Functional Reconstitution of Vascular Smooth Muscle Cells With cGMP-Dependent Protein Kinase I Isoforms
The cGMP-dependent protein kinase type I (cGKI) is a major mediator of NO/cGMP-induced vasorelaxation. Smooth muscle expresses two isoforms of cGKI, cGKIα and cGKIβ, 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 cGKIα and cGKIβ. 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 cGKIα and cGKIβ. The defective Ca2+ regulation in cGKI-knockout cells could be rescued by transfection of a fusion construct consisting of cGKIα and enhanced green fluorescent protein (EGFP) but not by a cGKIβ-EGFP construct. Fluorescence imaging indicated that the cGKIα-EGFP fusion protein was concentrated in the perinuclear/endoplasmic reticulum region of live VSMCs, whereas the cGKIβ-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 cGKIα isoform, which decreases the noradrenaline-stimulated cytosolic Ca2+ level.
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, cGKIα and cGKIβ, 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 cGKIα interacts specifically with the myosin-binding subunit of myosin phosphatase,12 whereas the amino terminus of cGKIβ 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 cGKIα and cGKIβ 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 cGKIα and cGKIβ 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 cGKIα but not cGKIβ.
Materials and Methods
Construction and Transfection of Plasmids
A BamHI site was introduced into the region encompassing the stop codon of the bovine cGKIα 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 cGKIα cDNA was inserted into the EcoRI-BamHI sites of pEGFP-N1 (Clontech), where EGFP indicates enhanced green fluorescent protein, resulting in the vector pCMV-cGKIαEGFP, where CMV indicates cytomegalovirus. The vector pCMV-cGKIβEGFP was constructed by replacing the 1.8-kb EcoRI-PacI fragment of pCMV-cGKIαEGFP with the 1.8-kb EcoRI-PacI fragment of the bovine cGKIβ cDNA27 encompassing the 5′ region, which differs between the α and β isoform. The CMV promoter–driven expression vectors pCMV-cGKIαEGFP and pCMV-cGKIβEGFP encode in-frame fusions of EGFP to the carboxy termini of cGKIα and cGKIβ, 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 α-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).
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 ×40 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.
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 cGKIα or cGKIβ (J.S., F.H., unpublished data, 2002), IRAG,26 and phospho-Ser157- VASP (Alexis), where VASP is vasodilator-stimulated phosphoprotein.
VSMCs were isolated from aortas of cGKI+/+ mice and cGKI−/− mice, which are deficient for both the cGKIα and cGKIβ 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.
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 cGKIα and cGKIβ 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.
To confirm the role of cGKI in NO/cGMP-dependent Ca2+ regulation and to analyze the specific functions of the cGKIα and cGKIβ 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 cGKIα-EGFP and cGKIβ-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 cGKIα-EGFP that had constitutive (cGMP-independent) kinase activity. In contrast, the kinase activity of the cGKIα-EGFP as well as the cGKIβ-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.
In the next set of experiments, we investigated whether [Ca2+]i was regulated by cGKIα and/or cGKIβ 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 cGKIα-EGFP or cGKIβ-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 cGKIα-EGFP but not cGKIβ-EGFP (Figure 4). These results indicate that specifically the cGKIα isoform but not the cGKIβ isoform inhibits NA-induced Ca2+ transients in VSMCs.
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 cGKIα-EGFP than in cells transfected with cGKIβ-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 cGKIα isoform is preferentially localized to the perinuclear/endoplasmic reticulum region, whereas the cGKIβ 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 cGKIα-EGFP fusion protein, which was detected in the perinuclear region but not in the nucleus of various non–smooth muscle cell lines.33
The present study analyzed, for the first time, the specific functions of cGKIα and cGKIβ 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 cGKIα and/or cGKIβ.
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 cGKIα-EGFP but not cGKIβ-EGFP, indicating that the inhibition of NA-induced Ca2+ transients by NO/cGMP is mediated via the activation of cGKIα but not cGKIβ. In agreement with this result, earlier work has shown that hormone-induced Ca2+ transients are inhibited by cGKIα in stably transfected CHO cells30,34⇓ but not by cGKIβ in stably transfected 293 cells and 3T6 cells.35 However, cGKIβ 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 cGKIα, respectively. These results are consistent with the notion that cGKIα 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 synthesis30,38,39⇓⇓ or inhibition of Ca2+ influx,7,40,41⇓⇓ should not be excluded.
The rescue experiments did not support a function for cGKIβ in the regulation of NA-induced Ca2+ transients, although both cGKIβ and IRAG were endogenously expressed in the VSMCs. Fluorescence imaging of native VSMCs indicated a relatively homogeneous distribution of cGKIβ-EGFP in the cytosol. It is conceivable that cGKIβ 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 cGKIβ 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 cGKIβ could be the translocation of the activated enzyme into the nucleus and activation of the fos promoter.43–45⇓⇓ By fluorescence imaging of cGKIα-EGFP and cGKIβ-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 cGKIα in NO/cGMP-dependent regulation of [Ca2+]i in VSMCs. Reduction of the [Ca2+]i level by cGKIα may be one component of signaling pathways regulating vascular tone in vivo. Additional cGKIα-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⇓
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.
Original received February 6, 2002; revision received April 9, 2002; accepted April 12, 2002.
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