 340/380 ) was calculated. Changes in this ratio were used as an index for changes in [Ca 2 ] i ( 6 ). Autofluorescence was measured in preliminary experiments in nonloaded preparations and was found to be ; e! V! `- Z1 H- i- o1 n
q/ o; e4 q# ]) ?+ BExperimental protocol. The experimental solutions at room temperature were added in a volume large enough to allow several total exchanges of the fluid in the experimental chamber. The volume in the experimental chamber was kept constant during replacement of fluids by a vacuum suction system. The viability of the preparation was assessed by stimulation with the NE solution. Vessels not responding promptly were discarded. The increase in [Ca 2 ] i was accompanied by a visible contraction of the vessel. The [Ca 2 ] i response to ANG II, NE, and a solution high in potassium (K100; obtained by replacing NaCl with KCl in the PSS solution to a final K concentration of 100 mM) was studied with and without 120 s of pretreatment with 50 µM niflumic acid. The effect of IAA-94 pretreatment (30 µM) on the [Ca 2 ] i responses to stimulation with ANG II and NE was tested in the same manner. Because of the autofluorescent properties of DIDS, we refrained from using this substance in the [Ca 2 ] i measurement experiments. Preliminary experiments were performed to ascertain that the dose of DMSO used to dissolve niflumic acid did not affect the responses to 0.1 µM ANG II and 5 µM NE. Responses to the vasoconstictors were recorded for 60-120 s. The responses obtained during niflumic acid or IAA-94 treatment were compared with the responses obtained when 0.1 µM ANG II, 5 µM NE, and K100 were added directly to the PSS in the absence of the inhibitor. The NE and K100 experiments were performed as paired studies, with control and pretreatment observations in the same vessel. Because of the tachyphylaxis elicited on ANG II administration, only one response per preparation could be obtained with this drug ( 15 ). We therefore made an unpaired comparison between control and pretreatment responses to ANG II. The experiments were performed in random order to establish reversibility and to exclude possible effects of a prolonged action of a particular pretreatment.
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8 ?! F1 \7 l; Z% k8 jStatistical Analyses& X/ M! t3 U0 x2 r! v
8 _7 x9 u0 ?% U+ R0 IValues are means ± SE. SigmaStat (Jandel Scientific/SPSS) software was used for data analysis. Statistical significance was evaluated within groups by Student's paired t -test or one-way ANOVA for repeated measurements with Student-Newman-Keuls post hoc test. Unpaired Student's t -test was used between groups. P 3 S3 u! t) r* x$ }: Y- Q, ]
, v, z+ @' r: cRESULTS
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Measurements of RBF
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In 49 Sprague-Dawley rats, RBF during euvolemic control conditions averaged 6.0 ± 0.15 ml/min. Arterial blood pressure and hematocrit averaged 111 ± 1.5 mmHg and 48.8 ± 0.5%, respectively.% M7 ]3 j! N# X& D0 H
, m' ^# M+ q1 X7 c' {Effects of DIDS on Maximal Responses Produced by NE and ANG II3 `% [) F5 l( R8 Q" o. r9 w* G
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Renal preinfusion of DIDS (0.6 and 1.25 µmol/min) attenuated the decrease in RBF mediated by ANG II (Figs. 1 and 2 ). In control experiments, 2-4 ng of ANG II elicited a transient 37 ± 5% maximum reduction in RBF ( P
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* I+ \. _0 D2 e& {$ o9 \( YFig. 1. Original recording of renal blood flow (RBF) and arterial blood pressure (BP). a : Bolus injection of 4 ng of ANG II. b : Infusion of DIDS (0.6 µmol/min). c : Infusion of DIDS (0.6 µmol/min) bolus injection of 4 ng of ANG II. d : Bolus injection of 4 ng of ANG II. DIDS attenuated the ANG II response, and the effect was reversible.
