Differential [Ca²⁺]_i signaling of vasoconstriction in mesenteric microvessels of normal and reduced uterine perfusion pregnant rats

Abstract

Vascular resistance and blood pressure (BP) are reduced during late normal pregnancy (Norm-Preg). In contrast, studies in human preeclampsia and in animal models of hypertension in pregnancy (HTN-Preg) have suggested that localized reduction in uterine perfusion pressure (RUPP) in late pregnancy is associated with increased systemic vascular resistance and BP; however, the vascular mechanisms involved are unclear. Because Ca²⁺ is a major determinant of vascular contraction, we hypothesized that the intracellular free calcium concentration ([Ca²⁺]_i) signaling of vasoconstriction is differentially regulated in systemic microvessels during normal and RUPP in late pregnancy. Pressurized mesenteric microvessels from Norm-Preg and RUPP rats were loaded with fura 2 in preparation for simultaneous measurement of diameter and [Ca²⁺]_i (presented as fura 2 340/380 ratio). Basal [Ca²⁺]_i was lower in RUPP (0.73 ± 0.03) compared with Norm-Preg rats (0.82 ± 0.03). Membrane depolarization by 96 mM KCl, phenylephrine (Phe, 10⁻⁵ M), angiotensin II (ANG II, 10⁻⁷ M), or endothelin-1 (ET-1, 10⁻⁷ M) caused an initial peak followed by maintained vasoconstriction and [Ca²⁺]_i. KCl caused similar peak vasoconstriction and [Ca²⁺]_i in Norm-Preg (45.5 ± 3.3 and 0.89 ± 0.02%) and RUPP rats (46.3 ± 2.1 and 0.87 ± 0.01%). Maximum vasoconstriction to Phe, ANG II, and ET-1 was not significantly different between Norm-Preg (28.6 ± 4.8, 32.5 ± 6.3, and 40 ± 4.6%, respectively) and RUPP rats (27.8 ± 5.9, 34.4 ± 4.3, and 38.8 ± 4.1%, respectively). In contrast, the initial Phe-, ANG II-, and ET-1-induced 340/380 ratio ([Ca²⁺]_i) was reduced in RUPP (0.83 ± 0.02, 0.82 ± 0.02, and 0.83 ± 0.03, respectively) compared with Norm-Preg rats (0.95 ± 0.04, 0.93 ± 0.01, and 0.92 ± 0.02, respectively). Also, the [Ca²⁺]_i-vasoconstriction relationship was similar in KCl-treated but shifted to the left in Phe-, ANG II-, and ET-1-treated microvessels of RUPP compared with Norm-Preg rats. The lower agonist-induced [Ca²⁺]_i signal of vasoconstriction and the leftward shift in the [Ca²⁺]_i-vasoconstriction relationship in microvessels of RUPP compared with Norm-Preg rats suggest activation of [Ca²⁺]_i sensitization pathway(s). The similarity in vasoconstriction in RUPP and Norm-Preg rats suggests that such a [Ca²⁺]_i sensitization pathway(s) may also provide a feedback effect on Ca²⁺ mobilization/homeostatic mechanisms to protect against excessive vasoconstriction in systemic microvessels during RUPP in late pregnancy.

during normal pregnancy(Norm-Preg) increases in plasma volume, heart rate, and renal blood flow, as well as decreases in systemic vascular resistance, blood pressure (BP), and vascular reactivity to circulating vasoconstrictors are often observed (8, 18, 37, 65). In contrast, in 5–7% of pregnancy, women develop a condition called preeclampsia, which is characterized by increased intravascular coagulation, proteinuria, increased systemic vascular resistance, and hypertension in pregnancy (HTN-Preg; Refs. 24, 25, 32, 59, 60, 63, 66). Although HTN-Preg is a major cause of maternal morbidity, fetal mortality, and low-birth-weight (2, 3, 27), its exact mechanism has not been clearly identified.

Because of the difficulty to perform mechanistic studies in women with preeclampsia, animal models of HTN-Preg have been developed (1, 13, 14, 44, 57, 58). Studies in these animal models have led to the concept that reduction in the uteroplacental perfusion pressure and the ensuing placental ischemia/hypoxia during late pregnancy may represent the initiating events that eventually lead to increased systemic vascular resistance and HTN-Preg. In support of this concept, studies (5, 6, 14, 26, 63) have demonstrated that reduction of uterine perfusion pressure (RUPP) in late pregnant rats is associated with significant increases in renal vascular resistance and BP; however, the vascular and cellular mechanisms involved have not been clearly elucidated.

