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Am J Physiol Regul Integr Comp Physiol 280: R87-R99, 2001;
0363-6119/01 $5.00
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Vol. 280, Issue 1, R87-R99, January 2001

[Ca2+]i signaling in renal arterial smooth muscle cells of pregnant rat is enhanced during inhibition of NOS

Jason G. Murphy, John B. Fleming, Kathy L. Cockrell, Joey P. Granger, and Raouf A. Khalil

Department of Physiology and Biophysics and Center for Excellence in Cardiovascular-Renal Research, University of Mississippi Medical Center, Jackson, Mississippi 39216 - 4505


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Vascular resistance and arterial pressure are reduced during normal pregnancy, but dangerously elevated during pregnancy-induced hypertension (PIH), and changes in nitric oxide (NO) synthesis have been hypothesized as one potential cause. In support of this hypothesis, chronic inhibition of NO synthesis in pregnant rats has been shown to cause significant increases in renal vascular resistance and hypertension; however, the cellular mechanisms involved are unclear. We tested the hypothesis that the pregnancy-associated changes in renal vascular resistance reflect changes in contractility and intracellular Ca2+ concentration ([Ca2+]i) of renal arterial smooth muscle. Smooth muscle cells were isolated from renal interlobular arteries of virgin and pregnant Sprague-Dawley rats untreated or treated with the NO synthase inhibitor nitro-L-arginine methyl ester (L-NAME; 4 mg · kg-1 · day-1 for 5 days), then loaded with fura 2. In cells of virgin rats incubated in Hanks' solution (1 mM Ca2+), the basal [Ca2+]i was 86 ± 6 nM. Phenylephrine (Phe, 10-5 M) caused a transient increase in [Ca2+]i to 417 ± 11 nM and maintained an increase to 183 ± 8 nM and 32 ± 3% cell contraction. Membrane depolarization by 51 mM KCl, which stimulates Ca2+ entry from the extracellular space, caused maintained increase in [Ca2+]i to 292 ± 12 nM and 31 ± 2% contraction. The maintained Phe- and KCl-induced [Ca2+]i and contractions were reduced in pregnant rats but significantly enhanced in pregnant rats treated with L-NAME. Phe- and KCl-induced contraction and [Ca2+]i were not significantly different between untreated and L-NAME-treated virgin rats or between untreated and L-NAME + L-arginine treated pregnant rats. In Ca2+-free Hanks', application of Phe or caffeine (10 mM), to stimulate Ca2+ release from the intracellular stores, caused a transient increase in [Ca2+]i and a small cell contraction that were not significantly different among the different groups. Thus renal interlobular smooth muscle of normal pregnant rats exhibits reduction in [Ca2+]i signaling that involves Ca2+ entry from the extracellular space but not Ca2+ release from the intracellular stores. The reduced renal smooth muscle cell contraction and [Ca2+]i in pregnant rats may explain the decreased renal vascular resistance associated with normal pregnancy, whereas the enhanced cell contraction and [Ca2+]i during inhibition of NO synthesis in pregnant rats may, in part, explain the increased renal vascular resistance associated with PIH.

vascular resistance; hypertension; calcium; contraction


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

NORMAL PREGNANCY IS OFTEN associated with increases in plasma volume and cardiac output and decreases in peripheral vascular resistance and mean arterial pressure (10, 16, 19, 23, 56). In addition to the pregnancy-associated changes in the systemic hemodynamics, specific regional changes in the renal circulation have been reported, including significant increases in renal plasma flow and glomerular filtration rate and reduction in renal vascular resistance (2, 10, 11, 17, 62). The pregnancy-associated changes in volume homeostasis, systemic hemodynamics, and regional renal blood flow may represent, to a certain extent, consecutive adjustments to a primary decrease in vascular tone (18). An increase in nitric oxide (NO) production by many cell types, including vascular endothelial cells, during normal pregnancy (5, 12, 14, 39, 53) has been suggested to bring about decreases in vascular tone, vascular resistance, and arterial pressure by direct vasodilatory actions and by blunting the vascular responsiveness to circulating vasoconstrictor agonists (41, 44, 53). This is supported by reports that the expression and specific actions of NO synthase (NOS) in several tissues, particularly those of the kidney, are elevated during late gestation (1, 12, 52, 60) and that the plasma level, metabolic production, and urinary excretion of cyclic cGMP, a second messenger of NO and a cellular mediator of vascular smooth muscle relaxation, are increased during pregnancy (13).

In ~5-10% of all pregnancies, women develop a condition called preeclampsia. Preeclampsia is characterized by severe edema, proteinuria, increased vascular resistance, and pregnancy-induced hypertension (PIH) (35, 36, 48, 54, 66). Although the exact mechanisms of PIH have not been clearly identified, abnormal reduction in NO synthesis during late pregnancy has been hypothesized as one potential cause (3, 6, 9, 21, 28). In support of this hypothesis, we and others have shown that chronic inhibition of NOS in rats during mid- to late gestation is associated with many of the pathological changes observed in women with PIH including significant increases in renal vascular resistance and arterial pressure (5, 28, 40, 47, 63). However, the intermediary vascular and cellular mechanisms involved in the increased renal vascular resistance observed during NOS inhibition in late pregnancy are not fully understood.