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Fig. 2. Effect of pretreatment with DIDS on RBF response to ANG II and norepinephrine (NE). ANG II (2-4 ng) and NE (20-40 ng) were administered as bolus doses into the renal artery. DIDS (0.6 and 1.25 µmol/min) was infused via the same catheter for 2 min before administration of agonists. RBF values were normalized and expressed as a percentage of baseline (see METHODS ). DIDS attenuated ANG II-induced vasoconstriction but did not affect the response to NE. * P
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& h% K0 S3 @! x/ X$ hAn intrarenal bolus of 20-40 ng of NE produced, on average, a transient 35 ± 2% maximum reduction in RBF ( n = 7). Renal preinfusion of DIDS (0.6 µmol/min) did not alter the increase in renal resistance induced by NE (33 ± 2%, P
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Effects of Niflumic Acid on Maximal Responses Produced by NE and ANG II+ Y5 S) R. e' T2 x1 d9 V$ X
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Renal arterial preinfusion of niflumic acid (0.14 and 0.28 µmol/min) did not affect the baseline RBF. In control experiments, 20-40 ng of NE caused a transient 45 ± 1% maximum reduction in RBF ( P
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Fig. 3. Lack of effect of pretreatment with niflumic acid on ANG II- and NE-induced reduction of RBF in paired experiments. ANG II (2-4 ng) and NE (20-40 ng) were administered as bolus doses into the renal artery. Niflumic acid (0.14 and 0.28 µmol/min) was infused via the same catheter for 2 min before administration of ANG II or NE. Neither ANG II nor niflumic acid was affected by preinfusion of niflumic acid.
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Also, the response to ANG II was unaffected by niflumic acid. An intrarenal ANG II bolus (2-4 ng) elicited a transient 45 ± 1% maximum reduction in RBF ( P 9 C3 F/ t7 `: p# Z
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Effects of IAA-94 on Maximal Responses Produced by NE and ANG II$ E. p/ K3 G# q6 E
t/ S* W; v: vIAA-94 preinfusion into the renal artery (0.045 and 0.09 µmol/min) had no effect on the baseline RBF. NE (20-40 ng) caused a maximum 39 ± 5% reduction in the RBF transient ( P
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+ [/ y) f7 ~4 t5 F3 SFig. 4. 2-[(2-Cyclopentenyl-6,7-dichloro-2,3-dihydro-2-methyl-1-oxo-1 H -inden-5-yl)oxy]acetic acid (IAA-94) did not affect ANG II- and NE-induced reduction of RBF in paired experiments. IAA-94 (0.045 and 0.09 µmol/min) was infused via the renal artery catheter for 2 min before bolus administration of ANG II (2-4 ng) or NE (20-40 ng).
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IAA-94 did not significantly affect the ANG II-induced reduction of RBF. An ANG II bolus injection (2-4 ng) elicited a 28 ± 4% maximum reduction in RBF ( P X6 E/ M0 S7 E5 P i3 b# K& o0 i
1 X. w2 F! ]5 d. w( F+ x9 u2 `: {Effect of Niflumic Acid, DIDS, and IAA-94 on Temporal Profiles of RBF Responses
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, x- i( C2 j! ITo examine whether the Cl - channel blockers had an effect on the temporal profile of the RBF responses to injection of NE or ANG II, we analyzed the time from initiation of the response to the maximal deflection of the RBF response. We found that none of the Cl - channel blockers had a significant effect on the temporal profile of the RBF response ( P 0.05 for all combinations of agonists and Cl - channel blockers; Table 1 ).