Vascular smooth muscle (VSM) contraction is triggered by an increase in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) due to initial Ca²⁺ release from the intracellular stores and maintained Ca²⁺ entry from the extracellular space (39–41, 51). Ca²⁺ binds calmodulin to form a complex that activates myosin light chain kinase, causes myosin light chain phosphorylation, initiates actin-myosin interaction, and produces VSM contraction (30, 62). Previous studies (51) in isolated VSM cells suggested that phenylephrine (Phe)-induced contraction and [Ca²⁺]_i are reduced in aortic VSM of female compared with male rats. Also, Phe-induced vascular contraction is reduced in aortic segments of Norm-Preg compared with virgin rats but significantly enhanced in rat models of HTN-Preg produced by administration of the nitric oxide synthase (NOS) inhibitor N^ω-nitro-l-arginine (l-NAME) or by inducing RUPP in late pregnancy (14, 15, 36, 45). We have also shown that both contraction and [Ca²⁺]_i are enhanced in renal arterial VSM cells isolated from RUPP rats compared with Norm-Preg rats (50). However, the vascular responses during pregnancy may not be uniform and may vary depending on the vascular bed studied and the vessel size down the arterial tree, i.e., large, intermediate, small, and microvessels. The differences in the responses of various blood vessels can be related to differences in vasoconstrictor receptor distribution, receptor-coupling mechanisms, and postreceptor mechanisms particularly [Ca²⁺]_i control mechanisms and [Ca²⁺]_i sensitization pathways (7, 31, 45). Previous studies (6) have shown that the mesenteric vascular resistance is elevated in rat models of HTN-Preg. Also, the mesenteric vascular resistance is reduced in Norm-Preg compared with nonpregnant normotensive rats (61, 68, 74) and in Norm-Preg compared with nonpregnant spontaneously hypertensive rats (12). However, little is known about the pregnancy-associated changes in the mechanisms of vasoconstriction in the small microvessels of the systemic circulation, which are directly relevant to the changes in BP. Also, in our previous studies (15, 49, 50), we examined the pregnancy-associated changes in vascular contraction and [Ca²⁺]_i in response to only one agonist, making it difficult to appreciate whether the observed alterations are specific to a particular agonist/receptor or represent changes in a common signaling mechanism downstream from receptor activation.

In the present study, we tested the hypothesis that the Ca²⁺-dependent mechanisms of vasoconstriction in the systemic microvessels are differentially regulated under conditions of normal and RUPP in late pregnancy. We used small mesenteric microvessels isolated from the well-described RUPP rat model of HTN-Preg and control Norm-Preg rats to determine the following: 1) whether the mesenteric microvessel reactivity to four different vasoconstrictor stimuli is altered in RUPP compared with Norm-Preg rats; 2) whether the alterations in microvessel reactivity in RUPP compared with Norm-Preg rats reflect differences in the microvessel [Ca²⁺]_i; and 3) whether the alterations in microvessel reactivity in RUPP compared with Norm-Preg rats reflect differences in the microvessel vasoconstriction sensitivity to [Ca²⁺]_i.

MATERIAL AND METHODS

Animals.

Time-pregnant (day 12) female Sprague-Dawley rats (12 wk of age) were purchased from Charles River Laboratories (Wilmington, MA). The rats were housed in the animal facility and maintained on ad libitum standard rat chow and tap water in 12-h light-dark cycle. All surgical procedures were performed using aseptic technique and proper anesthetics and analgesics in accordance with the National Institutes of Health Guide for the Care of Laboratory Animal Welfare Act, the guidelines of the Animal Care and Use Committee at Harvard Medical School, and the American Physiological Society.

Protocol for RUPP.

On day 13 of pregnancy, pregnant rats destined to be in the RUPP group were anesthetized by inhalation of isoflurane, the abdominal cavity was opened by a midline incision, the lower abdominal aorta was exposed, and a silver clip (0.203 mm ID) was placed around the aorta above the iliac bifurcation as previously described (1, 6, 14, 26, 50). This procedure has been shown to reduce uterine perfusion pressure in the gravid rat by ∼40% (20). Since compensation of blood flow to the placenta occurs through an adaptive increase in ovarian blood flow (53), a silver clip (0.1 mm ID) was also placed on the main uterine branches of both the right and left ovarian arteries. Norm-Preg rats were sham operated. RUPP rats in which the clipping procedure resulted in maternal death or total resorption of the fetuses were excluded from the data analyses. With the use of the same RUPP protocol, the BP was ∼25–35 mmHg greater in RUPP rats compared with Norm-Preg rats as previously reported (1, 5, 6, 14, 22, 26, 50, 56).

Tissue preparation.

On gestational day 19, the rats were euthanized by inhalation of CO₂. The abdominal cavity was opened, the pups and placentas were removed, the pups were weighed, and the litter size was recorded. The small intestine, adjacent mesentery, and mesenteric arterial arcade were excised and placed in ice-cold oxygenated Krebs solution. Small mesenteric arteries third or fourth order were dissected free of surrounding adipose tissue under microscopic visualization.

Pressurized microvessels.

A 1- to 2-mm microvessel segment was transferred to a temperature-controlled perfusion chamber (5 ml), mounted between two glass micropipettes (cannulas), and secured with 10–0 ophthalmic suture (Living Systems Instrumentation, Burlington, VT). The microvessel bath was placed on the stage of a Nikon inverted microscope with attached video camera. The lumen of the artery was filled with Krebs solution, one micropipette was clamped off, and the other micropipette was connected to a pressure servo control to maintain the intraluminal pressure at 60 mmHg. Applying the same constant pressure in the microvessels should limit potential fluctuations in endothelial cell production of vasodilators associated with changes in the microvessel pressure, flow, and sheer stress. The Krebs solution within the microvessel was not renewed during pressurization; however, the microvessel was bathed in 5 ml of Krebs and was continuously superfused with fresh Krebs at a rate of 1 ml/min using a peristaltic mini-pump (Master-Flex; Cole-Parmer, Vernon Hills, IL), which should maintain the ionic environment constant throughout the duration of the experiment. The temperature of Krebs solution was kept at 37°C. Drugs were added abluminally to the bath solution. Microvessels were unacceptable if they show leaks or if they fail to produce >30% constriction to 96 mM KCl or 80% dilation with sodium nitroprusside (10⁻⁵ M).

