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.

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).

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.

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.

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.

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.