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Table 1. Temporal profiles of RBF responses1 n' o- b5 d6 z4 h: {0 K1 U3 _+ {" r
# d* i5 S7 i% ]5 Y$ I' {. dEffect of Prazosin on the Acid RBF Response to NE( @9 n# y' v8 x/ \
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The validity of the experimental setup was tested in a paired fashion by the ability of preinfused prazosin to block the NE response. In the control experiments, NE caused a 46 ± 2% reduction of RBF ( n = 5, P
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) h# l& ?) D6 u- LMeasurement of [Ca 2 ] i
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$ ?7 w, j9 S. Y2 B4 o- mThe baseline R 340/380 in 35 preglomerular arterioles from 21 rats averaged 1.064 ± 0.02. Administration of 5 x 10 -6 M NE and K100 solution to the bath caused an abrupt, steep increase in vascular smooth muscle [Ca 2 ] i. The response pattern was normally a transient peak and a sustained plateau phase ( Fig. 5 ). The slope of the [Ca 2 ] i response after stimulation with 10 -7 M ANG II was somewhat more shallow, and no clear peak phase could be detected. For evaluation, we selected the time point of 30-35 s after the initiation of the response as representative of the sustained phase of the [Ca 2 ] i response pattern. When stimulated with NE after pretreatment with 0 Ca 2 , previous studies showed that the transient increase in [Ca 2 ] i is completed at 30-35 s and [Ca 2 ] i is not different from baseline ( 24, 25 ). Therefore, the [Ca 2 ] i response at this time point reflects Ca 2 entry from the extracellular space.; K1 r1 U. ?5 o+ x; L
* ]2 d, i; T4 P- h7 k6 B: e1 H. dFig. 5. Original recording of the ratio of fluorescence obtained with 340-nm excitation to that obtained with 380-nm excitation, reflecting cytosolic Ca 2 concentration in afferent arterioles, shows stimulatory effect of 5 µM NE and lack of antagonism afforded by pretreatment with 50 µM niflumic (Nifl) acid.5 u% J) M' L. L+ I) ?
; L- ]6 a. N, P9 y6 ?% V& \Effect of Niflumic Acid on [Ca 2 ] i Responses3 J0 Z) c0 D; x- t) e/ K7 v& x5 G
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Administration of 50 µM niflumic acid for 120 s caused a minor but significant decrease in R 340/380 (from 1.027 ± 0.018 to 1.007 ± 0.017, P
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+ @ d( [- J6 W% {Fig. 6. Summarized group data showing change in the ratio of fluorescence obtained with 340-nm excitation to that obtained with 380-nm excitation (R 340/380 ), reflecting cytosolic Ca 2 concentration in preglomerular vessels, after stimulation with 5 µM NE, 0.1 µM ANG II, and high K (K100) in control conditions and after 2 min of pretreatment with 50 µM niflumic acid. All stimulations are unaffected by niflumic acid. * P
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+ ]5 R6 l7 }9 w) K7 I/ s3 \We also evaluated whether niflumic acid affected the [Ca 2 ] i response to depolarization ( Fig. 6 ). After 120 s of pretreatment with niflumic acid, addition of the K100 solution induced an increase in R 340/380 from 0.995 ± 0.032 to 1.145 ± 0.027 ( P 6 b( W; H: {, @' ]' z
* Z" |$ }9 U9 \0 sFinally, the effect of niflumic acid on the [Ca 2 ] i response to ANG II was studied ( Fig. 6 ). These experiments were performed in an unpaired fashion. Because of strong tachyphylaxis, it is impossible to elicit more than one response to ANG II in isolated renal vessels ( 11, 15 ). In control vessels, 10 -7 M ANG II elicited a rise in R 340/380 from 1.013 ± 0.021 to 1.09 ± 0.026 ( P 4 X7 O$ ?8 o! w2 E% I
7 f: r3 T: @4 V2 e6 N0 ~9 wEffect of IAA-94 on [Ca 2 ] i Responses
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Pretreatment with 30 µM IAA-94 for 120 s caused a small but significant reduction in R 340/380 : from 1.122 ± 0.05 to 1.072 ± 0.04 ( P
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Fig. 7. Pretreatment with 30 µM IAA-94 for 2 min did not affect R 340/380 responses to 5 µM NE and 0.1 µM ANG II in isolated preglomerular vessels. All stimulations are unaffected by IAA-94. * P
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NE (5 µM) in the control period increased R 340/380 from 1.262 ± 0.076 to 1.538 ± 0.045 ( P , _+ L- B; B: S* v- o
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Next, we assessed whether IAA-94 affected the [Ca 2 ] i response to 10 -7 M ANG II ( Fig. 7 ). As in the niflumic acid experiments involving ANG II, these experiments were performed in an unpaired fashion. After 120 s of pretreatment with IAA-94, addition of the ANG II solution induced an increase in R 340/380 from 1.051 ± 0.056 to 1.245 ± 0.062 ( P ! Y7 s' o) W( _; s% j' u" l; Y6 u
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We tested the hypothesis that activation of Cl Ca channels is involved in the renal hemodynamic response to activation with ANG II or NE. An important aspect of this study is the assessment of the effect of Cl - channel blockers on renal vascular responses to agonists based on two different techniques. RBF was quantified in vivo and combined with measurements of [Ca 2 ] i by fura 2 photometry, in isolated afferent arterioles from Sprague-Dawley rats.