Simultaneous measurement of microvessel diameter and [Ca²⁺]_i.

The mesenteric microvessles were continuously monitored using a video camera connected to a television monitor, and the microvessel diameter was measured using automatic edge-detection system (Crescent Electronics, Sandy, UT) and digitized at 1 Hz using a personal computer. Snap-pictures of the microvessel were taken at specific time points using a digital camera (Cool-Snap, Photometrics, Tucson, AZ). For measurement of [Ca²⁺]_i, microvessels were incubated in Krebs solution containing the Ca²⁺ indicator fura 2/AM (5 μM) and pluronic F-127 (0.01%) for 60 min. The microvessel was excited alternately at 340 and 380 nm, and the emitted light was collected at 510 nm using Felix Fluorescence data acquisition and analysis software (Photon Technology International, Birmingham, NJ). The 340/380 ratio was calculated and represented the changes in [Ca²⁺]_i. The signal-to-noise ratio was improved by averaging 10 consecutive 340/380 fluorescence ratio readings.

The sensitivity of the contractile response to KCl and Phe has previously been published by our group and other laboratories in the aorta, uterine, and mesenteric arteries of Norm-Preg and RUPP rats (5, 6, 14, 23). Our initial experiments demonstrated that the KCl and Phe-induced changes in [Ca²⁺]_i were rather small, and we could not detect with confidence concentration-dependent changes in [Ca²⁺]_i. Also, the angiotensin II (ANG II) response in rat tissue is notoriously tachyphylactic, making it difficult to construct a cumulative concentration-constriction or concentration-[Ca²⁺]_i curve. Additionally, the endothelin-1 (ET-1) response was relatively slow in onset, particularly at low concentrations, and a cumulative-constriction response curve would require prolonged exposure to excitation light, which would cause significant photobleaching of fura 2 and affect the accuracy of [Ca²⁺]_i measurements. Therefore, to accurately compare the [Ca²⁺]_i-dependent constriction induced by various agonists, we used maximal concentrations and a 10-min exposure time. The maximal concentrations of KCl, Phe, ANG II, and ET-1 used were based on previous reports (4–6, 14, 23) from our laboratory and other groups, which examined the concentration-constriction curves for KCl, Phe, ANG II, and ET-1 in the aorta, uterine, and mesenteric vessels of nonpregnant, Norm-Preg, and RUPP rats. In all experiments, the microvessel was first stimulated with 96 mM KCI and the initial and maintained vasoconstriction and 340/380 ratio were measured. Once the constriction reached a plateau, the microvessel was washed with Krebs solution for 15 min. The microvessel was then stimulated with either Phe (10⁻⁵ M), ANG II (10⁻⁶ M), or ET-1 (10⁻⁷ M), and the initial and maintained vasoconstriction and 340/380 ratio were recorded and used to construct the 340/380 ratio ([Ca²⁺]_i)-vasoconstriction relationship for microvessels from Norm-Preg and RUPP rats.

Solutions and drugs.

Normal Krebs solution contained the following (in mM): 120 NaCl, 5.9 KCl, 25 NaHCO₃, 1.2 NaH₂PO₄, 11.5 dextrose, 2.5 CaCl₂, 1.2 MgCl₂ pH 7.4, and then it was bubbled with 95% O₂-5% CO₂. Ninety-six millimolars were prepared as Krebs solution with equimolar substitution of NaCl with KCl. Stock solutions of Phe, ANG II, and ET-1 (Sigma, St. Louis, MO) were prepared in distilledd water. All other chemicals were of reagent grade or better.

Statistical analysis.

Data from Norm-Preg (n = 10) and RUPP rats (n = 9) were analyzed and presented as means ± SE. Student's t-test for unpaired data was used for comparison of two means. Differences were considered statistically significant if P < 0.05.

RESULTS

Effect of RUPP on pups.

On the day of the experiment (day 19 of gestation), examination of the pup litter demonstrated that there was a significant reduction in litter size in RUPP (7.7 ± 1.3 pups) compared with Norm-Preg (12.7 ± 0.5 pups; P = 0.002). Also, the average pup weight was significantly reduced in RUPP rats (2.14 ± 0.17 g) compared with that in Norm-Preg rats (2.68 ± 0.12 g; P = 0.017).

Resting diameter and basal [Ca²⁺]_i.

Cumulative data in unstimulated pressurized mesenteric microvessels demonstrated that the resting internal diameters were 240.8 ± 11.2 μm in Norm-Preg rats and were not significantly different from that in RUPP rats (237.9 ± 18.1 μm; P = 0.575). The thickness of the microvessel wall was 40.4 ± 3.3 μm in Norm-Preg rats and was not significantly different in RUPP rats (45.7 ± 5.5 μm). Also, the wall thickness to luminal diameter was not significantly different in Norm-Preg compared with RUPP rats. On the other hand, the basal 340/380 ratio ([Ca²⁺]_i) was significantly reduced in RUPP rats (0.73 ± 0.03) compared with Norm-Preg rats (0.82 ± 0.03; P < 0.05).

Effect of KCl.

