Role of NHE1 in calcium oscillation and cell proliferation in human CNS pericytes

Background and aims: Central nervous system (CNS) pericytes located at the abluminal side of microvessels, such as arterioles, venules, and particularly capillaries, appear to play an important role in angiogenesis, regulation of blood flow, immune responses, and maintenance of blood-brain barrier (BBB). Na+/H+ exchanger isoform 1 (NHE1) ubiquitously expressed in plasma membrane has been implicated to have a role in proliferation of non-vascular cells through regulation of intracellular pH, Na+ and cell volume; however, the relationship between NHE1 and pericyte growth is not known. Thus, the aim of the present study was to elucidate the role of NHE1 in the proliferation of human CNS pericytes. We have found on intracellular Ca2+ change, an essential signal of cell growth. Methods: Human brain microvascular pericytes were cultured and cytosolic Ca2+ concentration and pH were measured by fluorescent indicators, fura-2 and 2', 7'-bis-2-carboxyethyl-5-(6)-carboxyfluorescein (BCECF), respectively. Reverse transcription and polymerase chain reaction (RT-PCR) was used to examine expression level of each mRNA. Knockdown of NHE1 mRNA was done by RNA interference (RNAi) with the double-strand siRNAs targeting NHE1 specifically. Cell proliferation was evaluated by cell count. Results: Human microvascular pericytes showed spontaneous Ca2+ oscillation in the presence of extracellular Na+ (132mM). A decrease in extracellular Na+ (0-99mM) evoked transient Ca2+ rise followed by Ca2+ plateau or Ca2+ oscillation, whereas increase in extracellular Na+ to 166mM eliminated the Ca2+ responses. A decrease in extracellular pH to 6.5 induced similar cytosolic Ca2+ change as that by low extracellular Na+. Low Na+-induced Ca2+ oscillation was inhibited by hexamethylene amiloride (HMA, an inhibitor of Na+/H+ exchanger) dosedependently (5-50μM). The Ca2+ oscillation was also inhibited by amiloride (50μM) or benzamil (50μM). Nicardipine (1μM), Gd3+ (100μM), La3+ (100μM) or omission of external Ca2+ did not affect the Ca2+ oscillation. KB-R7943 (10μM; a selective inhibitor of Na+/Ca2+ exchanger), carbonyl cyanide p-trifluoro-methoxyphenylhydrazone (1μM; FCCP, the mitochondrial uncoupler), or changes in the external osmolarity did not affect the Ca2+ oscillation. On the other side, the Ca2+ oscillation was completely abolished by pretreatment with thapsigargin (1μM; an inhibitor of sarco/endoplasmic reticulum Ca2+ ATPase), suggesting that Ca2+ oscillation was originated from endoplasmic reticulum. RT-PCR revealed that human CNS pericytes expressed NHE1 and NHE7. Low extracellular Na+ could not induce the Ca2+ oscillation in the cells transfected with specific siRNA targeting NHE1. Proliferation of the pericytes was significantly attenuated by addition of HMA (5μM) to the medium. Knockdown of NHE1 by transfecting mRNA also inhibited the proliferation of pericytes. Conclusions: These results indicate that NHE1 plays an important role in Ca2+ signaling via modulation of endoplasmic reticulum and thereby contributes to the regulation of proliferation in CNS pericytes. This novel role of NHE1 in the pericytes may have pathophysiological relevance to angiogenesis or BBB disruption in the cerebral ischemia.