We have previously reported that the vascular reactivity of large-conduit arteries, such as the thoracic aorta, is decreased in pregnant rats but significantly enhanced in pregnant rats chronically treated with NOS inhibitors, and alteration of one or more of the signaling mechanisms of vascular smooth muscle contraction has been suggested as one possible cause (15, 29). It is generally accepted that vascular smooth muscle contraction is triggered by increases in intracellular free Ca2+ concentration ([Ca2+]i) due to Ca2+ release from the intracellular stores and Ca2+ entry from the extracellular space (32). In addition, several protein kinases and phosphatases, such as myosin light chain kinase, protein kinase C, Rho-kinase, and mitogen-activated protein kinase, have been suggested to contribute to smooth muscle contraction (25, 27, 55). Although previous studies from our laboratory and others' have suggested pregnancy-associated changes in the vascular Ca2+-mobilization mechanisms (15, 49), these studies were performed on large-conduit arteries, such as the thoracic aorta, and therefore, the findings of these studies may not apply to the physiologically more relevant small resistance arteries particularly those of the kidney. Also, in these previous studies, radioactive 45Ca2+ was used to provide a rather crude estimate of the net aortic uptake of all lumped forms of Ca2+, both bound and free, and therefore did not provide accurate measurements of the pregnancy-associated changes in the more pertinent activator [Ca2+]i in vascular smooth muscle. In effect, to our knowledge, no information is available on the pregnancy-associated changes in [Ca2+]i in vascular smooth muscle, in general, or in the smooth muscle of the physiologically more relevant small resistance arteries of the kidney, in particular. Furthermore, because pregnancy may be associated with multiple vascular changes in various types of vascular cells, studying the pregnancy-associated changes in the Ca2+-mobilization mechanisms of vascular smooth muscle contraction in a multicellular vascular preparation such as the aorta could be difficult, making it necessary to measure [Ca2+]i in single vascular smooth muscle cells. Although [Ca2+]i has previously been measured in single preglomerular arterial smooth muscle cells of male rats (51), no information is available on the pregnancy-associated changes in [Ca2+]i in single renal interlobular smooth muscle cells of female rats.

The purpose of the present study was to test the hypothesis that the reduction of renal vascular resistance during normal pregnancy and its increase during NOS inhibition in late pregnancy are associated with changes in contractility and [Ca2+]i of renal arterial smooth muscle cells. The specific aims were 1) to determine whether normal pregnancy in rats is associated with reduction in contractility and [Ca2+]i of single renal interlobular arterial smooth muscle cells, 2) to determine whether chronic reduction in NO synthesis during late pregnancy is associated with increases in contractility and [Ca2+]i of renal arterial smooth muscle cells, and 3) to determine the source of the pregnancy-associated changes in [Ca2+]i of renal arterial smooth muscle cells, if any, and whether it is due to changes in the Ca2+-release mechanisms from the intracellular stores and/or the Ca2+-entry pathways from the extracellular space.

The resting cell length and basal [Ca2+]i and the changes in cell contraction and [Ca2+]i in response to smooth muscle activators with different mechanisms of action were measured in single smooth muscle cells freshly isolated from renal interlobular arteries of virgin and pregnant Sprague-Dawley rats untreated or treated with the NOS inhibitor nitro-L-arginine methyl ester (L-NAME), then loaded with the Ca2+ indicator fura 2.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Female virgin (10-12 wk of age) and time-pregnant (day 12 of gestation) Sprague-Dawley rats were purchased from Harlan Sprague Dawley (Indianapolis, IN). The rats were housed individually in the animal facility and maintained on ad libitum standard rat chow on a 12:12-h light-dark cycle. Virgin rats were either untreated (n = 24) or treated with L-NAME (n = 24). Late-pregnant rats were studied at days 19-21 of gestation (n = 24). Other late-pregnant rats were treated with L-NAME (n = 24) or with L-NAME plus the NOS substrate L-arginine (n = 12). The average weight of virgin rats was 240 ± 5.8 g compared with 349 ± 2.9 g in late-pregnant rats. All procedures were performed in accordance with the guidelines of the Animal Care and Use Committee at the University of Mississippi Medical Center and the American Physiological Society.

Protocol for L-NAME treatment. Pregnant and virgin rats in the untreated groups received drinking water. Pregnant and virgin rats in the treated groups received L-NAME (Sigma, St. Louis, MO) at a dose of ~4 mg · kg-1 · day-1. This relatively small dose of L-NAME has been shown to cause significant elevation of blood pressure in pregnant rats, whereas it has a minimal effect in virgin rats (15, 29, 40). L-NAME treatment of the pregnant rats began at day 15 of gestation and continued for 4-6 days before the rats were killed and the tissues harvested at days 19-21 of gestation. Because water intake in pregnant rats was approximately two times that in virgin rats, the amount of L-NAME in the drinking water was adjusted to maintain a daily dose of ~4 mg · kg-1 · day-1 in both pregnant and virgin rats. Some of the L-NAME-treated pregnant rats simultaneously received L-arginine (Sigma) in the drinking water at a dose of ~80 mg · kg-1 · day-1 for the same period of time (4-6 days). L-Arginine did not significantly affect the amount of drinking water in pregnant rats. Therefore, the amount of L-NAME the animals ingested was similar between pregnant rats treated with L-NAME and pregnant rats treated with L-NAME plus L-arginine. With the use of this protocol, the recorded systolic blood pressure on the day of the experiment, using an automated sphygmomanometer with a tail cuff device, was 118 ± 3 mmHg in virgin rats, 125 ± 6 mmHg in virgin rats treated with L-NAME, 113 ± 5 mmHg in late-pregnant rats, 172 ± 6 mmHg in late-pregnant rats treated with L-NAME, and 124 ± 3 mmHg in late-pregnant rats simultaneously treated with L-NAME plus L-arginine.

