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(Circulation Research. 2002;90:1080.)
© 2002 American Heart Association, Inc.

Molecular Medicine

Functional Reconstitution of Vascular Smooth Muscle Cells With cGMP-Dependent Protein Kinase I Isoforms

Robert Feil, Nicolai Gappa, Mark Rutz, Jens Schlossmann, Christine R. Rose, Arthur Konnerth, Sabine Brummer, Susanne Kühbandner, Franz Hofmann

From the Institut für Pharmakologie und Toxikologie der Technischen Universität München (R.F., N.G., M.R., J.S., S.B., S.K., F.H.) and Physiologisches Institut (C.R.R., A.K.), Universität München, München, Germany.

Correspondence to Robert Feil, Institut für Pharmakologie und Toxikologie der Technischen Universität München, Biedersteiner Str. 29, 80802 München, Germany. E-mail feil{at}ipt.med.tu-muenchen.de

	Abstract

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The cGMP-dependent protein kinase type I (cGKI) is a major mediator of NO/cGMP-induced vasorelaxation. Smooth muscle expresses two isoforms of cGKI, cGKI and cGKIß, but the specific role of each isoform in vascular smooth muscle cells (VSMCs) is poorly understood. We have used a genetic deletion/rescue strategy to analyze the functional significance of cGKI isoforms in the regulation of the cytosolic Ca²⁺ concentration by NO/cGMP in VSMCs. Cultured mouse aortic VSMCs endogenously expressed both cGKI and cGKIß. The NO donor diethylamine NONOate (DEA-NO) and the membrane-permeable cGMP analogue 8-bromo-cGMP inhibited noradrenaline-induced Ca²⁺ transients in wild-type VSMCs but not in VSMCs genetically deficient for both cGKI and cGKIß. The defective Ca²⁺ regulation in cGKI-knockout cells could be rescued by transfection of a fusion construct consisting of cGKI and enhanced green fluorescent protein (EGFP) but not by a cGKIß-EGFP construct. Fluorescence imaging indicated that the cGKI-EGFP fusion protein was concentrated in the perinuclear/endoplasmic reticulum region of live VSMCs, whereas the cGKIß-EGFP protein was more homogeneously distributed in the cytoplasm. These results suggest that one component of NO/cGMP-induced smooth muscle relaxation is the activation of the cGKI isoform, which decreases the noradrenaline-stimulated cytosolic Ca²⁺ level.

Key Words: smooth muscle • nitric oxide • calcium • gene targeting • cGMP kinase

	Introduction

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The mechanism by which NO and NO-generating drugs lead to vasodilatation is not well understood.^1–5 Recent studies with knockout mice have shown that NO can induce the relaxation of vascular smooth muscle by activation of soluble guanylyl cyclase, production of cGMP, and activation of cGMP-dependent protein kinase I (cGKI).^6,7 Furthermore, NO and cGMP may regulate vascular tone by signaling pathways that do not require cGKI.^7,8 The identity of the cellular substrates and mechanisms regulated by cGKI has been discussed controversially. cGKI-mediated smooth muscle relaxation may involve a decrease in the cytosolic Ca²⁺ concentration ([Ca²⁺]_i)^6,9,10 as well as a modulation of the activity of myosin phosphatase,^11–14 RhoA,¹⁵ or telokin.¹⁶ Overexpression of cGKI in non–smooth muscle cells suggests additional targets.^1,17 At present, the functional significance of these targets in the regulation of vascular tone is unclear. Moreover, the interpretation of some results obtained with intact vascular smooth muscle cells (VSMCs) may be limited by the fact that the extensively used "specific cGKI inhibitor" KT5823 may not inhibit cGKI activity in intact cells¹⁸ and that cGKI expression decreases during cell culture.^9,19

The cGKI gene encodes two isoforms, cGKI and cGKIß, which differ in their amino termini (100 amino acids).^20,21 Both isoforms are expressed in vascular and nonvascular smooth muscle.^22–24 The amino terminus is involved in homodimerization, regulation of cGMP affinity and kinase activation, and target protein recognition.^1,2,25 Recent experiments have shown that the amino terminus of cGKI interacts specifically with the myosin-binding subunit of myosin phosphatase,¹² whereas the amino terminus of cGKIß interacts specifically with inositol 1,4,5-trisphosphate receptor–associated cGMP kinase substrate (IRAG), a cGKI substrate protein.²⁶ In contrast to this detailed information on the biochemistry of the cGKI isoforms, the specific cellular functions of cGKI and cGKIß in VSMCs are not known.

