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(Circulation Research. 1996;79:765-772.)
© 1996 American Heart Association, Inc.

Articles

Effects of Intracellular Angiotensin II in Vascular Smooth Muscle Cells

Hermann Haller, Carsten Lindschau, Bettina Erdmann, Petra Quass, Friedrich C. Luft

the Franz Volhard Clinic and the Max Delbruck Center for Molecular Medicine, Virchow Klinikum, Humboldt University of Berlin (Germany).

Correspondence to Hermann Haller, MD, Franz Volhard Clinic, Wiltberg Strasse 50, 13122 Berlin, Germany.

	Abstract

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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.

Key Words: angiotensin II • intracellular receptor • vascular smooth muscle cell • cytosolic Ca²⁺ • gap junction

	Introduction

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Angiotensin II is a potent vasoconstrictor and growth factor for VSMCs.¹ Ang II effects are mediated via specific binding to the AT₁ receptor. Subsequently, a cascade of signaling events results in increased [Ca²⁺]_i,^{2 3} activation of protein kinase C,⁴ and activation of MAP kinase.⁵ However, Ang II may exert its cellular effects via other pathways as well. For instance, internalization of Ang II occurs through receptor-mediated uptake of the peptide and its receptor.⁶ This receptor-mediated uptake seems to play a role in signal transduction.⁷ Furthermore, intracellular delivery of Ang II, or possibly an active Ang II metabolite, may mediate the Ang II–induced growth of VSMCs. Intracellular high-affinity Ang II binding proteins have been described in the cytosol.⁸ Anderson et al⁶ found Ang II to be present in endosomes of the cytosol. Specific binding of Ang II has also been described in isolated nuclei and nuclear chromatin.^{9 10} Despite the published evidence on the intracellular localization of Ang II, the possible physiological consequences of intracellular Ang II are not understood. To elucidate some of these issues, we tested the hypothesis that intracellular Ang II is capable of eliciting a [Ca²⁺]_i second messenger response. We used microinjection and confocal microscopy of single VSMCs to address this question. We showed a specific intracellular Ang II effect on intracellular Ca²⁺ signaling, which was mediated via a cytosolic AT₁ receptor binding site. We also observed an Ang II–induced communication with neighboring VSMCs and showed that intracellular Ang II generates an intracellular Ca²⁺ signal in adjacent cells, possibly via IP₃ transport through gap junctions.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
The fluorescent probe fluo 3 was purchased from Molecular Probes. FITC-conjugated Ang II was obtained from RBI. Ang II receptor antagonist CV-11974 was supplied from Takeda Chemical. All other materials, if not stated otherwise, were purchased from Sigma Chemical Co. Male Sprague-Dawley rats aged 12 weeks were obtained from Ivanovas, Kisslegg, Germany. Care of animals was in accord with institutional and American Physiological Society guidelines.

VSMC Cultures
Rat aortic VSMCs were cultured by procedures modified from Chamley-Campbell et al¹¹ and Haller et al.¹² Briefly, the rats (12 to 14 weeks old) were killed instantly, and their thoracic aortas were excised. After adherent fat and connective tissue were removed, the aortas were cut longitudinally, and the endothelial cells were removed by gentle scraping with fine forceps. The aortas were then minced into small pieces and were incubated at 37°C for 2 hours in PBS without Ca²⁺ but with 1 mg/mL collagenase (type I, 150 IU/mg, Worthington Biochemical Corp), 0.5 mg/mL elastase (type III, 40 IU/mg, Sigma), and 0.5 mg/mL trypsin inhibitor (Sigma). After 2 hours, DMEM/F-12 containing 10% FBS (GIBCO) was added to the suspension to inactivate enzymes. The cells were then centrifuged at 120g for 10 minutes, and the pellet was resuspended in DMEM/F-12 with 10% FBS. The cells were then seeded at a density of 3 to 5x10⁵/cm² and were cultured at 37°C in 95% air/5% CO₂. Cells from passages 2 to 4 were used in all experiments. The phenotype of the cultured VSMCs was determined by staining the cells for -actin and desmin. Antibodies for muscle-specific -actin and desmin were obtained from Boehringer-Mannheim.