Single cell isolation. Rats were anesthetized by inhalation of isoflurane. The kidneys were rapidly removed and placed in oxygenated Krebs solution. The main branches of the right and left renal arteries were carefully dissected under microscopic visualization down to the interlobular renal arteries (<= 150 µm diameter). Single renal arterial smooth muscle cells were freshly isolated using a gentle procedure, specifically avoiding aspiration through a pipette or centrifugation as previously described (31, 42). Interlobular renal arterial strips (50 mg) were placed in a siliconized flask containing a tissue digestion mixture of collagenase type II (236 U/mg protein activity, Worthington, Freehold, NJ), elastase grade II (3.25 U/mg protein activity, Boehringer Mannheim, Indianapolis, IN), and trypsin inhibitor type II-soybean (10,000 U/ml, Sigma) in 7.5 ml of Ca2+- and Mg2+-free Hanks' solution supplemented with 30% bovine serum albumin (Sigma). The tissue was incubated three consecutive times in the tissue digestion mixture to yield three batches of cells. For the first batch, the tissue was incubated with 5 mg collagenase, 4 mg elastase, and 147 µl trypsin inhibitor for 60 min. For batches 2 and 3, the collagenase was reduced to 2.5 mg, the trypsin inhibitor was reduced to 122 µl, and the incubation period was reduced to 30 min. The tissue preparation was placed in a shaking water bath at 34°C in an atmosphere of 95% O2 and 5% CO2. At the end of each incubation period, the preparation was rinsed with 12.5 ml Hanks' with albumin and poured over glass coverslips placed in wells and cooled to 2°C. By using the gravitational force, the cells were allowed to settle and adhere to the glass coverslips. Ca2+ was gradually added back to the preparation to avoid the "calcium paradox" (45).

Contractility studies. Coverslips with the attached cells were mounted on a square-shaped opening in the middle of Plexiglas holders using a vaseline seal to prevent leakage of the incubation solution. The coverslip holders were placed on the stage of an inverted Nikon (Diaphot-300) microscope, and the cells were viewed using a Nikon 100× oil-immersion objective. The cell-isolation procedure yielded smooth muscle cells of variable lengths. Only viable, healthy, and spindle-shaped cells >= 40 µm in length were selected in this study. Viable, healthy cells adhered to the glass coverslips and appeared bright, with a halo along the periphery, and without a visible nucleus when viewed with phase-contrast optics. The viability of the smooth muscle cells was confirmed by their consistent and significant contraction in response to phenylephrine (Phe) and KCl. The cells were further characterized and consistently showed significant immunofluorescence signal when labeled with anti-smooth muscle myosin antibody. Cell images were acquired using a PXL charge-coupled device camera (Photometrics, Tucson, AZ) and displayed on a personal computer using PMIS image analysis software (Photometrics). The number of pixels corresponding to the cell length in the cell image was transformed into micrometers using a calibration bar.

Cell length was measured in isolated renal arterial smooth muscle cells of different groups of rats. Three different smooth muscle activators were used. Phe was used to stimulate both Ca2+ release from the intracellular stores and Ca2+ entry from the extracellular space (32, 33). Caffeine was used to activate the Ca2+-induced Ca2+-release mechanism in Ca2+-free solution (34). Membrane depolarization by high KCl was used to activate Ca2+ entry from the extracellular space (32, 33). The changes in cell length in response to Phe (10-5 M), caffeine (10 mM), and KCl (51 mM) were measured, and the magnitude of cell contraction was expressed as [(Li - L)/Li] × 100, where Li is the initial cell length and L is the final cell length.

Measurement of [Ca2+]i. [Ca2+]i was measured in fura 2-loaded single renal arterial smooth muscle cells using the ratio method as previously described (30, 42, 61). The cells were incubated in the fura 2 loading solution for 30 min at 34°C. The loading solution was made of normal Hanks' solution supplemented with 1 µM of the cell permeant fura 2-acetoxymethyl ester (fura 2-AM; Molecular Probes, Eugene, OR) and 0.01% Pluronic F-127 (Sigma). The fura 2-loaded cells were washed twice and further incubated in normal Hanks' solution for at least 30 min to allow complete deesterification of the dye. Nonspecific intracellular esterases hydrolyze the fura 2-AM ester and liberate the Ca2+-sensitive indicator fura 2 (24). Due to the photosensitivity of the fura 2 molecule, precautionary measures were taken throughout the procedure to avoid extensive photobleaching.

The fura 2-loaded cells were viewed through a Nikon Fluor 100× oil-immersion objective (NA 1.3) on an inverted Nikon (Diaphot-300) microscope. The Ca2+ indicator was excited alternately at 340 ± 5 and 380 ± 6 nm using a filter wheel that alternates between the two filters at a frequency of 0.5 Hz. The emitted light was collected at 510 nm to a photomultiplier tube R928 (Ludl Electronic Products, Hawthorne, NY) through a pinhole aperture 1 µm in diameter positioned 1 µm from the plasma membrane and 1 µm from the nucleus. The fluorescent signal was digitized using a module (Mac 2000, Ludl) and analyzed on a personal computer using data-analysis software. The signal-to-noise ratio was improved by averaging eight consecutive fluorescent intensity readings collected by the photomultiplier tube. The fluorescent signal was background subtracted. Spectral shifts that result from binding of the Ca ion allow the fura 2 indicator to be used ratiometrically, making the measurement of [Ca2+]i less sensitive to changes in cell thickness or the extent of dye loading and photobleaching. The 340/380 fluorescence ratio (R) was calculated from the fluorescence intensity at 340 nm divided by that at 380 nm and transformed to the corresponding levels of [Ca2+]i as described by Grynkiewicz et al. (24): [Ca2+]i = Kd(Sf2/Sb2)[(R - Rmin)/(Rmax - R)], where Rmin and Rmax represent the minimal and maximal fluorescence ratios and were measured by adding fura 2-pentapotassium salt (50 µM) into Ca2+-free (10 mM) and Ca2+-replete (2 mM) EGTA solutions, respectively, using Ca2+-EGTA buffers. Kd is the dissociation constant of fura 2 for Ca2+. Sf2/Sb2 is the ratio of the 380 signal in Ca2+-free and Ca2+-replete solutions, respectively. Because the basal and maintained agonist-stimulated changes in [Ca2+]i showed significant fluctuations, 10 consecutive measurements were averaged in each individual cell.

