Role of Ca²⁺ channels in NE-induced increase in [Ca²⁺](i) and tension in fetal and adult cerebral arteries

In vascular smooth muscle, elevation of agonist-induced intracellular Ca²⁺ concentration ([Ca²⁺](i)) occurs via both Ca²⁺ release from intracellular stores and Ca²⁺ influx across the plasma membrane. In the cerebral vasculature of the fetus and adult the relative roles of these mechanisms have not been defined. To test the hypothesis that plasma membrane L-type and receptor-operated Ca²⁺ channels play a key role in NE-induced vasoconstriction via alterations in plasma membrane Ca²⁺ flux and that this may change with developmental age, we performed the following study. In main branch middle cerebral arteries (MCA) from near-term fetal (~140 days) and nonpregnant adult sheep, we quantified NE-induced responses of vascular tension and [Ca²⁺](i) (by use of fura 2) under standard conditions in response to several Ca²⁺ channel blockers and in response to zero extracellular Ca²⁺. In fetal and adult MCA, maximal NE-induced tensions (g) were 0.91 ± 0.12 (n = 10) and 1.61 ± 0.13 (n = 12), respectively. The pD₂ values for NE-induced tension were both 6.0 ± 0.1, whereas the fetal and adult maximum responses (%K(max)) were 107 ± 16 and 119 ± 7, respectively. The fetal and adult pD₂ values for NE-induced increase of [Ca²⁺](i) were 6.2 ± 0.1 and 6.4 ± 0.1, respectively, whereas maximum [Ca²⁺](i) responses were 81 ± 9 and 103 ± 15% of K(max), respectively. After 10^-5 M NE-induced contraction, nifedipine resulted in dose-dependent decrease in vessel tone and [Ca²⁺](i) with pIC₅₀ values for fetal and adult tensions of 7.3 ± 0.1 and 6.6 ± 0.1, respectively (P < 0.01; n = 4 each), whereas pIC₅₀ for [Ca²⁺](i) responses were 7.2 ± 0.1 and 6.9 ± 0.1, respectively. The pIC₅₀ values for tension for diltiazem and verapamil were somewhat lower but showed a similar relationship. The receptor-operated Ca²⁺ channel blocker 2-nitro-4 carboxyphenyl-N,N-diphenyl carbamate showed little effect on NE-induced vessel contractility or [Ca²⁺](i). In the absence of extracellular Ca²⁺ for 2 min, 10^-5 M NE resulted in markedly attenuated responses of adult MCA tension and [Ca²⁺](i) to 39 ± 7 and 73 ± 8% of control values (n = 4). For fetal MCA, exposure to extracellular Ca²⁺ concentration resulted in essentially no contractile or [Ca²⁺](i) response (n = 4). Similar blunting of NE-induced tension and [Ca²⁺](i) was seen in response to 10^-3 M lanthanum ion. These findings provide evidence to suggest that especially in fetal, but also in adult, ovine MCA, Ca²⁺ flux via L-type calcium channels plays a key role in NE-induced contraction. In contrast, Ca²⁺ flux via receptor-operated Ca²⁺ channels is of less importance. This developmental difference in the role of cerebrovascular plasma membrane Ca²⁺ channels may be an important association with increased Ca²⁺ sensitivity of the fetal vessels.