Maturation of intracellular calcium homeostasis in sheep pulmonary arterial smooth muscle cells

Abstract

Cytosolic Ca(2+) signaling dynamics are important to pulmonary arterial reactivity, and alterations are implicated in pulmonary vascular disorders. Yet, adaptations in cellular Ca(2+) homeostasis and receptor-mediated Ca(2+) signaling with maturation from fetal to adult life in pulmonary arterial smooth muscle cells (PASMCs) are not known. The present study tested the hypothesis that cytosolic Ca(2+) homeostasis and receptor-generated Ca(2+) signaling adapt with maturation in sheep PASMCs. Digitalized fluorescence microscopy was performed using isolated PASMCs from fetal and adult sheep that were loaded with the Ca(2+) indicator fura 2. The results show that basal cytosolic and sarcoplasmic reticulum Ca(2+) levels are attained before birth. Similarly, Ca(2+) efflux pathways from the cytosol and basal as well as capacitative Ca(2+) entry (CCE) are also developed before birth. However, receptor-mediated Ca(2+) signaling adapts with maturation. Prominently, serotonin stimulation elicited Ca(2+) elevations in very few fetal compared with adult PASMCs; in contrast, phenylephrine elevated Ca(2+) in a similar percentage of fetal and adult PASMCs. Serotonin and phenylephrine elicited Ca(2+) increases of a similar magnitude in reactive cells of fetus and adult, supporting the assertion that inositol trisphosphate signaling is intact. Caffeine and ATP elevated Ca(2+) in equivalent numbers of fetal and adult PASMCs. However, the caffeine-induced cytosolic Ca(2+) increase was significantly greater in fetal PASMCs, whereas the ATP-elicited increase was greater in adult cells. Overall, the results of this study demonstrate selective adaptations in receptor-mediated Ca(2+) signaling, but not in cellular Ca(2+) homeostasis.

Fig. 1.

Basal intracellular Ca²⁺ concentration ([Ca²⁺]_i) is unchanged in pulmonary arterial smooth muscle cells (PASMCs) with maturation. Values are means ± SE. No significant difference was observed (P = 0.16 by unpaired t-test).

Fig. 2.

Activation of capacitative Ca²⁺ entry (CCE) in fetal and adult PASMCs. Representative traces of [Ca²⁺]_i of myocytes from fetal (A) and adult (B) sheep show effect of extracellular Ca²⁺ removal, addition of 10 μM cyclopiazonic acid (CPA), and sequential exposure to 10 mM caffeine (Caf) and readdition of extracellular Ca²⁺ on cytosolic Ca²⁺. Dashed horizontal line shows basal [Ca²⁺]_i. C: average change in [Ca²⁺]_i from basal levels during readdition of Ca²⁺ in cells from fetal and adult sheep. Values are means ± SE. No significant difference was observed (P = 0.4 by unpaired t-test).

Fig. 3.

Rate of plasma membrane Ca²⁺ extrusion is unchanged in PASMCs with maturation. Representative [Ca²⁺]_i recordings from myocytes of fetal (A) and adult (B) sheep show effect of extracellular Ca²⁺ removal in the presence of 10 μM CPA upon depletion of sarcoplasmic reticulum (SR) Ca²⁺ stores using the protocol described in Fig. 2. Solid curves show exponential curve-fit analyses. C: average plasma membrane clearance rate constant (m_clear) for cells from fetal and adult sheep. Values are means ± SE. No significant difference was observed (P = 0.37 by unpaired t-test).

Fig. 4.

SR Ca²⁺ storage undergoes early maturation. Representative [Ca²⁺]_i recordings and integrated Ca²⁺ response (dashed curve) of myocytes from fetal (A) and adult (B) sheep show response to extracellular Ca²⁺ removal and addition of 10 μM CPA. C: average of integrated Ca²⁺ concentration, which is equivalent to Ca²⁺ content of SR for cells from fetal and adult sheep. Values are means ± SE. No significant difference was observed (P = 0.49 by unpaired t-test).

Fig. 5.

Rate constant for Ca²⁺ leak from SR is unchanged in PASMCs with maturation. SR Ca²⁺ leak rate [Q(t)] for myocytes from fetal (A) and adult (B) sheep is shown as semilogarithmic plots. C: mean clearance rate for passive Ca²⁺ leak from SR (1/τ_SR) for cells from fetal and adult sheep. Values are means ± SE. No significant differences were found (P = 0.34 by unpaired t-test).

Fig. 6.

Ca²⁺ influx rate at rest and during CCE is unchanged in PASMCs with maturation. Average Ca²⁺ influx rates under basal conditions (A) and during CCE (B) are shown for cells from fetal and adult sheep. Values are means ± SE. No significant difference was observed for basal (P = 0.55 by unpaired t-test) or CCE (P = 0.27 by unpaired t-test) Ca²⁺ influx.

Fig. 7.

Serotonin (5-HT)-mediated Ca²⁺ signaling increases with maturation in PASMCs. Representative traces show 5-HT-mediated [Ca²⁺]_i elevation in myocytes from fetal (A) and adult (B) sheep. C: percentage of PASMCs from fetal and adult sheep showing Ca²⁺ elevation in response to 10 μM 5-HT. Responses of 78 cells from fetal and 43 cells from adult sheep were recorded. D: average increase in [Ca²⁺]_i due to 10 μM 5-HT in cells from fetal and adult sheep. Values are means ± SE. No significant difference was observed (P = 0.6 by unpaired t-test). *P < 0.001 (by χ² test).

Fig. 8.

Caffeine (Caf)-dependent Ca²⁺ release is augmented in PASMCs from fetus. Representative traces show caffeine-mediated [Ca²⁺]_i elevation in myocytes from fetal (A) and adult (B) sheep. C: percentage of PASMCs from fetal and adult sheep showing Ca²⁺ elevation in response to 10 mM caffeine. No significant difference was observed (P = 0.7 by χ² test). Responses of 55 cells from fetal and 41 cells from adult sheep were recorded. D: mean caffeine-elicited increase in [Ca²⁺]_i in cells from fetal and adult sheep. Values are means ± SE. *P = 0.01 (by unpaired t-test).

Fig. 9.

P2 receptor-dependent Ca²⁺ elevations are higher in adult PASMCs. Representative traces show 100 μM ATP-mediated [Ca²⁺]_i elevation in myocytes from fetal (A) and adult (B) sheep. C: percentage of PASMCs from fetal and adult sheep showing Ca²⁺ elevation in response to 100 μM ATP. No significant difference was observed (P = 0.15 by χ² test). D: ATP-mediated increases in [Ca²⁺]_i in cells from fetal and adult sheep. Values are means ± SE. *P = 0.02 (by unpaired t-test).

Fig. 10.

α-Adrenergic receptor-dependent Ca²⁺ responses are unchanged with maturation in PASMCs. Representative traces show 10 μM phenylephrine (PE)-mediated [Ca²⁺]_i elevations in myocytes from fetal (A) and adult (B) sheep. C: percentage of PASMCs from fetal and adult sheep showing Ca²⁺ elevation in response to 10 μM PE. No significant difference was observed (P = 0.2 by χ² test). D: average PE-mediated increase in [Ca²⁺]_i in cells from fetal and adult sheep. Values are means ± SE. No significant difference was observed (P = 0.6 by unpaired Student's t-test).