The effects of Phe, caffeine, and membrane depolarization by high KCl on [Ca2+]i were measured in cells of different groups of rats. To further identify the mechanisms of Phe-induced increases in [Ca2+]i, some cells were treated with phentolamine to block alpha -adrenergic receptors, neomycin and 1-(6-((17beta -3-methoxestra-1,3,5(10)-trien-17-yl)amino)hexyl)- 1H-pyrole-2,5-dione (U-73122) to inhibit phospholipase C, or NiCl2 and diltiazem to block Ca2+ entry through plasmalemmal Ca2+ channels.

Solutions. Krebs solution was used for dissecting the tissue and contained (in mM) 120 NaCl, 5.9 KCl, 25 NaHCO3, 1.2 NaH2PO4, 11.5 dextrose, 2.5 CaCl2, and 1.2 MgCl2 at pH 7.4. Hanks' solution was used for cell isolation and for performing the experiments and contained (in mM): 137 NaCl, 5.4 KCl, 0.44 KH2PO4, 0.42 Na2HPO4, 4.17 NaHCO3, 5.55 dextrose and 10 HEPES. The solution was bubbled for 30 min with a 95% O2-5% CO2 mixture, and the pH was adjusted to 7.4. For Ca2+- and Mg2+-containing Hanks', 1 mM CaCl2 and 1.2 mM MgCl2 were added. For Ca2+-free Hanks', CaCl2 was omitted and replaced with 2 mM EGTA.

Drugs and chemicals. Stock solutions of Phe (10-1 M, Sigma) and phentolamine (10-2 M, Sigma) were prepared in distilled water. Caffeine (Sigma) was prepared as 10 mM in Ca2+-free (2 mM EGTA) Hanks' solution. Neomycin sulfate and U-73122 were purchased from Biomol (Plymouth Meeting, PA). Diltiazem was purchased from Calbiochem (La Jolla, CA). All other chemicals were of reagent grade or better.

Statistical analysis. The data are presented as the means ± SE. The raw data were analyzed using ANOVA with multiple classification criteria [pregnancy (pregnant vs. virgin) and L-NAME treatment (treated vs. untreated)] followed by Bonferroni's posttest to compare selected groups or Dunnett's posttest to compare all groups with the control virgin group. Differences were considered statistically significant if P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Resting cell length and Phe-induced contraction in renal arterial smooth muscle cells. The cell-isolation procedure produced cells of variable lengths. Only spindle-shaped cells >= 40 µm in length were selected in this study (Fig. 1A, top). In resting cells of virgin rats, the average cell length was 57.5 ± 1.2 µm (n = 25; Fig. 1B). The resting cell length was longer in pregnant rats (64.5 ± 1.5 µm, n = 25) but shorter in pregnant rats treated with L-NAME (46.5 ± 1.3 µm, n = 20) compared with virgin rats (Fig. 1, A and B). The resting cell length was not significantly different between virgin rats untreated or treated with L-NAME or between pregnant rats untreated or treated with L-NAME + L-arginine (Fig. 1, A and B).


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  		Fig. 1.   Resting cell length (A, top, and B) and phenylephrine (Phe; 10-5 M)-induced contraction (A, bottom, and C) in renal arterial smooth muscle cells isolated from virgin and pregnant rats untreated or treated with nitro-L-arginine methyl ester (L-NAME) and pregnant rats treated with L-NAME + L-arginine and incubated in normal Hanks' (1 mM Ca2+). Data bars represent the means ± SE of measurements in 20-25 cells from 6-10 rats of each group. *Significantly different (P < 0.05) from respective measurements in virgin rats. dagger Not significantly different from respective measurements in untreated pregnant rats. Li, initial cell length.

Freshly isolated renal arterial smooth muscle cells from both virgin and pregnant rats were responsive to contractile stimuli. In normal Hanks' solution (1 mM Ca2+), Phe (10-5 M) caused contraction of the cells that reached a plateau after ~5 min (Fig. 1A, bottom). The average Phe-induced cell contraction after 5 min of stimulation was compared in the different groups of rats. In cells of virgin rats, Phe (10-5 M) caused 32 ± 3% (n = 25) cell contraction (Fig. 1C). The Phe-stimulated cell contraction was reduced in pregnant rats (21 ± 1.5%, n = 25) but significantly enhanced in pregnant rats treated with L-NAME (46 ± 2%, n = 20) compared with virgin rats (Fig. 1C). The Phe-induced cell contraction was not significantly different between virgin rats untreated or treated with L-NAME or between pregnant rats untreated or treated with L-NAME + L-arginine (Fig. 1C).

