Effects of intracellular angiotensin II in vascular smooth muscle cells

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 Ca2+ ([Ca2+]i) in VSMCs. Ang II was administered via microinjection. Intracellular Ang II localization was demonstrated by fluorescein-labeled Ang II and electron microscopy. [Ca2+]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 [Ca2+]i in the cytosol and in the nucleus. The [Ca2+]i increase was due to the influx of extracellular Ca2+ 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 [Ca2+]i was observed not only in the microinjected cell but also in directly adjacent VSMCs. In contrast to the microinjected cells, the [Ca2+]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 [Ca2+]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 [Ca2+]i surge in adjacent cells. We conclude that intracellular Ang II binds to intracellular Ang II receptors and elicits an increased [Ca2+]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.