We have used cGKI-deficient primary cells to study the functional significance of the endogenous NO/cGMP/cGKI signaling pathway and the specific roles of the cGKI and cGKIß isoforms in the regulation of [Ca²⁺]_i in VSMCs. This strategy overcomes the limitations of experiments that are based solely on pharmacological tools to dissect cGKI-dependent and cGKI-independent NO/cGMP signaling pathways in intact cells. Our results indicate that NO/cGMP inhibits noradrenaline (NA)-induced Ca²⁺ transients in VSMCs via activation of cGKI but not cGKIß.

	Materials and Methods

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Construction and Transfection of Plasmids
A BamHI site was introduced into the region encompassing the stop codon of the bovine cGKI cDNA²⁰ by polymerase chain reaction–mediated mutagenesis, such that the wild-type sequence GAC TTC TAA TGT encoding Asp Phe Stop was changed into GAC TTG GAT CCA encoding Asp Leu Asp Pro. The 2.1-kb EcoRI-BamHI fragment of the mutated cGKI cDNA was inserted into the EcoRI-BamHI sites of pEGFP-N1 (Clontech), where EGFP indicates enhanced green fluorescent protein, resulting in the vector pCMV-cGKIEGFP, where CMV indicates cytomegalovirus. The vector pCMV-cGKIßEGFP was constructed by replacing the 1.8-kb EcoRI-PacI fragment of pCMV-cGKIEGFP with the 1.8-kb EcoRI-PacI fragment of the bovine cGKIß cDNA²⁷ encompassing the 5' region, which differs between the and ß isoform. The CMV promoter–driven expression vectors pCMV-cGKIEGFP and pCMV-cGKIßEGFP encode in-frame fusions of EGFP to the carboxy termini of cGKI and cGKIß, respectively. The identities of all constructs were verified by DNA sequence analysis. VSMCs were transiently transfected with the use of LipofectAMINE (Life Technologies) and then maintained in serum-free medium for 2 days before analysis. COS-7 cells were transiently transfected by using the calcium phosphate method and were analyzed 2 days after transfection.

Cell Culture and Measurement of [Ca²⁺]_i
VSMCs were isolated from aortas of 3- to 6-week-old litter-matched wild-type (cGKI^+/+) and cGKI-knockout (cGKI^-/-) mice²⁸ by enzymatic digestion and grown in DMEM (Life Technologies) supplemented with 10% FCS.²⁹ The identity of the cells was confirmed by staining with a smooth muscle -actin antibody (Sigma Chemical Co). Subconfluent cells in their first passage were maintained in serum-free medium for 2 days and then in buffer A (mmol/L: NaCl 140, KCl 5.0, MgSO₄ 1.2, CaCl₂ 2.0, glucose 10, and HEPES 5.0, pH 7.4) for experiments. [Ca²⁺]_i was analyzed in single fura 2–loaded cells,³⁰ with the investigator being unaware of the genotype of the cells and the identity of the transfected plasmids. Ca²⁺ transients were elicited at room temperature by local application of NA (pipette solution containing 1 µmol/L NA in buffer A) in the absence or presence of 10 µmol/L diethylamine NONOate (DEA-NO; Alexis) or 1 mmol/L 8-bromo-cGMP (8-Br-cGMP, Biolog).

Fluorescence Imaging
Imaging of EGFP fluorescence was performed by use of a custom-built two-photon laser-scanning microscope based on a mode-locked titanium-sapphire laser system operated at 790-nm center wave length, 80-MHz pulse repeat, and <100-femtosecond pulse width (Tsunami and Millenia, Spectra Physics) and a laser-scanning system (MRC 1024, Bio-Rad) mounted on an upright microscope (BX50WI, Olympus) equipped with a x40 water immersion objective (Olympus). Cells were perfused with buffer A or buffer A containing 1 mmol/L 8-Br-cGMP. Every 5 to 10 minutes, 14 to 20 focal planes 0.3 to 0.5 µm apart were scanned. These were later projected into a single image. Images were analyzed offline with NIH Image software.

Miscellaneous Methods
Cyclic nucleotides and cGKI activity were determined by using enzyme immunoassay kits (Cayman Chemical) and an in vitro kinase assay,³¹ respectively. Western blot analysis and immunocytochemistry were performed by using polyclonal rabbit antisera detecting cGKI,⁶ specifically cGKI or cGKIß (J.S., F.H., unpublished data, 2002), IRAG,²⁶ and phospho-Ser¹⁵⁷- VASP (Alexis), where VASP is vasodilator-stimulated phosphoprotein.