Basal and Phe-induced [Ca2+]i in normal Hanks' solution (1 mM Ca2+). The basal and Phe-stimulated [Ca2+]i were measured in cells of the different groups of rats. To avoid the fluctuations in the basal [Ca2+]i measurements (Fig. 2), 10 consecutive measurements were averaged in each individual cell. In resting cells of virgin rats, the average basal [Ca2+]i was 86 ± 6 nM (n = 25). Phe caused a transient initial peak in [Ca2+]i followed by a steady-state increase that was maintained for at least 5 min (Fig. 2). Both the initial and maintained increase in [Ca2+]i were abolished in cells treated with the alpha -adrenergic blocker phentolamine (1 µM; Fig. 2A). Also, the Phe-induced increases in [Ca2+]i were significantly inhibited in cells treated with the phospholipase C inhibitor neomycin (0.5 mM; Fig. 2B) or U-73122 (10 µM; data not shown).


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  		Fig. 2.   Effect of phenylephrine (Phe) (10-5 M) on intracellular Ca2+ concentration ([Ca2+]i) in renal arterial smooth muscle cells isolated from virgin rats and incubated in Hanks' solution (1 mM Ca2+). Both the basal and Phe-induced [Ca2+]i were recorded. A: phentolamine (1 µM) was applied on top of Phe-maintained response (left), or the cells were pretreated with phentolamine for 15 min before eliciting the Phe response (right). B: neomycin (0.5 mM) was applied on top of Phe-maintained response (left), or the cells were pretreated with neomycin for 15 min before eliciting the Phe response (right). The figure shows representative traces of experiments in 5-7 cells from 5-7 virgin rats.

The sources of the Phe-induced increases in [Ca2+]i were investigated. The maintained Phe-induced increases in [Ca2+]i were completely abolished in Ca2+-free (2 mM EGTA) Hanks' solution (Fig. 3A) and in the presence of the Ca2+-channel blockers NiCl2 (1 mM; Fig. 3B) or diltiazem (1 µM; Fig. 3C), suggesting that the maintained increases in [Ca2+]i are due to Ca2+ entry from the extracellular space. On the other hand, the transient Phe-induced increase in [Ca2+]i was still present under these conditions (Fig. 3), suggesting that it is mainly due to Ca2+ release from the intracellular stores.


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  		Fig. 3.   Effect of removal of extracellular Ca2+ (A) and Ca2+-entry blockers (B and C) on Phe (10-5 M) induced [Ca2+]i in renal arterial smooth muscle cells isolated from virgin rats. Cells were incubated in Hanks' solution (1 mM Ca2+), then stimulated with Phe for 5 min. A: the bathing solution was rapidly changed to Ca2+-free (2 mM EGTA) Hanks' in the continuous presence of Phe (left), or the cells were incubated in Ca2+-free Hanks' for 1 min before eliciting the Phe response (right). B: NiCl2 (1 mM) was applied on top of Phe-maintained response (left), or the cells were pretreated with NiCl2 for 1 min before eliciting the Phe response (right). C: diltiazem (1 µM) was applied on top of Phe-maintained response (left), or the cells were pretreated with diltiazem for 5 min before eliciting the Phe response (right). The figure shows representative traces of experiments in 6-7 cells from 6-7 virgin rats.

The basal, transient, and maintained Phe-induced increases in [Ca2+]i were compared in cells of different groups of rats. The basal [Ca2+]i was reduced in pregnant rats (63 ± 5 nM, n = 25) but significantly elevated in pregnant rats treated with L-NAME (109 ± 8 nM, n = 20) compared with virgin rats (Fig. 4, A and B). The basal [Ca2+]i was not significantly different between virgin rats untreated or treated with L-NAME or between pregnant rats untreated or treated with L-NAME + L-arginine (Fig. 4, A and B).


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  		Fig. 4.   Basal (A and B) and Phe (10-5 M)-induced transient (A and C) and maintained [Ca2+]i (A and D) in renal arterial smooth muscle cells isolated from virgin and pregnant rats untreated or treated with L-NAME and pregnant rats treated with L-NAME + L-arginine and incubated in Hanks' solution (1 mM Ca2+). The traces are representative of [Ca2+]i measurements. Dashed lines were drawn at the smallest basal and maintained Phe-induced increase in [Ca2+]i recorded in cells of pregnant rats to facilitate comparison with the other groups of rats. Horizontal bars below the traces represent time of application of Phe. The data bars represent the means ± SE of the basal (B) and Phe-induced initial peak [Ca2+]i (C) and maintained [Ca2+]i (D) in 20-25 cells from 6-10 rats of each group. *Significantly different (P < 0.05) from respective measurements in virgin rats. dagger Not significantly different from respective measurements in untreated pregnant rats.

The average Phe-induced initial peak (Fig. 4, A and C) and steady-state increase in [Ca2+]i after 5 min of stimulation (Fig. 4, A and D) were compared in the different groups of rats. To avoid the fluctuations in the maintained Phe-induced increase in [Ca2+]i measurements (Fig. 4A), 10 consecutive measurements were averaged in each individual cell. In cells of virgin rats, Phe (10-5 M) caused an initial peak in [Ca2+]i to 417 ± 11 nM (n = 25; Fig. 4, A and C) and a maintained increase in [Ca2+]i to 183 ± 8 nM (n = 25; Fig. 4, A and D). The Phe-induced initial peak [Ca2+]i was not significantly different among the different groups of rats (Fig. 4C), suggesting no difference in Ca2+ release from the intracellular stores. In contrast, the Phe-induced and maintained [Ca2+]i (Fig. 4D) was significantly reduced in pregnant rats (149 ± 8 nM, n = 25) but significantly enhanced in pregnant rats treated with L-NAME (234 ± 11 nM, n = 20) compared with virgin rats, suggesting pregnancy-associated differences in Ca2+ entry from the extracellular space. On the other hand, the Phe-induced, maintained [Ca2+]i was not significantly different between virgin rats untreated or treated with L-NAME or between pregnant rats untreated or treated with L-NAME + L-arginine (Fig. 4D).