KCl (96 mM) caused a significant decrease in the diameter of microvessels of both Norm-Preg (Fig. 1A) and RUPP rats (Fig. 1B). The KCl-induced response demonstrated an initial maximum followed by maintained vasoconstriction (Fig. 1, C and D). Also, in microvessels of both Norm-Preg and RUPP rats, KCl caused a slight change in the 340-nm fura 2 fluorescence signal, a significant decrease in the 380-nm fluorescence signal (Fig. 1, E and F), and an increase in the 340/380 fluorescence ratio (Fig. 1, G and H), indicating a simultaneous increase in [Ca²⁺]_i during KCl-induced vasoconstriction. In both Norm-Preg and RUPP rats, the KCl-induced [Ca²⁺]_i preceded and reached peak before the maximum vasoconstriction (Table 1). Also, in microvessels of Norm-Preg and RUPP rats stimulated with KCl, the time to steady-state [Ca²⁺]_i coincided with the time to steady-state vasoconstriction (Table 1). Cumulative data indicated that the KCl-induced initial and maintained vasoconstriction (Fig. 1, I and J) and [Ca²⁺]_i (Fig. 1, K and L) was not significantly different between microvessels of Norm-Preg and RUPP rats.

Fig. 1.

Effect of KCl (96 mM) on vasoconstriction and intracellular free calcium concentration ([Ca²⁺]_i) in mesenteric microvessels of late normal pregnancy (Norm-Preg) and reduction in uterine perfusion pressure (RUPP) rats. Images of fura 2 loaded microvessel from Norm-Preg (A) and RUPP rat (B) were taken at rest and after stimulation with KCl. Simultaneous measurements of diameter, 340- and 380-nm fluorescence signal, and 340/380 ratio were recorded in the isolated microvessel of Norm-Preg (C, E, G) and RUPP rat (D, F, H). Bar graphs represent means ± SE of vasoconstriction and [Ca²⁺]_i measurements in 10 microvessels of Norm-Preg (I, K) and 9 microvessels of RUPP rats (J, L).

Table 1.

Time-to-peak and time-to-steady-state vasoconstriction and [Ca²⁺]_i in response to KCl (96 mM), Phe (10⁻⁵ M), ANG II (10⁻⁷ M), and ET-1 (10⁻⁷ M) in Norm-Preg and RUPP rats

	Norm-Preg				RUPP
	KCl	Phe	ANG II	ET-1	KCl	Phe	ANG II	ET-1
Time-to-maximum constriction, s	38.4±2.4	34.4±6.2	33.3±5.8	46.5±6.5	26.8±4.3	29.4±9.2	33.8±7.8	39.7±11.1
Time-to-peak [Ca2+]i, s	19.8±3.1	10.8±0.9	9.3±1.9	13.5±1.5	22.1±6.6	12.6±2.4	12.8±1.5	16.5±4.4
Time-to-steady-state constriction, s	106.4±20.1	86.6±15.7	125±20.7	97±9	115.8±12.1	88.8±19.9	135±12.1	98±16.8
Time-to-steady-state [Ca2+]i, s	105.3±8.8	85.8±14.2	108.7±6.9	148.8±11.8	114.3±12.9	89.5±16.1	92.2±2.2	147.3±16.8

[Ca²⁺]_i, intracellular free calcium concentration; ED-1, endothelin-1; Norm-Preg, late normal pregnancy. RUPP, reduction in uterine perfusion pressure; Phe, phenylalanine.

Effect of Phe.

Mesenteric microvessels of both Norm-Preg and RUPP rats showed an initial vasoconstriction followed by maintained decrease in diameter in response to the α-adrenergic agonist Phe (10⁻⁵ M; Fig. 2, A and B). The Phe-induced vasoconstriction was preceded by an initial spike followed by a smaller, but maintained, increase in [Ca²⁺]_i (Fig. 2, C and D). In microvessels of both Norm-Preg and RUPP rats, the Phe-induced [Ca²⁺]_i preceded and reached peak before the maximum vasoconstriction (Table 1). Also, in microvessels of Norm-Preg and RUPP rats stimulated with Phe, the time to steady-state [Ca²⁺]_i coincided with the time to steady-state vasoconstriction (Table 1). Cumulative data demonstrated no significant difference in Phe-induced initial or maintained vasoconstriction between Norm-Preg and RUPP rats (Fig. 2, E and F). In contrast, the Phe-induced initial and maintained [Ca²⁺]_i presented as the 340/380 ratio was significantly reduced in RUPP compared with Norm-Preg rats (P < 0.05; Fig. 2, G and H).

Fig. 2.

Effect of phenylephrine (Phe; 10⁻⁵ M) on vasoconstriction and [Ca²⁺]_i in mesenteric microvessels of Norm-Preg and RUPP rats. Simultaneous measurements of Phe-induced changes in diameter and 340/380 fluorescence ratio were recorded in isolated microvessels from Norm-Preg (A, C) and RUPP rat (B, D). Bar graphs represent means ± SE of vasoconstriction and [Ca²⁺]_i measurements in 10 microvessels of Norm-Preg (E, G) and 9 microvessels of RUPP rats (F, H). *,†[Ca²⁺]_i (340/380 ratio) measurements in RUPP rats are significantly different (P < 0.05) from corresponding measurements in Norm-Preg rats.

Effect of ANG II.

