Calcium influx pathways in rat CNS pericytes

In central nervous system (CNS), pericytes have been proposed to play a role in broad functional activities including blood-brain barrier, microcirculation, and macrophage activity. However, contractile responses and Ca²⁺ signaling in CNS pericytes have not been elucidated. The aim of the present study is to investigate contractility and Ca²⁺ influx pathway in CNS pericytes. CNS pericytes were cultured from rat brain. Contraction of the pericytes in response to various stimuli was evaluated by the change in surface area measured by a light microscope with a digital camera. Reverse transcription and polymerase chain reaction (RT-PCR) was performed to examine the expression of mRNA of α-smooth muscle actin. Intracellular Ca²⁺ was measured using fura-2 fluorescence spectroscopy. A23187 (Ca²⁺ ionophore), high external K⁺ (4×10 ^-2 mol/l), endothelin-1, and serotonin induced contraction of CNS pericytes. RT-PCR analysis revealed the expression of α-smooth muscle actin in CNS pericytes. Cytosolic Ca²⁺ ([Ca²⁺]i) increased after application of high concentration of external K⁺, tetraethylammonium, and charybdotoxin, which was inhibited by nicardipine and removal of external Ca²⁺. Angiotensin-II, serotonin, acetylcholine, ATP, and endothelin-1 caused biphasic response in [Ca²⁺]i. In response to these agents, [Ca²⁺]i rapidly increased and then decayed to a relatively constant Ca²⁺ plateau. The Ca²⁺ plateau was partially inhibited by nicardipine and completely abolished by omission of external Ca²⁺. After intracellular Ca²⁺ store was depleted by the removal of external Ca²⁺ and addition of thapsigargin, reapplication of external Ca²⁺ evoked increases in [Ca ²⁺]i. These results indicate that CNS pericytes express mRNA of α-smooth muscle actin and possess contractile ability. In CNS pericytes, resting membrane potential is regulated by large conductance Ca ²⁺-activated K⁺ channels and Ca²⁺ enters into the cells via L-type voltage-dependent Ca²⁺ channels, agonist-activated Ca²⁺ permeable channels, and capacitative Ca ²⁺ entry pathways.