Electron Microscopy
Electron microscopy was carried out as previously described.¹³ Cultured cells were fixed in a mixture of freshly prepared 4% formaldehyde/0.5% glutaraldehyde in 0.1 mol/L phosphate buffer and 0.18 mol/L sucrose for 2 hours. After several washes in phosphate buffer and 0.18 mol/L sucrose, they were immersed in a mixture of 1.8 mol/L sucrose/20% polyvinylpyrrolidone (K15, Fluka) overnight and then frozen in liquid nitrogen. Ultrathin (40-nm) cryosections were cut with a Reichert-Jung Ultracut S attached to a cryosystem FC4S at -110°C, according to the method of Tokuyasu.¹⁴ Sections were then mounted on Formvar carbon-coated copper grids. Immunolabeling was performed as recently described.¹³ Briefly, monoclonal antibodies against Ang II¹⁵ were diluted in washing buffer (PBS containing 0.12% glycine and 1% BSA) to a final concentration of 5 µg/mL and detected with 12-nm goat anti-mouse gold conjugates (Dianova). The sections were contrasted and stabilized using a mixture of 0.3% uranyl acetate and 2% methylcellulose (25 cps, Sigma). Nonspecific staining was assessed by omission or preadsorption of the primary antibody. Electron micrographs were taken with an EM 400T (Philips) at an acceleration voltage of 80 kV.

Microinjection
VSMC microinjection was carried out as described elsewhere.¹² Ang II was dissolved in a buffer (mmol/L: HEPES 10, NaCl 145, KCl 5, Na₂HPO₄ 0.5, glucose 6, and MgSO₄ 0.5, pH 7.4) at a concentration of 10^-9 mol/L. Microinjection was carried out using Narishige micromanipulators and a Nikon TLI-188 microinjector. Ang II was injected at a concentration of 10^-9 mol/L in 40 to 70 fL per cell. VSMCs were microinjected 2 days after plating.

[Ca²⁺]_i Imaging With Confocal Microscopy
For [Ca²⁺]_i imaging, VSMCs were transferred onto coverslips and incubated at 37°C for 1 to 2 days.^{3 16} The cells were then loaded with fluo 3 by a 20-minute incubation in PBS containing 2 µmol/L fluo 3-AM (added from a 5 mmol/L stock solution in DMSO and 2% BSA) at 37°C. Before the measurements, cells were rinsed three times with PBS and once with 1.5 mmol/L CaCl₂/HEPES buffer and mounted on a Nikon Diaphot inverted microscope. Measurements were performed with an MRC 600 confocal imaging system (Bio-Rad Laboratories) with an argon laser. We used a Nikon Plan Apo 100 objective with a numerical aperture of 1.4, resulting in a theoretical Z-width half maximum of 0.23 mm. As the optical section depth depends on the confocal aperture, we selected a small aperture 1.8 mm (range of opening width, 0.7 to 8 mm). We then assessed the Z-width half maximum for this pinhole setting by measuring the distance between maximal and half-maximal intensity of a fluo 3 solution. The measured Z-width half maximum was 0.45 mm.

Measurements were performed using the 488-nm wavelength of an argon laser by time mode and line-scanning mode. A perinuclear area was assessed in all experiments in which we used the line-scan mode. Ca²⁺-free conditions were achieved by adding 2.5 mmol/L EGTA to all solutions. F values were obtained through the Bio-Rad Comos software. After exposure to Ang II, picture frames were recorded in 3-second intervals. At least 10 to 18 cells from each of at least seven separate experiments were examined for each experimental condition.