	Results

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VSMCs were isolated from aortas of cGKI^+/+ mice and cGKI^-/- mice, which are deficient for both the cGKI and cGKIß isoform.²⁸ Cells were studied in their first passage to limit phenotypic modulation, downregulation of cGKI expression,^9,19 and potential deregulation of other components of the NO/cGMP pathway, which may occur after repeated passaging of VSMCs. The morphology and general growth characteristics of cGKI^-/- cells were indistinguishable from those of cGKI^+/+ cells (Figure 1A and data not shown). Basal levels of cGMP as well as cAMP were similar in cGKI^+/+ and cGKI^-/- cells (Figure 1B). Treatment with the NO donor DEA-NO (10 µmol/L) increased cGMP levels 50-fold in cGKI^+/+ and cGKI^-/- cells, whereas cAMP levels were not increased by treatment with DEA-NO (10 µmol/L) or the membrane-permeable cGMP analogue 8-Br-cGMP (1 mmol/L) (Figure 1B). Western blot analysis showed that cGKI was endogenously expressed in cGKI^+/+ but not in cGKI^-/- cells (Figure 1C, top panels). DEA-NO (10 µmol/L) as well as 8-Br-cGMP (1 mmol/L) stimulated the phosphorylation of VASP, a substrate for both cGKI- and cAMP-dependent protein kinases,³² in cGKI^+/+ but not in cGKI^-/- cells (Figure 1C, bottom panels). These results indicated that the cGKI^+/+ VSMCs analyzed in the present study endogenously expressed a functional NO/cGMP/cGKI pathway and that DEA-NO as well as 8-Br-cGMP activated the endogenous cGKI in intact cells without increasing cAMP levels or cAMP-dependent protein kinase activity. Furthermore, the lack of functional cGKI in the cGKI^-/- cells did not affect the general growth properties and cyclic nucleotide levels compared with those in the cGKI^+/+ cells.

View larger version (37K): [in this window] [in a new window]   Figure 1. Characterization of endogenous NO/cGMP/cGKI signaling in wild-type and cGKI-deficient VSMCs. A, Phase-contrast photomicrographs of confluent cGKI+/+ cells (left) and cGKI-/- cells (right) grown in medium containing 10% FCS (original magnification x100). B, cGMP levels (left) and cAMP levels (right) in cGKI+/+ cells (open bars) and cGKI-/- cells (solid bars) in the absence (control) and presence of 10 µmol/L DEA-NO or 1 mmol/L 8-Br-cGMP for 10 minutes. Values are means of triplicate determinations (n=3). C, Western blot analysis of cGKI+/+ cells (left) and cGKI-/- cells (right) with antibodies detecting both cGKI isoforms (cGKI, top) or phospho-Ser157-VASP (p-VASP, bottom). Loaded cell lysates were from untreated cells (control) or cells incubated with 10 µmol/L DEA-NO or 1 mmol/L 8-Br-cGMP for 10 minutes. D, Immunocytochemical detection of endogenous cGKI in subconfluent cGKI+/+ cells grown in serum-free medium with the use of an antibody that detects both cGKI isoforms (original magnification x400). The arrow indicates perinuclear staining. No staining was observed in cGKI-/- cells (not shown). E, Western blot analysis of endogenous cGKI, cGKIß, and IRAG expression in cGKI+/+ cells with the use of specific antibodies.

Immunocytochemistry with a cGKI-specific antibody indicated that the endogenous cGKI was concentrated in the perinuclear/endoplasmic reticulum region (Figure 1D). Western blot analysis (Figure 1E) showed that the VSMCs expressed both the cGKI and cGKIß isoform as well as the recently identified cGKI substrate, IRAG.¹⁰

To evaluate the functional significance of the endogenous NO/cGMP/cGKI pathway for the regulation of [Ca²⁺]_i in VSMCs, NA-induced Ca²⁺ transients were measured in single cells loaded with the Ca²⁺ indicator fura 2. NA elicited Ca²⁺ transients in cGKI^+/+ and cGKI^-/- cells, and the magnitude of the Ca²⁺ transients varied from cell to cell in preparations of both genotypes (Figures 2A through 2C). A second stimulation of a cell after the first Ca²⁺ transient reproducibly elicited a second Ca²⁺ transient of similar magnitude (Figures 2A and 2D). Preincubation with DEA-NO (10 µmol/L) or 8-Br-cGMP (1 mmol/L) for 10 minutes significantly reduced the magnitude of the second Ca²⁺ transient in cGKI^+/+ cells but not in cGKI^-/- cells (Figures 2B through 2D), indicating that NO/cGMP suppressed the NA-stimulated increase in [Ca²⁺]_i by activation of endogenous cGKI.