In mesenteric microvessels of both Norm-Preg and RUPP rats ANG II (10⁻⁷ M) caused a rapid decrease in diameter that reached a maximum in ∼30 s (Fig. 3, A and B). ANG II also caused an initial peak followed by a maintained increase in [Ca²⁺]_i in microvessels of both Norm-Preg and RUPP rats (Fig. 3, C and D). In both Norm-Preg and RUPP rats the ANG II-induced [Ca²⁺]_i preceded and reached peak and steady-state levels before the maximum and steady-state vasoconstriction, respectively (Table 1). Cumulative data demonstrated no significant difference in the ANG II-induced initial and maintained vasoconstriction (Fig. 3, E and F). In contrast, the ANG II-induced initial and maintained [Ca²⁺]_i presented as the 340/380 ratio was significantly reduced (P < 0.05) in RUPP compared with Norm-Preg rats (Fig. 3, G and H).

Fig. 3.

Effect of ANG II (10⁻⁷ M) on vasoconstriction and [Ca²⁺]_i in mesenteric microvessels of Norm-Preg and RUPP rats. Simultaneous measurements of ANG II-induced changes in diameter and 340/380 fluorescence ratio were recorded in isolated microvessels from Norm-Preg (A, B) and RUPP rat (B, D). Bar graphs represent means ± SE of vasoconstriction and [Ca²⁺]_i measurements in 10 microvessels of Norm-Preg (E, G) and 9 microvessels of RUPP rats (F, H). *,†[Ca²⁺]_i (340/380 ratio) measurements in RUPP rat are significantly different (P < 0.05) from corresponding measurements in Norm-Preg rats.

Effect of ET-1.

ET-1 (10⁻⁷ M) caused a relatively slow developing decrease in diameter of microvessels of Norm-Preg and RUPP rats that reached a steady state in ∼45 s. The ET-1-induced vasoconstriction was prolonged and could not be reversed despite that the microvessels were washed several times with Krebs solution (Fig. 4, A and B). The ET-1-induced vasoconstriction was associated with an initial peak in [Ca²⁺]_i followed by smaller but maintained increase in [Ca²⁺]_i (Fig. 4, C and D). In both Norm-Preg and RUPP rats, the ET-1-induced [Ca²⁺]_i preceded and reached peak before the maximum vasoconstriction (Table 1). In contrast, in microvessels stimulated with ET-1 the time to steady-state [Ca²⁺]_i lagged behind the time to steady-state vasoconstriction in both Norm-Preg (P = 0.003) and RUPP rats (P = 0.054) (Table 1). Cumulative data demonstrated no significant difference in ET-1-induced initial or maintained vasoconstriction between microvessels of Norm-Preg and RUPP rats (Fig. 4, E and F). In contrast, the ET-1-induced initial and maintained [Ca²⁺]_i was lower in RUPP compared with Norm-Preg rats (Fig. 4, G and H).

Fig. 4.

Effect of endothelin-1 (ET-1; 10⁻⁷ M) on vasoconstriction and [Ca²⁺]_i in mesenteric microvessels of Norm-Preg and RUPP rats. Simultaneous measurements of ET-1-induced changes in diameter and 340/380 fluorescence ratio were recorded in isolated microvessels from Norm-Preg (A, C) and RUPP rat (B, D). Bar graphs represent means ± SE of vasoconstriction and [Ca²⁺]_i measurements in 10 microvessels of Norm-Preg (E, G) and 9 microvessels of RUPP rats (F, H). *,†[Ca²⁺]_i (340/380 ratio) measurements in RUPP rats are significantly different (P < 0.05) from corresponding measurements in Norm-Preg rats.

[Ca²⁺]_i-vasoconstriction relationship.

The initial and maintained 340/380 ratio ([Ca²⁺]_i) and vasoconstriction in response to KCl, Phe, ANG II, and ET-1 were used to construct the [Ca²⁺]_i-vasoconstriction relationship in microvessels of Norm-Preg and RUPP rats. Both the initial and maintained KCl [Ca²⁺]_i-vasoconstriction relationship were not different between Norm-Preg and RUPP rats. In contrast, the initial and maintained [Ca²⁺]_i-vasoconstriction relationship for Phe, ANG II, or ET-1 was shifted to the left in RUPP rats compared with Norm-Preg rats (Fig. 5).

Fig. 5.

[Ca²⁺]_i-vasoconstriction relationship in mesenteric microvessels from Norm-Preg and RUPP rats. Mesenteric microvessels from Norm-Preg and RUPP rats were stimulated with either KCl (96 mM), Phe (10⁻⁵M), ANG II (10⁻⁷M), or ET-1 (10⁻⁷M) and the initial (A) and maintained (B) changes in vasoconstriction and [Ca²⁺]_i (340/380 ratio) were used to construct a [Ca²⁺]_i-vasoconstriction relationship. The initial and maintained Phe, ANG II, and ET-1 induced [Ca²⁺]_i-vasoconstriction relationships were shifted to the left (less [Ca²⁺]_I) in RUPP rats compared with Norm-Preg rats. *,†[Ca²⁺]_i (340/380 ratio) measurements in RUPP rats are significantly different (P < 0.05) from corresponding measurements in Norm-Preg rats.

DISCUSSION

The main findings of the present study are as follows: 1) the maximal mesenteric microvessel vasoconstriction to KCl and the more physiological vasoconstrictor stimuli Phe, ANG II, and ET-1 is not different between RUPP and Norm-Preg rats; 2) the KCl-induced [Ca²⁺]_i is not different, but the basal and agonist-induced initial and maintained [Ca²⁺]_i is reduced in RUPP compared with Norm-Preg rats; and 3) the [Ca²⁺]_i-vasoconstriction relationship for KCl is similar, while that for Phe, ANG II, or ET-1 is shifted to the left (enhanced) in microvessels of RUPP compared with Norm-Preg rats.