Statistics
Statistical analysis was carried out on a Macintosh II computer (Apple Inc) using a commercially available statistical program (Statview, Cricket Software Inc). Since the data feature substantial variability and are not uniformly distributed, we used nonparametric statistical tests, such as the sign test and Mann-Whitney test, to analyze the data from the 7 to 10 separate experiments. A value of P<.05 was accepted as significant. References to increases or decreases in the following section are only so stated if statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
We first used electron microscopy with immunocytochemistry and colloidal gold to localize intracellular Ang II in cultured VSMCs. A representative transmission electron photomicrograph of these experiments is shown in Fig 1. Gold grains are visible (arrows) in the submembranal space, in vesicle-like structures, and in the nucleus. We then analyzed the intracellular distribution of the microinjected Ang II using fluorescently labeled Ang II. These results are shown in Fig 2. Microinjection of fluorescently labeled Ang II into the cytoplasm of a single VSMC was rapidly distributed throughout the cytoplasm. Increased concentrations of the hormone were observed in submembranous domains and in the perinuclear area. Ang II was prominently present in the nucleus of the injected cell. After 15 to 20 seconds, the fluorescence began to decline and almost completely disappeared after 30 seconds. No fluorescence was observed in adjacent neighboring cells at any time. The rapid disappearance of the signal from FITC-labeled Ang II may indicate a quenching of fluorescence due to receptor binding or changes in the local ionic composition. To further analyze the fate of the microinjected Ang II, we fixed microinjected VSMCs at different time points and stained them with an anti-FITC antibody (Sigma), followed by a secondary Cy3-labeled antibody. To rule out interference of FITC and Cy3, we showed in preliminary experiments that the anti-FITC antibody totally quenched the FITC fluorescence. We found that 0.5 minute after microinjection, Ang II was present within tiny spots in the perinuclear area; at 1 minute, spots of Ang II were visible in the cytosol. These cytosolic "spots" increased in intensity at 2.5 and 5 minutes, indicating uptake of the labeled Ang II into a subcellular compartment. These results indicate that after the initial distribution of microinjected Ang II in the cytosol and in the nucleus (see Figs 1 and 2), Ang II is localized in a cytosolic department.

View larger version (133K): [in this window] [in a new window]   Figure 1. Transmission electron photomicrograph of resting VSMC with immunocytochemical-labeled Ang II. Gold grains are visible (arrows) in the submembranal space, in vesicle-like structures of the endoplasmic reticulum, and in the nucleus (magnification x60 000, bar=0.5 µm).

View larger version (82K): [in this window] [in a new window]   Figure 2. Microinjection of fluorescently labeled Ang II into the cytoplasm of a VSMC at 0, 10, 20, and 30 seconds. Ang II was rapidly distributed in cytoplasmic domains near the plasma membrane and the nucleus of the injected cell. After 15 to 20 seconds, the fluorescence signal started to decline and disappeared after 30 seconds. No fluorescence was observed in adjacent neighboring cells.

We then used confocal microscopy to examine the effects of intracellular Ang II on [Ca²⁺]_i. Fig 3 (top panel) shows photomicrographs of the changes in [Ca²⁺]_i after Ang II microinjection into the cytoplasm of a VSMC at 0, 5, and 10 seconds. VSMCs were labeled with fluo 3. Ang II (10^-9 mol/L) induced a rapid and sustained increase in [Ca²⁺]_i and nuclear [Ca²⁺]_i. In the bottom panel, the time course of the [Ca²⁺]_i signal after intracellular Ang II is shown. We next carried out experiments to demonstrate the specificity of the intracellular Ang II response. These results are shown in Fig 4. The top left panel shows the intracellular Ca²⁺ signal after intracellular Ang II. The top right panel demonstrates the persistence of the intracellular Ca²⁺ signal after AT₁ receptor downregulation. We first exposed the cells to extracellular Ang II (10^-7 mol/L); a repeated exposure to extracellular Ang II after 60 seconds showed no signal. However, intracellular Ang II in the same VSMCs promptly elicited an intracellular Ca²⁺ signal. The bottom panels show experiments with the extracellular AT₁ receptor blocker (CV-11974). Extracellular CV-11974 (10^-7 mol/L) inhibited the [Ca²⁺]_i response to extracellular Ang II but did not influence the intracellular effects of Ang II (bottom left panel). Microinjection of CV-11974 (10^-9 mol/L), on the other hand, inhibited the [Ca²⁺]_i response to intracellular Ang II (bottom right panel). We also used an anti–Ang II peptide, Glu-Gly-Val-Tyr-Val-His-Pro-Val (Bachem), to demonstrate the specificity of the observed intracellular effects of Ang II. The microinjection of the anti-peptide blocked the subsequent intracellular response to Ang II (data not shown).

