Intracellular angiotensin II inhibits heterologous receptor stimulated Ca 2+ entry

Recent studies show that angiotensin II (AngII) can act from within the cell, possibly via intracellular receptors pharmacologically different from typical plasma membrane AngII receptors. The role of this intracellular AngII (AngII(i)) is unclear. Besides direct effects of AngII(i) on cellular processes one could hypothesise a possible role of AngII(i) in modulation of cellular responses induced after heterologous receptor stimulation. We therefore examined if AngIIi influences [Ca2+](i) in A7r5 smooth muscle cells after serotonin (5HT) or UTP receptor stimulation. Application of AngIIi using liposomes, markedly inhibited Ca-45(2+) influx after receptor stimulation with 5HT or UTP. This inhibition was reversible by intracellular administration of the AT(1)-antagonist losartan and not influenced by the AT(2)-antagonist PD123319. Similar results were obtained in single cell [Ca2+](i) measurements, showing that AngII(i) predominantly influences Ca2+ influx and not Ca2+ release via AT(1)-like receptors. It is concluded that AngII(i) modulates signal transduction activated by heterologous receptor stimulation. (C) 2001 Elsevier Science Inc. All rights reserved

TGF-beta inhibits Ang II-induced MAPK p44/42 signaling in vascular smooth muscle cells by Ang II type 1 receptor downregulation.

Vascular changes in diabetes are characterized by reduced vasoconstriction and vascular remodeling. Previously, we demonstrated that TGF-beta1 impairs Ang II-induced contraction through reduced calcium mobilization. However, the effect of TGF-beta1 on Ang II-induced vascular remodeling is unknown. Therefore, we investigated the effect of TGF-beta1 on Ang II-induced activation of the MAPK p44/42 pathway in cultured rat aortic smooth muscle cells (RASMC). Activation of MAPK p44/42 was determined with a phospho-specific antibody. Angiotensin type 1 receptor (AT(1)) and AT(1) mRNA levels were measured by [(3)H]candesartan-binding and real-time PCR, respectively. AT(1) gene transcription activity was assessed using AT(1) promoter-reporter constructs and by a nuclear runoff assay. In TGF-beta1-pretreated cells, Ang II-induced phosphorylation of MAPK p44/42 was inhibited by 29 and 46% for p42 and p44, respectively, and AT(1) density was reduced by 31%. Furthermore, pretreatment with TGF-beta1 resulted in a 64% reduction in AT(1) mRNA levels and decreased AT(1) mRNA transcription rate by 42%. Pretreatment with TGF-beta1 blocked Ang II-induced proliferation of RASMC, while stimulating Ang II-induced upregulation of plasminogen activator inhibitor-1. In conclusion, TGF-beta1 attenuates Ang II-mediated MAPK p44/42 kinase signaling in RASMC through downregulation of AT(1) levels, which is mainly caused by the inhibition of transcription of the AT(1) gene

Ultrasound and microbubble-targeted delivery of therapeutic compounds

The molecular understanding of diseases has been accelerated in recent years, producing many new potential therapeutic targets. A noninvasive delivery system that can target specific anatomical sites would be a great boost for many therapies, particularly those based on manipulation of gene expression. The use of microbubbles controlled by ultrasound as a method for delivery of drugs or genes to specific tissues is promising. It has been shown by our group and others that ultrasound increases cell membrane permeability and enhances uptake of drugs and genes. One of the important mechanisms is that microbubbles act to focus ultrasound energy by lowering the threshold for ultrasound bioeffects. Therefore, clear understanding of the bioeffects and mechanisms underlying the membrane permeability in the presence of microbubbles and ultrasound is of paramount importance. (Neth Heart J 2009;17:82-6.

Ultrasound and microbubble-targeted delivery of therapeutic compounds: ICIN Report Project 49: Drug and gene delivery through ultrasound and microbubbles

The molecular understanding of diseases has been accelerated in recent years, producing many new potential therapeutic targets. A noninvasive delivery system that can target specific anatomical sites would be a great boost for many therapies, particularly those based on manipulation of gene expression. The use of microbubbles controlled by ultrasound as a method for delivery of drugs or genes to specific tissues is promising. It has been shown by our group and others that ultrasound increases cell membrane permeability and enhances uptake of drugs and genes. One of the important mechanisms is that microbubbles act to focus ultrasound energy by lowering the threshold for ultrasound bioeffects. Therefore, clear understanding of the bioeffects and mechanisms underlying the membrane permeability in the presence of microbubbles and ultrasound is of paramount importance. (Neth Heart J 2009;17:82-6.

Ultrasound and Microbubble-Targeted Delivery of Macromolecules Is Regulated by Induction of Endocytosis and Pore Formation

Contrast microbubbles in combination with ultrasound (US) are promising vehicles for local drug and gene delivery. However, the exact mechanisms behind intracellular delivery of therapeutic compounds remain to be resolved. We hypothesized that endocytosis and pore formation are involved during US and microbubble targeted delivery (UMTD) of therapeutic compounds. Therefore, primary endothelial cells were subjected to UMTD of fluorescent dextrans (4.4 to 500 kDa) using 1 MHz pulsed US with 0.22-MPa peak-negative pressure, during 30 seconds. Fluorescence microscopy showed homogeneous distribution of 4.4-and 70-kDa dextrans through the cytosol, and localization of 155-and 500-kDa dextrans in distinct vesicles after UMTD. After ATP depletion, reduced uptake of 4.4-kDa dextran and no uptake of 500-kDa dextran was observed after UMTD. Independently inhibiting clathrin-and caveolae-mediated endocytosis, as well as macropinocytosis significantly decreased intracellular delivery of 4.4-to 500-kDa dextrans. Furthermore, 3D fluorescence microscopy demonstrated dextran vesicles (500 kDa) to colocalize with caveolin-1 and especially clathrin. Finally, after UMTD of dextran (500 kDa) into rat femoral artery endothelium in vivo, dextran molecules were again localized in vesicles that partially colocalized with caveolin-1 and clathrin. Together, these data indicated uptake of molecules via endocytosis after UMTD. In addition to triggering endocytosis, UMTD also evoked transient pore formation, as demonstrated by the influx of calcium ions and cellular release of preloaded dextrans after US and microbubble exposure. In conclusion, these data demonstrate that endocytosis is a key mechanism in UMTD besides transient pore formation, with the contribution of endocytosis being dependent on molecular size. (Circ Res. 2009; 104: 679-687.