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. A% q7 h6 v2 Z& r6 E SWe found that basal RBF was unaffected by intrarenal infusion of DIDS, niflumic acid, or IAA-94. In another recent study, DIDS in increasing doses gave rise to a corresponding renal vasodilatation ( 16 ). In this study, however, DIDS was infused into the femoral artery, which makes it difficult to evaluate the role of a possible direct central action or compensatory mechanisms. Our RBF results after DIDS treatment are supported by findings from Jensen and Skott ( 11 ). They found that the basal diameter was not changed in isolated rabbit afferent arterioles after administration of 0.5 mM DIDS, which indicates that this compound does not affect the basal state of renal resistance vessels. Furthermore, in two different studies, one with a hydronephrotic rat kidney preparation and another with a juxtamedullary nephron preparation, using another Cl - channel blocker, IAA-94, no effect on afferent arteriolar baseline diameter could be detected ( 3, 29 ). In contrast, we found that 30 µM IAA-94 administered to isolated preglomerular vessels for 120 s caused a reduction in R 340/380, reflecting a decrease in [Ca 2 ] i. Also, when we pooled the data from all isolated vessels pretreated with 50 µM niflumic acid for 120 s, we found a small but significant decrease in baseline R 340/380. We have no complete explanation for this discrepancy between the in vivo and the in vitro studies. It is possible that both compounds inhibit a Cl - channel significant for the maintenance of the basal membrane potential under the prevailing experimental conditions. Niflumic acid has also been reported to activate Ca 2 -activated and ATP-dependent K channels ( 14, 31 ). The concomitant hyperpolarization might, at least partly, explain the fall in baseline [Ca 2 ] i. However, it seems clear that this minor change in R 340/380 observed in vitro is not reflected by a change in vascular tone of major physiological importance in vivo. One possibility is that the decreased [Ca 2 ] i in vitro is not reflected by a vasodilation in vivo, because it might be counteracted by compensatory physiological mechanisms.9 o d8 L1 x; ]1 m
8 ^7 ^3 P5 K( t: E3 J6 A- FIn the present study, NE and ANG II caused a prompt reduction in RBF in vivo, as well as a sustained [Ca 2 ] i elevation in isolated afferent arterioles. This is established in several studies where NE and ANG II induced a dose-dependent renal vasoconstrictor response in vivo ( 23, 24 ). Also, it has previously been shown that the same drugs elevate [Ca 2 ] i in smooth muscle cells from preglomerular resistance vessels ( 8, 15, 24 ). Earlier work has established that the vasoconstrictor actions of NE and ANG II involve Ca 2 entry, which is antagonized by blockers of voltage-sensitive L-type Ca 2 channels ( 8, 23, 24 ). This implies that activation of cell-surface receptors eventually leads to depolarization of the cell membrane of the smooth muscle cells of the afferent arteriole. This notion is supported by the observations in this study that depolarization of the smooth muscle cell membrane of afferent arterioles by KCl causes an increase in [Ca 2 ] i of the same magnitude as that caused by stimulation with ANG II or NE. These findings are also in accord with previous observations ( 24, 25 ). However, the exact mechanism behind the agonist-induced depolarization remains unresolved.
