Effects of Intracellular Angiotensin II in Vascular Smooth Muscle Cells

Abstract

Angiotensin (Ang) II is present inside vascular smooth muscle cells (VSMCs); however, its intracellular functions, if any, are unknown. We tested the hypothesis that intracellular Ang II exerts effects on cytosolic Ca 2+ ([Ca 2+ ] i ) in VSMCs. Ang II was administered via microinjection. Intracellular Ang II localization was demonstrated by fluorescein-labeled Ang II and electron microscopy. [Ca 2+ ] i was monitored by confocal microscopy with fluo 3. Ang II was identified in endosomes and in the nucleus by both localizing techniques. Microinjection of Ang II (10 −10 mol/L) led to a rapid increase in [Ca 2+ ] i in the cytosol and in the nucleus. The [Ca 2+ ] i increase was due to the influx of extracellular Ca 2+ ions. The intracellular Ang II effect was totally inhibited by the concomitant injection of the Ang II antagonist CV-11947. Desensitization of extracellular Ang II receptors, on the other hand, did not influence the intracellular effects, nor did extracellular CV-11947. The increase in [Ca 2+ ] i was observed not only in the microinjected cell but also in directly adjacent VSMCs. In contrast to the microinjected cells, the [Ca 2+ ] i increase in the adjacent cells was mostly due to release from intracellular stores. Pretreatment with thapsigargin abolished the Ang II response in adjacent cells. Microinjection of inositol tris-phosphate induced a [Ca 2+ ] i response in adjacent cells that was similar to the Ang II–induced effects. Preincubation of VSMCs with the uncoupling substances dimethyl sulfoxide and heptanol did not decrease the Ang II response but instead prevented a [Ca 2+ ] i surge in adjacent cells. We conclude that intracellular Ang II binds to intracellular Ang II receptors and elicits an increased [Ca 2+ ] i in the injected cell and, thereafter, cells in the immediate neighborhood. Cell-cell contact is necessary for the Ang II–mediated effects. The data suggest that intracellular Ang II may stimulate a cluster of VSMCs from a single cell via the release of second messengers.