Cell contraction and [Ca2+]i in Ca2+-free medium. To further investigate the role of the intracellular Ca2+ release mechanisms, the Phe response in Ca2+-free (2 mM EGTA) Hanks' solution was measured. In cells of virgin rats incubated in Ca2+-free (2 mM EGTA) Hanks' for 1 min, the basal [Ca2+]i was reduced to 33 ± 2 (n = 32), which was not significantly different from that in cells isolated from the other groups of virgin and pregnant rats. Also, in cells of virgin rats, Phe (10-5 M) caused 10 ± 1% (n = 16) cell contraction (Fig. 5A) and a transient increase in [Ca2+]i to 329 ± 15 nM (n = 16), which were not significantly different from the respective measurements in the other groups of rats (Fig. 5, B and C). Similarly, in Ca2+-free (2 mM EGTA) Hanks' solution, caffeine (10 mM) caused 8 ± 1.5% (n = 16) cell contraction (Fig. 6A) and a transient increase in [Ca2+]i to 434 ± 14 (n = 16) in cells of virgin rats, which were not significantly different from the respective measurements in the other groups of virgin and pregnant rats (Fig. 6, B and C).


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  		Fig. 5.   Phe (10-5 M)-induced cell contraction (A) and [Ca2+]i transient (B and C) in renal arterial smooth muscle cells isolated from virgin and pregnant rats untreated or treated with L-NAME and pregnant rats treated with L-NAME + L-arginine and incubated in Ca2+-free (2 mM EGTA) Hanks' solution. The figure shows data bars of the means ± SE and representative [Ca2+]i traces of measurements in 15 or 16 cells from 5-8 rats of each group. Horizontal bars below the traces represent time of application of Phe.



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  		Fig. 6.   Caffeine (10 mM)-induced cell contraction (A) and [Ca2+]i transient (B and C) in renal arterial smooth muscle cells isolated from virgin and pregnant rats untreated or treated with L-NAME and pregnant rats treated with L-NAME + L-arginine and incubated in Ca2+-free (2 mM EGTA) Hanks' solution. The figure shows data bars of the means ± SE and representative [Ca2+]i traces of measurements in 14-16 cells from 7 or 8 rats of each group. Horizontal bars below the traces represent time of application of caffeine.

Effect of membrane depolarization by high KCl. To further investigate whether the observed pregnancy-associated changes in cell contraction and [Ca2+]i reflect changes in Ca2+ entry from the extracellular space, the response to high KCl solution was measured. Membrane depolarization by high KCl is known to stimulate Ca2+ entry through voltage-gated Ca2+ channels (32, 33). In cells of virgin rats, KCl (51 mM) caused cell contraction that reached a plateau after ~5 min. KCl also caused an increase in [Ca2+]i that was maintained for at least 5 min (Fig. 7, A and B). The KCl-induced increase in [Ca2+]i was completely abolished by the nonselective Ca2+ entry blocker NiCl2 (1 mM; Fig. 7A) or the Ca2+-channel blocker diltiazem (1 µM; Fig. 7B), suggesting that it is due to Ca2+ entry from the extracellular space.


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  		Fig. 7.   Effect of membrane depolarization by 51 mM KCl on [Ca2+]i in renal arterial smooth muscle cells isolated from virgin rats. Both the basal and KCl-induced [Ca2+]i were recorded. A: NiCl2 (1 mM) was applied on top of KCl response (left), or the cells were pretreated with NiCl2 for 1 min before eliciting the KCl response (right). B: diltiazem (1 µM) was applied on top of KCl response (left), or the cells were pretreated with diltiazem for 5 min before eliciting the KCl response (right). The figure shows representative traces of experiments in 6 or 7 cells from 6 or 7 virgin rats.

The average KCl-induced cell contraction and [Ca2+]i after 5 min of stimulation were compared in the different groups of rats. To avoid the fluctuations in the KCl-stimulated [Ca2+]i measurements (Fig. 8), 10 consecutive measurements were averaged in each individual cell. In cells of virgin rats, KCl caused 31 ± 2% (n = 25) cell contraction (Fig. 8A) and increased [Ca2+]i to 292 ± 12 nM (n = 25; Fig. 8, B and C). The KCl-induced cell contraction (Fig. 8A) and [Ca2+]i (Fig. 8C) were significantly reduced in pregnant rats but significantly enhanced in pregnant rats treated with L-NAME compared with virgin rats or virgin rats treated with L-NAME, suggesting differences in Ca2+ entry from the extracellular space through voltage-gated Ca2+ channels. The KCl-induced responses were not significantly different between virgin rats untreated or treated with L-NAME or between pregnant rats untreated or treated with L-NAME + L-arginine (Fig. 8).


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  		Fig. 8.   Effect of 51 mM KCl on cell contraction (A) and [Ca2+]i (B and C) in renal arterial smooth muscle cells isolated from virgin and pregnant rats untreated or treated with L-NAME and pregnant rats treated with L-NAME + L-arginine. The figure shows data bars of the means ± SE and representative traces of [Ca2+]i measurements in 20-25 cells from 7-10 rats of each group. Dashed lines were drawn at the smallest basal and KCl-induced increase in [Ca2+]i recorded in cells of pregnant rats to facilitate comparison with the other groups of rats. Horizontal bars below the traces represent time of application of KCl. *Significantly different (P < 0.05) from respective measurements in virgin rats. dagger Not significantly different from respective measurements in untreated pregnant rats.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study showed that the resting cell length of resistance renal arterial smooth muscle cells was longer in pregnant rats than in virgin rats, suggesting reduction in basal smooth muscle tone in resistance renal arteries during pregnancy. These data are consistent with other reports that the intrinsic basal tone of isolated resistance mesenteric arteries and renal interlobar arteries is reduced during late pregnancy in rats (22, 38). We also found that the contraction of renal arterial smooth muscle cells in response to alpha -adrenergic stimulation by Phe was reduced in pregnant rats compared with virgin rats. Although the observed decrease in renal arterial smooth muscle cell contraction to Phe in pregnant rats can be explained by a decrease in the sensitivity to Phe at the alpha -adrenergic receptor level, it could also be due to inhibition of signaling mechanisms downstream from receptor activation.