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It has been suggested that the increase in [Ca 2 ] i that results from IP 3 -activated release of Ca 2 from the sarcoplasmic reticulum or, possibly, Ca 2 entry from the extracellular space might activate Cl Ca channels ( 17 ). Because intracellular cytosolic free Cl - concentration in smooth muscle cells supposedly is higher than the electrochemical equilibrium concentration, an opening of Cl - channels causes efflux of Cl -. This would result in cell membrane depolarization and opening of voltage-sensitive Ca 2 channels, allowing influx of Ca 2 and subsequent contraction. This mode of action has been described for vascular smooth muscle cells of different origin ( 17 ). There are also reports in the literature that such a mechanism would, at least partially, play a role in agonist-induced contraction of the renal vasculature. Okuda and co-workers ( 20 ) suggested that ANG II and vasopressin induced opening of Cl Ca channels as a consequence of elevation of [Ca 2 ] i via intracellular release.
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In the rat hydronephrotic kidney, Takenaka et al. ( 28 ) found that IAA-94, a nonspecific blocker of Cl - channels, significantly attenuated the afferent arteriolar vasoconstriction elicited by endothelin. They also reported that IAA-94 decreased the [Ca 2 ] i response and depolarization to endothelin in cultured vascular smooth muscle cells.
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3 T3 O5 I: L# V7 O; LAlso, in the rat juxtamedullary nephron preparation, Carmines ( 3 ) found that IAA-94 attenuated the afferent arteriolar vasoconstriction elicited by ANG II.* O- a0 S( w% c. |
m- p2 F. I. g0 y3 n! XIn the hydronephrotic kidney, in a similar study, Takenaka et al. ( 29 ) found that IAA-94 abolished the afferent arteriolar vasoconstriction elicited by NE and ANG II. In isolated afferent arterioles from the rabbit, Jensen and Skott ( 11 ) also found that the ANG II-induced reduction in vessel diameter was attenuated by administration of DIDS, which blocks Cl - channels, including Cl Ca channels. On the other hand, they found no significant effect of DIDS on the magnitude of NE-induced contraction ( 11 ). In accord with these findings, we found that the ANG II-induced reduction in RBF was attenuated by administration of DIDS, whereas the RBF response to NE was unaltered.
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The different effects of DIDS on the renal vasoconstriction induced by ANG II and NE indicate that these agonists activate different intracellular pathways leading to contraction. Support for this notion has previously been described. For example, in vivo and in vitro studies indicate that the ANG II response is more dependent on Ca 2 entry via the voltage-sensitive Ca 2 channel than the response to NE ( 9, 23, 24 ). One study indicated that the NE-induced increase in renal vascular resistance depends on mobilization of [Ca 2 ] i from internal stores, because TMB-8, an agent that is believed to block IP 3 -mediated Ca 2 release, attenuated the NE response by 70-80% ( 24 ). In another study, TMB-8 maximally antagonized 50% of the renal vasoconstriction produced by ANG II ( 23 ). It is therefore reasonable to conclude that Ca 2 mobilization from intracellular stores plays a more important role in the renal vascular response elicited by NE than by ANG II. Also, in one study comparing the effects of another Ca 2 channel blocker, verapamil, on the action of ANG II and NE on RBF in dogs, verapamil had a stronger attenuating effect on the ANG II response than on the NE response ( 19 ). In accord with this observation, Ca 2 measurements on smooth muscle cells from rat preglomerular vessels indicate that nifedipine totally eliminates the [Ca 2 ] i response to ANG II, whereas only 50% of this response to NE is affected by nifedipine ( 9, 24, 25; unpublished observations). From these results, it is tempting to attribute the effect of DIDS on the ANG II response to a higher dependence on Ca 2 entry via voltage-sensitive Ca 2 channels than to NE. This entry might in turn be triggered by the depolarization induced by the opening of Cl - channels that might belong to the Cl Ca channel subtype.