View larger version (18K): [in this window] [in a new window]   Figure 2. Regulation of NA-induced Ca2+ transients by NO/cGMP in wild-type and cGKI-deficient VSMCs. Cells were stimulated with NA (1 µmol/L in the pipette) for 1 second and then superfused with buffer A for 15 minutes; the last 10 minutes was with or without 10 µmol/L DEA-NO or 1 mmol/L 8-Br-cGMP and was followed by a second stimulation with NA. A through C, Representative original recordings of [Ca2+]i in single cells. The arrows mark the application of NA. The horizontal and vertical bars indicate 50 seconds and 500 nmol/L [Ca2+]i, respectively. A, cGKI+/+ cell stimulated 2 times under control conditions. B, cGKI+/+ cell preincubated with DEA-NO before the second stimulation. C, cGKI-/- cell preincubated with DEA-NO before the second stimulation. D, Histogram summarizing the effects of DEA-NO and 8-Br-cGMP on NA-induced Ca2+ transients in cGKI+/+ cells (open bars) and cGKI-/- cells (solid bars). Data are expressed as the ratio of the area under the curve (AUC) of the second over the first transient. The second transient was elicited in the absence of drugs (control) or in the presence of DEA-NO or 8-Br-cGMP. Shown are mean±SEM values. The number of cells (n) measured under each condition is given inside each bar. *P<0.001 vs control (Student t test).

To confirm the role of cGKI in NO/cGMP-dependent Ca²⁺ regulation and to analyze the specific functions of the cGKI and cGKIß isoforms, cells were transfected with expression plasmids encoding the cGKI isoforms. EGFP was fused to the carboxy terminus of each cGKI isoform to identify transfected cells as well as the intracellular localization of each isoform. Both the cGKI-EGFP and cGKIß-EGFP fusion constructs were expressed with the expected apparent molecular weights (Figure 3A) and had cGMP-dependent kinase activities and activation constants similar to the respective wild-type isoforms (Figure 3B). Browning et al³³ recently reported a fusion construct similar to cGKI-EGFP that had constitutive (cGMP-independent) kinase activity. In contrast, the kinase activity of the cGKI-EGFP as well as the cGKIß-EGFP fusion protein generated in the present study was stimulated 10-fold by cGMP (Figure 3B), allowing for the functional analysis of the cGKI isoforms before and after activation by cGMP in living cells.

View larger version (22K): [in this window] [in a new window]   Figure 3. Expression and activity of cGKI-EGFP fusion proteins in mammalian cells. Cell extracts from COS cells transiently transfected with expression plasmids encoding cGKI, cGKI-EGFP, cGKIß, or cGKIß-EGFP were analyzed. A, Western blot analysis of cGKI-EGFP and cGKIß-EGFP expression with the use of cGKI-specific antibodies. As controls, cell extracts from mock-transfected cells and from cells expressing cGKI or cGKIß were also loaded. The positions of molecular weight markers are indicated. B, In vitro assay of the cGMP-dependent kinase activity of cGKI-EGFP () and cGKIß-EGFP () compared with that of wild-type cGKI () and cGKIß (). The kinase activity in the presence of increasing concentrations of cGMP was normalized to the activity in the presence of 10 µmol/L cGMP. Shown are mean values of triplicate determinations (n=3). A sigmoidal function was used to fit the activation curves and to determine the activation constants (Ka values, inset). In extracts from mock-transfected cells, the total phosphotransferase activity in the absence/presence of 10 µmol/L cGMP was 0.32/0.41 nmol · min-1 · mg-1. In extracts from cells transfected with cGKI, cGKI-EGFP, cGKIß, and cGKIß-EGFP constructs, the total phosphotransferase activity in the absence/presence of 10 µmol/L cGMP was 0.44/5.20, 0.47/3.52, 0.31/2.63, and 0.33/3.23 nmol · min-1 · mg-1, respectively.