View larger version (62K): [in this window] [in a new window]   Figure 3. Top, Microinjection of Ang II into the cytoplasm of a VSMC at 0, 5, and 10 seconds. VSMCs were labeled with fluo 3. Ang II induced a rapid and sustained increase in cytosolic and nuclear [Ca2+]i. Bottom, Time course of the intracellular Ca2+ signal after intracellular Ang II administration (see "Materials and Methods").

View larger version (24K): [in this window] [in a new window]   Figure 4. Top left, Intracellular Ca2+ signal in VSMC after intracellular Ang II (Ang II intra). Top right, Intracellular Ca2+ signal in VSMC after extracellular Ang II (Ang II extra), repeat Ang II extra at 60 seconds (AT1 receptor downregulation, no signal), and Ang II intra in the same VSMC (repeat signal). Bottom left, Intracellular Ca2+ signal with extracellular AT1 receptor blocker (CV-11974 extra) alone in VSMC (no signal), Ang II extra and CV-11974 extra together (no signal), and CV-11974 extra with Ang II intra (signal). Bottom right, Intracellular Ca2+ signal with intracellular AT1 receptor blocker (CV-11974 intra) alone in VSMC (no signal), and intracellular Ang II intra and CV-11974 intra (no signal).

We then investigated the effects of smaller peptides on [Ca²⁺]_i in VSMCs. We used the microinjection of Ang 1-7, Ang 3-8 (both Bachem), and Ang 1-4, as well as Ang 5-8 (American Peptides). Only microinjection of Ang 3-8 induced a rise in [Ca²⁺]_i, which was similar to Ang II. The other peptides had no effect on the influx or release of Ca²⁺. These results argue that the intracellular receptor(s) for Ang II also recognizes Ang 3-8.

In order to rule out an influence by heterologous desensitization, we exposed the cells to bradykinin extracellularly (10^-7 mol/L) and intracellularly (10^-9 mol/L) before exposure to Ang II. The response to Ang II was not influenced by preincubation or by microinjection of bradykinin (data not shown).

During the course of the microinjection experiments with Ang II, we observed that not only the microinjected cell but also adjacent neighboring cells displayed an intracellular Ca²⁺ signal. These results are shown in Fig 5. In the top panel is shown a photomicrograph after microinjection of Ang II into the cytoplasm of a VSMC at 0, 5, and 10 seconds. An intracellular Ca²⁺ signal was observed in the injected cell and in adjacent neighboring cells. In the bottom panel, the time course of the intracellular Ca²⁺ signal after Ang II in an injected VSMC (A) and adjacent VSMCs (B and C) is shown. Adjacent VSMCs showed a typical delay in the [Ca²⁺]_i response in the range of 2 to 8 seconds. From this time lag and the distance between the points where the intracellular Ca²⁺ signal was recorded, we can calculate an approximate diffusion velocity of 5 to 10 µm/s. The pattern of the intracellular Ca²⁺ signal appeared similar between the microinjected and the adjacent cells.