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1 M( |& B2 n$ _' ZIn the present study, niflumic acid and IAA-94 failed to attenuate the RBF responses to ANG II as well as to NE. Also, niflumic acid does not affect the L-type Ca 2 channels, as indicated by the observation that the R 340/380 response, reflecting [Ca 2 ] i, to a depolarizing concentration of KCl was unaffected by niflumic acid. A failed effect of a blocker in the RBF experiments might be attributed to uneven distribution of the drug within the kidney. In our hands, this seems less likely, because we found an attenuating effect of DIDS on the ANG II response. We also performed experiments where we almost completely blocked the effect of bolus injection of NE by continuous preinfusion of prazosin. Furthermore, the in vitro experiments support an absent effect of niflumic acid and IAA-94 in this respect. We found that the R 340 /R 380 response to NE and ANG II was unaffected by pretreatment with 50 µM niflumic acid, a concentration that is supposed to inhibit Cl Ca channels (see below). Taken together, these findings indicate that the putative Cl Ca channels blockers DIDS and niflumic acid might interact with different pathways. In contrast to earlier studies, we did not find that IAA-94 attenuated the renal vascular responses to NE and ANG II ( 3, 29 ). However, conflicting results in this regard have previously been reported. In one study, IAA-94, in the same concentration used in the present study (30 µM), attenuated the response to 0.1 µM ANG II by 60% in the juxtamedullary nephron preparation ( 3 ). On the other hand, it was recently reported, in the hydronephrotic kidney preparation, that IAA-94 reversed the renal vasoconstriction to 50 pM ANG II but was without effect on the response to higher ANG II concentrations (3 nM) ( 30 ). Because of different methodology, it is difficult to provide a satisfactory explanation for these discrepant results. One possibility, however, might be the use of different vessels. Although the present study and the hydronephrotic kidney preparation utilize more cortical vessels, nephrons close to the medulla are observed in the juxtamedullary nephron preparation. The vessels of these nephrons give rise to the vasa recta perfusing the medulla. An attenuating effect of IAA-94 has indeed been observed on isolated ANG II-constricted vasa recta (see below) ( 32 ). Thus it is possible that Cl - channels play different roles in cortical and juxtamedullary nephrons.! ^* X: d, k0 P, b1 T F
: x8 e. {; D& n1 rA different mode of action by the different Cl - channel blockers has been reported. In one study, IAA-94 and DIDS dilated pressurized rat posterior cerebral arteries, whereas niflumic acid was without effect ( 18 ). DIDS and IAA-94 have low selectivity for different Cl - channel subtypes ( 12 ). Niflumic acid, on the other hand, is considered to be a more specific blocker of Cl Ca channels ( 13, 17, 21 ). This blocker has a reported IC 50 for the Cl Ca channel from as low as 2.3 µM in rabbit portal vein, whereas the IC 50 for the swelling-activated Cl - channel is between 100 µM and 2 mM ( 7, 13 ). DIDS and IAA-94, on the other hand, appear to have a higher affinity for the swelling-activated Cl - channel than for the Cl Ca channel; this finding is consistent with those from the rat cerebral vessels ( 13, 18 ). In addition, DIDS affects the pH regulation of the smooth muscle cells, inasmuch as it blocks acidifying HCO 3 - exchangers and the alkalization via Na -HCO 3 - co-transport ( 2 ). The baseline RBF is not affected by DIDS, but it is still possible that the effect of ANG II, at least in part, is affected by changes in intracellular pH that do not affect the baseline ( 27 ). For example, stimulation with NE and vasopressin is augmented under intracellular acidification ( 27 ).1 W7 _% a6 e4 _