In the next set of experiments, we investigated whether [Ca²⁺]_i was regulated by cGKI and/or cGKIß in VSMCs. Control experiments with cGKI^+/+ cells expressing EGFP alone demonstrated that the presence of EGFP, per se, did not grossly affect the determination of [Ca²⁺]_i or the [Ca²⁺]_i-lowering effect of 8-Br-cGMP (Figure 4). VSMCs transfected with the cGKI-EGFP or cGKIß-EGFP construct showed similar intensities of EGFP fluorescence, indicating a similar expression level of the two fusion proteins. Interestingly, the defective Ca²⁺ regulation in cGKI^-/- cells (Figure 2D) could be rescued by transfection of cGKI-EGFP but not cGKIß-EGFP (Figure 4). These results indicate that specifically the cGKI isoform but not the cGKIß isoform inhibits NA-induced Ca²⁺ transients in VSMCs.

View larger version (41K): [in this window] [in a new window]   Figure 4. Rescue of the defective Ca2+ regulation in cGKI-deficient VSMCs by transfection of cGKI-EGFP fusion constructs. Transfected cells were identified by their EGFP fluorescence. NA-induced Ca2+ transients were measured in the absence (-) and presence (+) of 1 mmol/L 8-Br-cGMP as detailed in the legend to Figure 2. As a control, cGKI+/+ cells (open bars) were transfected with an expression plasmid encoding EGFP. cGKI-/- cells (solid bars) were transfected with expression plasmids encoding cGKI-EGFP or cGKIß-EGFP. Shown are mean±SEM values. The number of cells (n) measured under each condition is given inside each column. *P<0.01 vs control in the absence of 8-Br-cGMP (Student t test).

The intracellular localization of the cGKI-EGFP fusion proteins was studied in live VSMCs by fluorescence imaging of the EGFP fluorescence with the use of two-photon microscopy. As illustrated in Figure 5, the relative fluorescence intensity within the perinuclear region was significantly higher in cells transfected with cGKI-EGFP than in cells transfected with cGKIß-EGFP. Addition of 8-Br-cGMP (1 mmol/L for up to 60 minutes) did not alter the distribution of cellular EGFP fluorescence (Figure 5C and data not shown). These results suggest that the cGKI isoform is preferentially localized to the perinuclear/endoplasmic reticulum region, whereas the cGKIß isoform is more homogeneously distributed in the cytosol. Neither cGKI isoform had translocated into the nucleus nor had either shown any other redistribution in the presence of 8-Br-cGMP. Similar results were obtained with a constitutively active cGKI-EGFP fusion protein, which was detected in the perinuclear region but not in the nucleus of various non–smooth muscle cell lines.³³

View larger version (47K): [in this window] [in a new window]   Figure 5. Intracellular localization of cGKI-EGFP fusion proteins in VSMCs. cGKI-/- cells were transfected with expression plasmids encoding cGKI-EGFP or cGKIß-EGFP. A, Representative color-coded fluorescence images of cells expressing cGKI-EGFP (left) or cGKIß-EGFP (right) in the absence of 8-Br-cGMP. Blue colors represent low fluorescence intensity; red colors represent high fluorescence intensity (bar=50 µm). B, Schematic illustration of the analysis of fluorescence intensity. The perinuclear region was defined as the concentric area around the nucleus with a radius twice the radius of the nucleus, excluding the nuclear area. The fluorescence intensity within the perinuclear region was then normalized to the average fluorescence intensity within the entire cell to account for potential variations in the expression level of EGFP fusion proteins and/or laser excitation intensity. C, Histogram summarizing the normalized fluorescence intensity (NFI) of EGFP fluorescence within the perinuclear area of cells expressing cGKI-EGFP or cGKIß-EGFP in the absence (-) or presence (+) of 1 mmol/L 8-Br-cGMP for 45 minutes. Shown are mean±SEM values. The number of cells (n) measured under each condition is given inside each column. *P<0.005 vs cells transfected with cGKI-EGFP (Student t test).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study analyzed, for the first time, the specific functions of cGKI and cGKIß for vascular NO/cGMP/cGKI signaling. Both cGKI isoforms are endogenously expressed in VSMCs. We developed a genetic deletion/rescue strategy that appeared to be superior to the use of inhibitors, because isoform-specific cGKI inhibitors are not available and the in vivo efficiency of the cGKI-specific inhibitor KT5823 is variable.¹⁸ DEA-NO stimulated endogenous cGMP synthesis in both cGKI^+/+ and cGKI^-/- VSMCs, whereas DEA-NO as well as 8-Br-cGMP inhibited NA-induced Ca²⁺ transients in cGKI^+/+ but not in cGKI^-/- cells. These results indicate that the regulation of [Ca²⁺]_i by NO is mediated via the stimulation of cGMP synthesis and the activation of cGKI and/or cGKIß.