View larger version (73K): [in this window] [in a new window]   Figure 5. Top, Microinjection of Ang II into the cytoplasm of a VSMC at 0, 5, and 10 seconds. An intracellular Ca2+ signal was observed in the injected cell and in adjacent neighboring cells. Bottom, Time course of intracellular Ca2+ signal after Ang II in injected VSMC (A) and adjacent VSMCs (B and C).

We next investigated the source of the Ca²⁺ ions in the Ang II–elicited [Ca²⁺]_i response. In Fig 6 (top panel) is shown the effect of EGTA (10 mmol/L) on the time course of the intracellular Ca²⁺ signal in an Ang II–microinjected VSMC (A) and in adjacent VSMCs (B and C). Extracellular Ca²⁺-free medium attenuated the intracellular Ca²⁺ signal in the injected cell but had no effect on the intracellular Ca²⁺ signals of adjacent cells. Preincubation of the VSMCs with the L-type Ca²⁺ channel antagonist nitrendipine (10^-7 mol/L) also diminished the Ang II–induced intracellular Ca²⁺ signal in the microinjected cell but had no effect on the [Ca²⁺]_i increase in adjacent cells. These findings indicate that Ca²⁺ influx in the microinjected cells is via voltage-operated Ca²⁺ channels but does not play an important role in the [Ca²⁺]_i response of the adjacent VSMCs. In the bottom panel of Fig 6, the effect of thapsigargin on the time course of the intracellular Ca²⁺ signal in a microinjected VSMC (A) and in adjacent VSMCs (B and C) is shown. Thapsigargin attenuated the initial intracellular Ca²⁺ signal in the injected cell and abolished the intracellular Ca²⁺ signals in adjacent cells. Using preincubation with ryanodine (10^-7 mol/L), we also abolished the intracellular Ca²⁺ signal in the adjacent cells, whereas the microinjected cell was only slightly affected. These findings support the assumption that the intracellular Ca²⁺ signal in the adjacent cells is mediated via the release of Ca²⁺ from intracellular stores. We subsequently tested this hypothesis directly by microinjecting IP₃ into VSMCs. These experiments are shown in the top panel of Fig 7. Microinjection of IP₃ into the cytoplasm of a VSMC at 0, 5, and 10 seconds induced an intracellular Ca²⁺ signal in adjacent cells that was similar to the Ang II–induced effects. The time course comparison between intracellular Ang II and intracellular IP₃ in an injected VSMC (A) and in adjacent cells (B and C) is shown in the bottom panel of Fig 7. The same cell was injected twice, and the same adjacent cells were observed with a 5-minute interval. From these experiments, we calculated a diffusion time for IP₃ of 5 to 10 µm/s. The Ang II and IP₃ effects were similar with regard to the cells that responded to the microinjection and in terms of the shape of the [Ca²⁺]_i response.

View larger version (21K): [in this window] [in a new window]   Figure 6. Top, Effect of EGTA. Time course of intracellular Ca2+ signal in microinjected VSMC (A) and adjacent VSMCs (B and C). Extracellular Ca2+-free medium attenuated the intracellular Ca2+ signal in the injected cell but had no effect on the intracellular Ca2+ signals of adjacent cells. Bottom, Effect of thapsigargin. Time course of intracellular Ca2+ signal in microinjected VSMC (A) and adjacent VSMCs (B and C). Thapsigargin attenuated the initial intracellular Ca2+ signal in the injected cell and abolished the intracellular Ca2+ signals in adjacent cells.

View larger version (92K): [in this window] [in a new window]   Figure 7. Top, Microinjection of IP3 into the cytoplasm of a VSMC at 0, 5, and 10 seconds. Intracellular Ca2+ signals were observed in the injected cell and in adjacent cells. Bottom, Time course comparison between intracellular Ang II and intracellular IP3 in injected VSMC (A) and in adjacent cells (B and C). The same cell was injected twice, and the same adjacent cells were observed with a 5-minute interval. The Ang II and IP3 effects were similar.