Previous studies (6) have shown that the mesenteric vascular resistance is elevated in rat models of HTN-Preg. Studies have also shown that the mesenteric vascular resistance is reduced in Norm-Preg compared with nonpregnant normotensive rats (61, 68, 74) and in Norm-Preg compared with nonpregnant spontaneously hypertensive rats (12). Studies (11) have also shown that the vascular reactivity to electrical stimulation or intraarterial norepinephrine, ANG II, and arginine vasopressin are decreased in the in situ blood perfused mesenteries of Norm-Preg rats compared with nonpregnant controls. Other studies (12) in the in situ blood perfused mesenteric resistance vessels of 18- to 20-day pregnant spontaneously hypertensive rats have shown much lower vascular response to electrical stimulation or intraarterial norepinephrine than either pregnant or nonpregnant spontaneously hypertensive rats. Although the in situ blood-perfused mesenteries could provide important physiological vascular reactivity information, they may not allow further investigation of underlying cellular mechanisms, particularly measurement of [Ca²⁺]_i.

One goal of the present study was to investigate the mechanisms of vasoconstriction in small systemic microvessels of Norm-Preg and RUPP rats. Arteries of internal diameter <300 μm are by and large considered resistance vessels (47, 48, 75). Specifically, the small mesenteric feed arteries and microcirculatory vessels have been used in several studies (6, 21, 52) as representative of resistance vessels. These small resistance size arteries have a significant myogenic tone that primarily maintains constant blood flow and provide a baseline diameter that is modulated by vasoconstrictors and vasodilators (10, 16, 75). Therefore, we used isolated mesenteric microvessels to determine the [Ca²⁺]_i-signaling mechanism underlying the vascular changes in Norm-Preg and RUPP rats. The average internal diameter of the mesenteric microvessels used in the present study was 241 in Norm-Preg and 238 μm in RUPP rats, well in the range of resistance arteries. Also, all microvessels tested produced ∼45% constriction in response to KCl and significant vasoconstriction to three different physiological agonists namely Phe, ANG II, and ET-1, confirming viability of the microvessel preparation. Furthermore, during stimulation by KCl and other agonists the time to peak [Ca²⁺]_i always preceded the time to maximum vasoconstriction (Table 1), supporting the contention that the increased [Ca²⁺]_i triggers the vasoconstriction.

Previous studies (1, 14, 22, 26) have shown that RUPP in late pregnant rats is associated with significant increases in renal vascular resistance and BP. Also, we (14) have shown that Phe-induced vascular contraction is greater in aortic strips isolated from RUPP compared with Norm-Preg rats. Although the differences in aortic contraction were partially related to reduced endothelium-dependent nitric oxide-mediated vascular relaxation in RUPP rats, differences in contraction were still observed in endothelium-denuded aortic strips of RUPP compared with Norm-Preg rats, suggesting additional differences in the mechanisms of aortic VSM contraction (14). In support of this view, studies (5, 70) have shown enhanced Phe-induced contraction in isolated uterine arteries from RUPP or transgenic preeclampsia rats compared with Norm-Preg rats.

In search for the cellular mechanisms involved in the enhanced vasoconstriction during HTN-Preg, our previous experiments on the aorta have shown that the Phe- and caffeine-induced contraction in Ca²⁺-free solution are not different in RUPP rats compared with Norm-Preg rats, suggesting that the IP₃-sensitive and the Ca²⁺-induced Ca²⁺ release mechanisms from the intracellular stores are not different between the RUPP rat model of HTN-Preg and Norm-Preg rats (23). On the other hand, the aortic contractile response to membrane depolarization by KCl, which stimulates Ca²⁺ entry from the extracellular space, is reduced in Norm-Preg compared with virgin rats but significantly enhanced in RUPP rats (15, 23, 36). Also, our studies (50) in isolated renal arterial VSM cells have shown that the basal and ANG II-stimulated [Ca²⁺]_I is reduced in Norm-Preg compared with virgin rats but significantly elevated in RUPP rats. These data suggest that the Ca²⁺ entry mechanisms of vascular contraction are enhanced in the aorta and renal artery of RUPP rats compared with Norm-Preg rats.

Based on previous measurements of VSM contraction and Ca²⁺ in the aorta, renal, and uterine arteries (5, 15, 36, 50), we hypothesized that the Ca²⁺-dependent mechanisms of vasoconstriction are most likely enhanced in small mesenteric microvessels of RUPP compared with Norm-Preg rats. Contrary to our prediction, the KCl-induced vasoconstriction and [Ca²⁺]_i were not different in mesenteric microvessels of RUPP and Norm-Preg rats. Because KCl largely stimulates Ca²⁺ influx through voltage-gated Ca²⁺ channels, the present data suggest that this Ca²⁺ entry mechanism of vasoconstriction is not different in mesenteric microvessels of Norm-Preg and RUPP rats. We should note that while the KCl-induced response is thought to be mainly due to Ca²⁺ entry from the extracellular space, the KCl-induced Ca²⁺ entry can also activate Ca²⁺ release from internal stores by Ca²⁺-induced Ca²⁺ release mechanism and the contribution of this mechanism to the observed KCl response cannot be ruled out.