The interaction of Phe with its receptor is believed to cause activation of phospholipase C and to increase the hydrolysis of phosphatidlylinositol 4,5-bisphosphate into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (7). IP3 stimulates Ca2+ release from intracellular stores (57), and diacylglycerol stimulates protein kinase C (27, 46). In addition, Phe may enhance Ca2+ entry through plasma membrane Ca2+ channels (32). We found that the Phe-induced contraction and [Ca2+]i in renal arterial smooth muscle cells were completely abolished by the alpha -adrenergic blocker phentolamine, suggesting that the Phe-induced contraction and [Ca2+]i are mediated via alpha -adrenergic receptors. Also, the significant inhibition of Phe-induced contraction and [Ca2+]i by the phospholipase C inhibitors neomycin and U-73122 provides evidence that the Phe responses involve activation of PLC, which, in turn, increases the turnover of plasma membrane phospholipids. The observation that the initial Phe-induced Ca2+ transient was still observed in Ca2+-free solution and in cells treated with the Ca2+-channel blockers NiCl2 and diltiazem provides evidence that the initial peak [Ca2+]i is mainly due to Ca2+ release from the intracellular stores. On the other hand, the complete inhibition of the maintained Phe-induced increase in [Ca2+]i in Ca2+-free solution or in cells treated with the Ca2+-channel blockers NiCl2 and diltiazaem provides evidence that the maintained increase in [Ca2+]i involves Ca2+ entry from the extracellular space.

We found that the Phe-induced cell contraction and transient increase in [Ca2+]i in Ca2+-free solution, which are often used as measures of IP3-releasable intracellular Ca2+ stores (32, 33), were not significantly different in pregnant rats compared with virgin rats, suggesting that the reduced smooth muscle cell contraction and [Ca2+]i observed in pregnant rats are not due to changes in Ca2+ uptake to or Ca2+ release from the IP3-releasable intracellular Ca2+ stores. Also, the caffeine-induced contraction and transient increase in [Ca2+]i in Ca2+-free solution, which are often used as measures of the Ca2+-induced Ca2+-release mechanism from the intracellular Ca2+ stores (34), were not significantly different in pregnant rats compared with virgin rats, providing further evidence that the reduced smooth muscle cell contraction and [Ca2+]i observed in pregnant rats are less likely due to changes in Ca2+ uptake to or Ca2+ release from the intracellular stores.

We found that the Phe-induced, maintained [Ca2+]i in Ca2+-containing medium was reduced in pregnant rats compared with virgin rats, suggesting that a Ca2+-entry pathway may be reduced in renal arterial smooth muscle during pregnancy. To investigate the possible Ca2+-entry pathways involved, we compared the Phe response with that induced by KCl. Membrane depolarization by high KCl is known to stimulate Ca2+ entry through voltage-gated Ca2+ channels (32, 33). The present study showed that the KCl-induced cell contraction and [Ca2+]i were completely inhibited by the Ca2+-channel blockers NiCl2 and diltiazem, supporting the contention that the KCl responses involve Ca2+ entry from the extracellular space. Also, the KCl-induced cell contraction and [Ca2+]i were reduced in normal pregnant rats, providing evidence that Ca2+ entry from the extracellular space, possibly through voltage-gated Ca2+ channels, and into renal arterial smooth muscle is reduced during pregnancy. The cause of the reduced Ca2+ entry from the extracellular space is unclear at the present time but could be related to the possibility that either the Ca2+ permeability or the number of voltage-gated Ca2+ channels is reduced during pregnancy and should represent an important area for future investigations. However, the present results cannot exclude the possibility that other types of Ca2+ channels may also be involved in the observed decrease in Phe-induced cell contraction and [Ca2+]i during pregnancy. These Ca2+ channels have been described in several types of smooth muscle and have been termed receptor-operated Ca2+ channels (32).