The fact that not all adjacent cells but only certain cells near the microinjected VSMC reacted with an intracellular Ca²⁺ signal suggested that specific cell-cell communications were responsible for this effect. Therefore, we tested whether or not communication via gap junctions was responsible for the effect of intracellular Ang II. In Fig 8 is shown the effect of extracellular DMSO (10^-8 mol/L) on cell-cell communication. VSMCs were preincubated with DMSO for 5 minutes. Microinjection of VSMCs with Ang II was followed by an intracellular Ca²⁺ signal, but only in the injected cell. The adjacent cells showed no intracellular Ca²⁺ signal. Similar responses were observed when we applied heptanol (10^-8 mol/L). To further analyze the cell-cell communication between VSMCs, we also microinjected fluo 3 into the VSMCs. However, we were not able to detect a fluorescence signal in adjacent cells.

View larger version (35K): [in this window] [in a new window]   Figure 8. Effect of extracellular DMSO. Microinjection of VSMC with Ang II was followed by an intracellular Ca2+ signal, but only in the injected cell. The adjacent cells showed no intracellular Ca2+ signal.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The important findings in the present study were that Ang II is detectable within resting VSMCs and that intracellular Ang II elicits a response of second messengers within the cell in question and also in adjacent cells. We showed that Ang II is present in both the cytoplasm and nucleus of VSMCs. We found that intracellular Ang II induces a surge in [Ca²⁺]_i. The effect appears to be AT₁ receptor dependent, since the effect could be blocked with the Ang II receptor antagonist CV-11974. We also found that the response to intracellular Ang II spreads via cell-cell contacts to neighboring adjacent cells and that this effect may be mediated via IP₃. To our knowledge, this is the first report to demonstrate that intracellular Ang II induces second messengers in VSMCs directly and suggests that intracellular Ang II may stimulate a cluster of VSMCs via release of second messengers.

Our first experimental observation was that microinjection of Ang II generates an intracellular Ca²⁺ signal in the cytosol and the nucleus within a few seconds. This effect was not due to leakage of microinjected Ang II into the extracellular space, because blockade and downregulation of extracellular Ang II receptors did not influence the intracellularly elicited Ca²⁺ signal. The effect of intracellular Ang II on Ca²⁺ implies a specific binding of Ang II in the cytosol, since microinjection of an Ang II receptor blocker abolished the Ang II effect. This observation is in agreement with the results of Kiron and Soffer,⁸ who described a specific cytosolic Ang II binding protein. Our observations with fluorescent Ang II indicate that the microinjected hormone is distributed throughout the cytosol and then is rapidly taken up by endosomes,⁶ after which the fluorescent signal is quenched. The quenching effect is most likely related to changes in pH.

Intracellular Ang II could conceivably bind to endocytosed Ang II receptors. Recent work indicates that endocytosis of membrane-bound polypeptide receptors may not only be part of the cellular desensitization and receptor recycling apparatus but may also play an active role in intracellular signaling.¹⁷ Griendling et al⁷ have suggested that the internalized hormone-receptor complex activates signaling pathways. In our electron photomicrographs, Ang II was present in the cytosol within endosomes and to a large extent in the nucleus. Using fluorescence-labeled Ang II, we were able to demonstrate a prominent fluorescence signal in the cytosol and in the nucleus. These localizations are compatible with the hypothesis that subsequent to internalization (or after intracellular generation), Ang II exerts signaling in the cytosol and then translocates to the nucleus, where it could participate directly in regulating gene expression.¹⁷ We are aware that this suggestion is provocative; however, our findings are consistent with such a view. We are unable to state for certain on the basis of our data by which mechanism Ang II induces Ca²⁺ influx and IP₃-mediated intracellular Ca²⁺ release. Intracellular Ang II binding sites may be coupled to G proteins and phospholipases. Evidence for an intracellular signaling function of G proteins has been reported.¹⁸