The present data also demonstrate that the mesenteric microvessel reactivity to Phe was not significantly different between RUPP and Norm-Preg rats. Because different parts of the circulation may have different distribution of α-adrenergic receptors, we hypothesized that the lack of change in the responsiveness to Phe in mesenteric microvessels of RUPP rats compared with the previously reported enhanced sensitivity to Phe in the aorta and uterine artery may be related to decreased amount of α-adrenergic receptors in the mesenteric vessels. If this is the case, then the mesenteric microvessels of RUPP rats should be more responsive to other agonists/receptors. Thus a second goal of the present study was to investigate whether the differences in the mechanisms of vasoconstriction in systemic microvessels of Norm-Preg and RUPP rats are specific to a particular agonist/receptor or represent difference in a common signaling pathway downstream from receptor activation.

ANG II, which stimulates angiotensin type 1 receptor in VSM, caused significant vasoconstriction of mesenteric microvessels that was similar in RUPP and Norm-Preg rats. Also, ET-1, which stimulates ET_A and perhaps ET_B2 receptor in VSM, induced similar vasoconstriction in RUPP and Norm-Preg rats. Nevertheless, similar to the Phe response, the ANG II- and ET-1-induced [Ca²⁺]_i was lower in RUPP compared with Norm-Preg rats. These data suggest that the decreased [Ca²⁺]_i signaling of vasoconstriction in the RUPP rats in response to the α-adrenergic agonist Phe is shared by other agonists such as ANG II and ET-1, which act on different sets of receptors. Alternatively, the decreased [Ca²⁺]_i signaling of vasoconstriction in response to Phe, ANG II, and ET-1 in RUPP compared with Norm-Preg rats may represent a difference in a common [Ca²⁺]_i regulatory pathway downstream from receptor activation. Agonists such as Phe, ANG II, and ET-1 are known to activate Ca²⁺ influx through ligand-gated and store-operated Ca²⁺ channels in VSM (17). The decreased [Ca²⁺]_i signaling of vasoconstriction in response to Phe, ANG II, and ET-1 in RUPP compared with Norm-Preg rats may therefore reflect reduced expression/activity of ligand-gated and/or store-operated Ca²⁺ channels. However, if [Ca²⁺]_i is the sole regulator of the microvessel vasoconstriction, then the reduced [Ca²⁺]_i in microvessels of RUPP rats should be associated with reduced vasoconstriction. This is not the case, as the microvessel vasoconstriction was similar in magnitude in RUPP rats and Norm-Preg rats, suggesting activation of other control or signaling mechanisms in addition to [Ca²⁺]_i.

An important observation is that the basal [Ca²⁺]_i was lower in mesenteric microvessels of RUPP rats compared with Norm-Preg rats. Ca²⁺ homeostasis is controlled by Ca²⁺ extrusion mechanisms in the plasma membrane including the Ca²⁺-ATPase and Na⁺-Ca²⁺ exchanger (40). The decreased basal [Ca²⁺]_i in microvessels of RUPP rats may be related to enhanced Ca²⁺ extrusion mechanisms. Similarly, the reduced [Ca²⁺]_i response to vasoconstrictor agonists in RUPP rats could be partly explained by increased activity of Ca²⁺ extrusion mechanisms. Another possibility is that the vasoconstriction sensitivity to [Ca²⁺]_i is altered in microvessels of RUPP rats. We found that the [Ca²⁺]_i-vasoconstriction relationship was similar in KCl-stimulated microvessels of Norm-Preg and RUPP rats. In contrast, the [Ca²⁺]_i-vasoconstriction relationship was enhanced in Phe-, ANG II-, and ET-1-stimulated microvessels of RUPP compared with Norm-Preg rats. These findings suggest activation of a [Ca²⁺]_i regulatory pathway that increases the myofilament sensitivity to [Ca²⁺]_i. Several studies (28–30, 63, 73) have shown that in addition to the role of Ca²⁺, calmodulin and myosin light chain kinase, Rho kinase, and mitogen-activated protein kinase may contribute to VSM contraction. Also, PKC has been suggested to play an important role in the regulation of VSM contraction, in part by increasing the [Ca²⁺]_i sensitivity of the contractile proteins (38, 41, 54, 35, 62, 72). Additionally, previous studies in rat small mesenteric arteries have shown that norepinephrine-induced Ca²⁺ sensitization is associated with increased myosin light chain phosphorylation and suggested decreased myosin light chain phosphatase activity that is mediated through PKC (9, 55). Collectively, the present and previous studies suggest that while Ca²⁺-dependent myosin light chain phosphorylation is a major regulator of vasoconstriction in both Norm-Preg and RUPP rats, other [Ca²⁺]_i sensitization pathways such as PKC or Rho kinase may be involved in the regulation of vasoconstriction in microvessels of RUPP rats.