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The lack of effect of niflumic acid on the ANG II response in the present study indicates a minor role for the Cl Ca channel in ANG II-induced renal vasoconstriction. However, a role for this channel has been proposed in the ANG II-induced vasoconstriction of the rat descending vasa recta, because this was attenuated by niflumic acid (30 µM) and IAA-94 (30 µM) ( 32 ). This observation could well be in accord with the present lack of effect of niflumic acid and IAA-94 on the ANG II-induced reduction in RBF, because vasa recta blood flow accounts for only 10-15% of the total RBF. Thus a change in vasa recta flow might be too small to be detected using the present methods for RBF measurements. In one study dealing with the effect of an Na /Ca 2 exchanger on renal resistance in isolated perfused rat kidney, the vasoconstriction elicited by lowering extracellular Na was attenuated by niflumic acid ( 26 ). This might indicate that the increase in [Ca 2 ] i elicited by a decreased Na gradient might stimulate Cl Ca channels and, in turn, depolarization and opening of voltage-sensitive Ca 2 channels. However, in this study, a concentration of niflumic acid as high as 300 µM was utilized, which might affect Cl - channels other than the Cl Ca channels.% a' H. ?+ s5 {4 G0 X5 U- m5 p
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An inhibitory effect on cyclooxygenase has been attributed to niflumic acid ( 5 ). Theoretically, an inhibition of the synthesis of vasodilatory prostaglandins might mask an attenuating effect on the agonist-induced increase in [Ca 2 ] i by blocking Cl Ca channels. For example, another inhibitor of prostaglandin synthesis, indomethacin, causes an increased baseline [Ca 2 ] i in cultured cells emanating from afferent arteriolar smooth muscle cells ( 22 ). In contrast, in the present study, we found a small but significant decrease in R 340/380. Furthermore, if the activation of Cl Ca channels was crucial for the agonist-induced increase in [Ca 2 ] i and the subsequent contraction, it is less likely that inhibition of the modulatory influences of prostaglandins would fully compensate for blockade of these channels." ?9 ?1 W$ F! L, D
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It is difficult to fully evaluate the role of Cl Ca channels in renal resistance vessels because of lack of specific inhibitors. For example, even if niflumic acid is considered more specific for the Cl Ca channels than the other blockers described here, it is not sufficiently specific to distinguish between Cl Ca channels and swelling-activated Cl - channels (see IC 50 values above). However, the negative results obtained in this study using niflumic acid (and IAA-94) indicate that activation of Cl Ca channels does not play a major role in the chain of events leading to ANG II- and NE-induced renal vasoconstriction./ U3 S I. V1 R+ n
R: n& j: m3 K' J" p: RTo summarize, we have found that niflumic acid and IAA-94 do not affect the reduction in RBF induced by the vasoactive agents NE and ANG II in vivo in rats. These results were confirmed by the fact that these drugs did not affect the [Ca 2 ] i responses to the vasoconstrictors in isolated preglomerular rat vessels. On the other hand, in accord with previous findings, we found that the RBF response to ANG II was attenuated by DIDS. This indicates that these compounds might exert their actions via different pathways. It is concluded that Cl Ca channels do not play a major role in the NE- and ANG II-induced renal vascular responses., R6 C! H# c6 {4 p, s
, I) l% _* |/ z3 S& aDISCLOSURES. W1 L0 d" ?- }
% }% ^2 b: B: T8 n& d4 AThe present study was supported by grants from the Danish Medical Research Council, the Novo-Nordisk Foundation, the König-Petersen Foundation, and the Crafoord Foundation, Lund, Sweden.
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; L( l2 {8 L4 T0 Q9 y n: n. t5 u9 eACKNOWLEDGMENTS) f6 N0 R9 R4 ?6 M+ ?- n/ W
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The skillful technical assistance of Anni Salomonsson and Ian Godfrey is gratefully acknowledged.5 k: L7 U$ K; _( [
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发表于 2009-4-22 08:16