Ang II also induced an increase in nuclear [Ca²⁺]_i. The possibility that Ca²⁺ participates in nuclear signaling has been raised by us³ and by other investigators.¹⁹ Electron microscopy and the fluorescence-labeled Ang II both showed that Ang II is present in the nucleus. These data support the possibility that Ang II may have direct nuclear effects. Such a notion is consistent with earlier reports suggesting that Ang II induces transcription.²⁰

Our second observation was that the transmission of the Ang II induced an intracellular Ca²⁺ signal in adjacent neighboring VSMCs. The experiments with microinjected IP₃ suggest that this transmission is mediated via IP₃. That cell-cell communication is due to movement of IP₃ has been suggested by others.²¹ However, we cannot rule out the possibility that the Ca²⁺ ion itself may be part of the junctional signaling pathway.²² Previous studies have demonstrated that several cell types are able to propagate slow Ca²⁺ waves from cell to cell.^{23 24} Our experiments with uncoupling substances, such as DMSO and heptanol, demonstrated that the coupling between the VSMCs is due to gap junctions.^{25 26} The gap junctional proteins connexin43 and connexin40 have been described in VSMCs in vivo.²⁷ The observation that not all neighboring cells are connected is in agreement with the findings of Little et al,²⁷ who used dye tracers and confocal microscopy. They observed a heterogeneous coupling between VSMCs in intact vessels. Our results support the hypothesis that VSMCs form a syncytium and are functionally coupled within the vessel wall.²⁸ Excitation of a single cell would then result in the activation of adjacent cells. As in our experiments, an intracellularly acting stimulant could thereby activate a cluster of cells.^{29 30}

We were interested in the observation that the [Ca²⁺]_i surge observed with intracellular Ang II was primarily a function of extracellular Ca²⁺, since the surge was not observed in a Ca²⁺-free medium. On the other hand, the secondary intracellular Ca²⁺ signals were abolished by thapsigargin, suggesting that they were related to the release of intracellular Ca²⁺ from intracellular stores. These findings are consistent with the [Ca²⁺]_i responses observed with extracellular Ang II, namely a facilitated entry of Ca²⁺ through voltage-dependent Ca²⁺ channels.³¹ Our results suggest that the intracellular binding of Ang II has a direct effect on the membrane-bound Ca²⁺ channels. In addition to a rise in [Ca²⁺]_i, the intracellular response to Ang II requires activation of protein kinases, such as MAP kinase and protein kinase C.^{4 32 33 34} We have not yet investigated the effects of the intracellular Ang II on MAP kinase or protein kinase C. Since a rise in [Ca²⁺]_i is sufficient to translocate Ca²⁺-sensitive protein kinase C isoforms toward the nucleus (authors' unpublished data, 1996), it will be of interest to analyze the effects of the intracellular Ang II on kinase activation and cell proliferation.

We can only speculate about the relevance of the present findings under in vivo conditions. It is conceivable that Ang II is generated intracellularly. The enzymes necessary for its production are present within cells. Other growth factors, such as acidic fibroblast growth factor, have been shown to exert intracellular effects.¹⁷ However, as far as we are aware, intracellular generation of Ang II has not been described. An alternative possibility is that Ang II is internalized with its receptor. Our data suggest that Ang II exerts no effects independent of its receptor, since the [Ca²⁺]_i surge could be blocked with AT₁ receptor blockade. In any event, we believe our findings suggest that Ang II has unique intracellular actions.

	Selected Abbreviations and Acronyms

 

Ang = angiotensin DMSO = dimethyl sulfoxide IP3 = inositol tris-phosphate MAP = mitogen-activated protein VSMC = vascular smooth muscle cell

	Acknowledgments

 
This study was supported by a grant from the Deutsche Forschungsgemeinschaft.

	Footnotes

 
Previously presented in part at the Annual Meeting of the American Society of Nephrology, San Diego, Calif, November 1995.

Received May 6, 1996; accepted July 3, 1996.

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