If activation of a Ca²⁺ regulatory pathway such as PKC causes an increase in the vasoconstriction sensitivity to [Ca²⁺]_i in RUPP rats, then the question is why the initial and maintained vasoconstriction is similar in microvessels of Norm-Preg and RUPP rats? Studies (35, 62) have suggested that PKC may activate feedback mechanisms involving uncoupling of the surface receptors from the GTP-binding protein, inhibition of phospholipase C, inhibition of Ca²⁺ mobilization via the Ca²⁺ release or Ca²⁺ entry channels, activation of Ca²⁺ extrusion mechanisms, and phosphorylation and inhibition of myosin light chain kinase. Interestingly, during microvessel stimulation with most of the agonists tested in the present study the time to steady-state [Ca²⁺]_i preceded or coincided with the time to steady-state vasoconstriction, suggesting that [Ca²⁺]_i remains a major determinant of vasoconstriction during steady state. A clear exception was the ET-1 response in which the time to steady-state [Ca²⁺]_i lagged behind the time to steady-state vasoconstriction. We (46) have previously shown that ET-1 promotes VSM contraction via activation of PKC, raising the possibility that an ET-1-induced activation of PKC during maximal contraction would cause feedback control of Ca²⁺ entry and therefore a delay in the [Ca²⁺]_i steady state. PKC is a family of Ca²⁺-dependent and Ca²⁺-independent isoforms that exhibit different enzyme properties, substrates, functions, and subcellular distributions in various blood vessels (35, 55, 62), and therefore, activation of PKC may have different effects in various vascular beds particularly during pregnancy. We (15) have previously shown that the vascular sensitivity to Ca²⁺ entry was enhanced in the aorta of l-NAME treated compared with control Norm-Preg rats. We (33, 34) have also shown that the activity of the Ca²⁺-dependent α-PKC and the Ca²⁺-independent δ-PKC is enhanced in l-NAME-treated compared with control Norm-Preg rats. Although our previous data point to PKC as a possible regulator of Ca²⁺ signaling in the l-NAME treated rat model of HTN, the specific role of PKC or other Ca²⁺ sensitization pathways such as Rho kinase in the RUPP model of HTN-Preg is unclear at the present time and should be further examined in future studies.

We should note that previous wire myography studies in first order mesenteric arteries have shown enhanced vasoconstrictor responses to a range of KCl, Phe, and ANG II concentrations in RUPP rats compared with Norm-Preg rats (6). The differences in the results could be related to differences in recording techniques (wire myography vs. pressurized microvessels) or in the size of the vessels studied (1st order arteries compared with 3rd to 4th branch mesenteric microvessels). Our (69, 71) results in the RUPP rats are consistent with studies in human vessels thatdemonstrated no significant differences in basal myogenic tone or constrictor responses to KCl, Phe, or ANG II in subcutaneous resistance arteries from women with preeclampsia compared with those from Norm-Preg women. It has been suggested that the vasoconstrictor sensitivity of arteries may be different in isobaric vs. isometric conditions for reasons related to both the mounting technique and mechanical loading (9, 67). It has also been suggested that conditions associated with medial hypertrophy might result in a higher maximal tension development to vasoconstrictors in isometric arteries, while isobaric arteries may show similar maximal constrictions as long as the pressure load is not too high (69).

Thus the Ca²⁺ regulatory mechanisms in mesenteric microvessels appear to be different from those previously demonstrated in the aorta, uterine arteries, or main mesenteric arteries of RUPP rats compared with Norm-Preg rats (5, 6, 14, 23). Whether the difference represents different adaptation mechanisms in the mesenteric microvessels compared with other vascular beds or large conduit vessels needs to be further examined. The microvessel vasoconstriction to three different physiological agonists acting through different receptors is associated with smaller increases in [Ca²⁺]_i in RUPP compared with Norm-Preg rats. The smaller [Ca²⁺]_i signaling of vasoconstriction in microvessels of RUPP compared with Norm-Preg rats suggests activation of additional Ca²⁺ regulatory pathway(s) that increase the vasoconstriction sensitivity to [Ca²⁺]_i. The similarity in the initial and maintained vasoconstriction in RUPP and Norm-Preg rats suggests that such Ca²⁺ regulatory pathway(s) may have a feedback effect on Ca²⁺ mobilization/homeostatic mechanisms to protect against excessive vasoconstriction in systemic microvessels during RUPP in late pregnancy.

Perspectives

Preeclampsia is associated with increased total vascular resistance, which is thought to cause generalized organ hypoperfusion and multisystem disorder. Vascular resistance is determined by vascular tone, which depends on vascular smooth muscle [Ca²⁺]_i and the Ca²⁺ sensitivity of the contractile proteins. Studying the vascular reactivity and [Ca²⁺]_i in animal models of HTN-Preg should help to elucidate the mechanisms of preeclampsia in women. Studies in the aorta, uterine, and main mesenteric arteries suggest an increase in vascular reactivity in rat models of HTN-Preg (5, 6, 14, 23). In contrast, the present study suggests no difference in reactivity of mesenteric microvessels of RUPP rats compared with Norm-Preg rats, a finding that is consistent with previously reported lack of difference in vascular reactivity in subcutaneous resistance arteries from preeclamptic and Norm-Preg women (69, 71). The present results suggest that the vascular responses during HTN-Preg are not uniform and highlight the importance of studying other vascular beds including the renal, coronary, and cerebral resistance arteries in future studies. The study also highlights the importance of measuring [Ca²⁺]_i and Ca²⁺-sensitization pathways in blood vessels to demonstrate potential abnormalities in the underlying cellular mechanisms, despite apparent lack of changes in vascular reactivity. Many factors could affect vascular [Ca²⁺]_i in preeclampsia, including depolarization of the membrane potential, maladjustment of Ca²⁺ influx across the plasma membrane and Ca²⁺ release from intracellular stores in addition to abnormal transport of sodium and magnesium, and alteration of Ca²⁺ metabolism and plasma Ca²⁺ (19, 42, 71), and these factors should be thoroughly examined in future investigations.