Although a clear relationship between the changes in systemic hemodynamics and regional renal blood flow observed during late pregnancy and the renal arterial smooth muscle cell contraction and [Ca2+]i is difficult to discern at the present time, such a relationship is conceivable. It has been suggested that an increase in endogenous NO production during late pregnancy causes a decrease in vascular reactivity (39, 53), perhaps through increased formation of cGMP in vascular smooth muscle (12, 13). Other studies have shown that cGMP reduces [Ca2+]i by decreasing Ca2+ entry from the extracellular space through voltage-gated Ca2+ channels (37, 50, 58). On the basis of these premises, one would predict that blocking NO production during late pregnancy would bring the renal arterial smooth muscle cell contraction and [Ca2+]i back to the level observed in virgin rats. However, we observed that the Phe- and KCl-induced cell contraction and [Ca2+]i in pregnant rats treated with L-NAME were significantly greater than those in virgin rats and virgin rats treated with L-NAME. These results suggest that treatment of pregnant rats with L-NAME not only inhibits NO synthesis but may also increase the synthesis of, or sensitivity to, other vasoactive compounds that would increase the Phe- and KCl-induced cell contraction and [Ca2+]i. It has been hypothesized that abnormal reduction in uteroplacental blood flow during late pregnancy could trigger the release of certain cytokines from the placenta that, in turn, may alter the endothelial cell function leading to reduction in the synthesis of vasodilators such as NO or prostacyclin or, more importantly, increased production of vasoconstrictor factors such as endothelin or thromboxanes (4, 5, 40, 47). In support of this hypothesis, preliminary studies from our laboratory have shown that chronic NO-synthesis inhibition during pregnancy in rats is associated with an increase in the urinary excretion of thromboxanes (47). Also, it has been reported that long-term inhibition of NO synthesis during mid- to late gestation in rats is associated with increased blood pressure and elevated plasma levels of endothelin-1 (20). Thus evidence for an increase in the release of thromboxanes and endothelin has been observed during chronic inhibition of NO synthesis in pregnant rats. Thromboxanes and endothelin have also been shown to increase vascular reactivity by increasing Ca2+ entry through Ca2+ channels in vascular smooth muscle (8, 26, 43, 59, 64, 65), specifically in preglomerular renal arterial smooth muscle (51). However, the question remains whether the vascular reactivity to thromboxanes and endothelin is enhanced in isolated vascular smooth muscle preparations of pregnant rats chronically treated with NOS inhibitors compared with untreated control pregnant rats and should represent important areas for future investigations.

We found that the Phe- and caffeine-induced [Ca2+]i transient and cell contraction in Ca2+-free solution were not significantly changed in pregnant rats treated with L-NAME compared with pregnant rats, virgin rats, or virgin rats treated with L-NAME, suggesting that the enhanced cell contraction in pregnant rats treated with L-NAME is not due to changes in Ca2+ uptake to or Ca2+ release from the intracellular stores. On the other hand, the observed increase in the Phe-induced, maintained [Ca2+]i and contraction in pregnant rats treated with L-NAME suggest increased Ca2+ entry from the extracellular space through one or more type of excitable Ca2+ channel. The present results suggest that the enhanced cell contraction and [Ca2+]i in pregnant rats treated with L-NAME may involve stimulation of Ca2+ entry through voltage-gated Ca2+ channels, because the cell contraction and [Ca2+]i in response to KCl, a known activator of voltage-gated Ca2+ channels, were enhanced in pregnant rats treated with L-NAME. The enhanced Ca2+ entry could well be related to an increase in the permeability of the individual Ca2+ channel or to an increase in the number of voltage-gated Ca2+ channels. However, the present results cannot rule out the possibility that other types of excitable Ca2+ channels, such as the receptor-operated Ca2+ channels, may also be involved. Also, the enhanced Phe-induced cell contraction could be due to activation of other contractile mechanisms in addition to Ca2+ entry. For example, Phe may activate protein kinase C through increased formation of diacylglycerol (27, 31, 46).

Finally, the present study showed that, compared with the L-NAME-treated pregnant rats, in pregnant rats simultaneously treated with L-NAME + L-arginine, the Phe- and KCl-induced cell contraction and [Ca2+]i were significantly reduced to levels not significantly different from those observed in the untreated pregnant rats, lending support to the contention that the enhanced responses observed in the L-NAME-treated pregnant rats are possibly due to inhibition of the L-arginine-NO pathway.

In conclusion, the present results provide evidence that in renal arterial smooth muscle, cell contraction and [Ca2+]i are reduced during pregnancy but significantly enhanced during chronic inhibition of NO synthesis in pregnant rats. The pregnancy-associated changes in renal arterial smooth muscle cell contraction and [Ca2+]i involve Ca2+ entry from the extracellular space but not Ca2+ release from the intracellular stores. The reduced renal arterial smooth muscle cell contraction and [Ca2+]i in control pregnant rats may explain the decreased renal vascular resistance associated with normal pregnancy. The enhanced renal arterial smooth muscle cell contraction and [Ca2+]i during inhibition of NO synthesis in late-pregnant rats may, in part, explain the increased renal vascular resistance associated with PIH.

Perspectives

Understanding the changes in the cellular mechanisms of vascular smooth muscle contraction in animal models of PIH should help us to better understand the pathophysiological basis of preeclampsia in pregnant women. Abnormal reduction in uteroplacental blood flow during late pregnancy has been suggested as an initiating event that triggers a cascade of events leading to increased vascular resistance and hypertension. Several intermediary events have been suggested including the release of cytokines from the placenta, which may, in turn, alter endothelial cell function leading to reduction in the synthesis of vasodilators such as NO or prostacyclin or increased production of vasoconstrictor factors such as endothelin or thromboxanes. The present results in pregnant rats chronically treated with NOS inhibitor provide evidence that reduction of NO production during late pregnancy is associated with increased contractility and [Ca2+]i in renal interlobular arterial smooth muscle cells and may, therefore, represent one of the intermediary mechanisms of the increased renal vascular resistance associated with PIH. Other possible intermediary mechanisms, such as reduction in prostacyclin synthesis or increased production of endothelin and thromboxanes, are less clear and should be investigated in future studies.


    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grants HL-51971 and HL-33849 to J. P. Granger and HL-52696 to R. A. Khalil and a grant-in-aid from the American Heart Association, Mississippi affiliate.


    FOOTNOTES

Address for reprint requests and other correspondence: R. A. Khalil, Dept. of Physiology and Biophysics, Univ. of Mississippi Medical Center, 2500 North State St., Jackson, MS 39216-4505 (E-mail: rkhalil{at}physiology.umsmed.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 30 May 2000; accepted in final form 7 September 2000.


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DISCUSSION
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