The specific functions of the cGKI isoforms were dissected by using EGFP fusion proteins. The kinase activity of these fusion proteins was stimulated 10-fold by cGMP with activation constants similar to the respective wild-type isoforms, suggesting that the expressed fusion proteins retained the properties of the native enzymes. Interestingly, the defective Ca²⁺ regulation in cGKI^-/- VSMCs was restored by transfection of cGKI-EGFP but not cGKIß-EGFP, indicating that the inhibition of NA-induced Ca²⁺ transients by NO/cGMP is mediated via the activation of cGKI but not cGKIß. In agreement with this result, earlier work has shown that hormone-induced Ca²⁺ transients are inhibited by cGKI in stably transfected CHO cells^30,34 but not by cGKIß in stably transfected 293 cells and 3T6 cells.³⁵ However, cGKIß inhibited bradykinin-induced Ca²⁺ transients in the presence of its substrate IRAG in transiently transfected COS cells.^10,26 Thus, the effects of the different cGKI isoforms on Ca²⁺ transients may depend on the cell type and/or agonist used to mobilize Ca²⁺.

Immunocytochemistry and EGFP fluorescence imaging indicated a perinuclear concentration of cGKI and cGKI, respectively. These results are consistent with the notion that cGKI may modulate [Ca²⁺]_i mainly by inhibiting Ca²⁺ release from endoplasmic stores³⁶ and/or by increasing Ca²⁺ reuptake.³⁷ However, alternative mechanisms, such as a reduction of inositol 1,4,5-trisphosphate synthesis^30,38,39 or inhibition of Ca²⁺ influx,^7,40,41 should not be excluded.

The rescue experiments did not support a function for cGKIß in the regulation of NA-induced Ca²⁺ transients, although both cGKIß and IRAG were endogenously expressed in the VSMCs. Fluorescence imaging of native VSMCs indicated a relatively homogeneous distribution of cGKIß-EGFP in the cytosol. It is conceivable that cGKIß and IRAG have important physiological functions in VSMCs other than the modulation of NA-induced Ca²⁺ transients. cGKI has been implicated in the regulation of smooth muscle growth and phenotypic modulation.⁵ IRAG has been postulated to be a tumor suppressor in myeloid cells.⁴² These findings support the hypothesis that cGKIß and IRAG may regulate cell growth and differentiation, which may be affected by the release of Ca²⁺ from specific stores not involved in the regulation of vascular tone.

Another function of cGKIß could be the translocation of the activated enzyme into the nucleus and activation of the fos promoter.^43–45 By fluorescence imaging of cGKI-EGFP and cGKIß-EGFP fusion proteins in living cells (Figure 5) as well as by immunocytochemical analysis of endogenous cGKI in fixed cells (R.F., F.H., unpublished data, 2002), we did not detect nuclear translocation or any other redistribution of cGKI isoforms in VSMCs on treatment with 1 mmol/L 8-Br-cGMP for up to 60 minutes, although this drug concentration clearly activated cGKI in intact cells (Figure 1C). Similar results were obtained by other groups in other cells with the use of immunocytochemistry⁴⁶ or EGFP fusion constructs.³³ According to these findings, it seems unlikely that either cGKI isoform translocates into the nucleus of VSMCs.

Taken together, the present study identifies a specific role for cGKI in NO/cGMP-dependent regulation of [Ca²⁺]_i in VSMCs. Reduction of the [Ca²⁺]_i level by cGKI may be one component of signaling pathways regulating vascular tone in vivo. Additional cGKI-dependent mechanisms may involve changes in the Ca²⁺ sensitivity of the contractile machinery,⁴ the translocation of Rho A,¹⁵ the activity of myosin phosphatase,¹² and the membrane potential.^3,41

	Acknowledgments

 
This study was supported by grants from the Deutsche Forschungsgemeinschaft, Volkswagen Stiftung, and Fonds der Chemischen Industrie. We thank Thomas Kleppisch and Wiebke Wolfsgruber for help with computer software and immunocytochemistry, respectively.

Received February 6, 2002; revision received April 9, 2002; accepted April 12, 2002.

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