Cyclic guanosine monophosphate-dependent protein kinase I promotes adhesion of primary vascular smooth muscle cells

The cyclic guanosine monophosphate (cGMP)/cGMP-dependent protein kinase type I (cGKI) pathway regulates many cellular functions. The current study shows that 8-Br-cGMP stimulates the number of attached primary but not that of subcultured murine vascular smooth muscle cells (VSMCs). These effects of 8-Br-cGMP require the presence of cGKI. In agreement with previous studies, cGKI inhibited the number of cells in repeatedly passaged murine VSMCs. Activation of the cGMP/cGKI pathway in freshly isolated primary VSMCs slightly decreased apoptosis and strongly increased cell adhesion. The stimulation of cell adhesion by cGKI involves an inhibition of the RhoA/Rho kinase pathway and increased exposure of β₁ and β₃ integrins on the cell surface. Together, these results identify a novel proadhesive function of cGMP/cGKI signaling in primary VSMCs and suggest that the opposing effects of this pathway on VSMC number depend on the phenotypic context of the cells.     

Defective smooth muscle regulation in cGMP kinase I-deficient mice.

Regulation of smooth muscle contractility is essential for many important biological processes such as tissue perfusion, cardiovascular haemostasis and gastrointestinal motility. While an increase in calcium initiates smooth muscle contraction, relaxation can be induced by cGMP or cAMP. cGMP-dependent protein kinase I (cGKI) has been suggested as a major mediator of the relaxant effects of both nucleotides. To study the biological role of cGKI and its postulated cross-activation by cAMP, we inactivated the gene coding for cGKI in mice. Loss of cGKI abolishes nitric oxide (NO)/cGMP-dependent relaxation of smooth muscle, resulting in severe vascular and intestinal dysfunctions. However, cGKI-deficient smooth muscle responded normally to cAMP, indicating that cAMP and cGMP signal via independent pathways, with cGKI being the specific mediator of the NO/cGMP effects in murine smooth muscle.     

cGMP-mediated signaling via cGKIalpha is required for the guidance and connectivity of sensory axons

Previous in vitro studies using cGMP or cAMP revealed a cross-talk between signaling mechanisms activated by axonal guidance receptors. However, the molecular elements modulated by cyclic nucleotides in growth cones are not well understood. cGMP is a second messenger with several distinct targets including cGMP-dependent protein kinase I (cGKI). Our studies indicated that the alpha isoform of cGKI is predominantly expressed by sensory axons during developmental stages, whereas most spinal cord neurons are negative for cGKI. Analysis of the trajectories of axons within the spinal cord showed a longitudinal guidance defect of sensory axons within the developing dorsal root entry zone in the absence of cGKI. Consequently, in cGKI-deficient mice, fewer axons grow within the dorsal funiculus of the spinal cord, and lamina-specific innervation, especially by nociceptive sensory neurons, is strongly reduced as deduced from anti-trkA staining. These axon guidance defects in cGKI-deficient mice lead to a substantial impairment in nociceptive flexion reflexes, shown using electrophysiology. In vitro studies revealed that activation of cGKI in embryonic dorsal root ganglia counteracts semaphorin 3A-induced growth cone collapse. Our studies therefore reveal that cGMP signaling is important for axonal growth in vivo and in vitro.     

Functional characteristics of urinary tract smooth muscles in mice lacking cGMP protein kinase type I

Nitric oxide (NO)-mediated smooth muscle relaxation is mediated by cGMP through activation of cGMP-dependent protein kinase I (cGKI). We studied the importance of cGKI for lower urinary tract function in mice lacking the gene for cGKI (cGKI-/-) and in litter-matched wild-type mice (cGKI+/+) in vitro and in vivo. cGKI deficiency did not result in any changes in bladder gross morphology or weight. Urethral strips from cGKI-/- mice showed an impaired relaxant response to nerve-derived NO. The cGMP analog 8-bromo-cGMP (8-BrcGMP) and the NO-donor SIN-1 relaxed the wild-type urethra (50-60%) but had only marginal effects in the cGKI-deficient urethra. Bladder strips from cGKI-/- mice responded normally to electrical field stimulation and to carbachol but not to 8-BrcGMP. In vivo, the cGKI-deficient mice showed bladder hyperactivity characterized by decreased intercontraction intervals and nonvoiding bladder contractions. Loss of cGKI abolishes NO-cGMP-dependent relaxations of urethral smooth muscle and results in hyperactive voiding. These data suggest that certain voiding disturbances may be associated with impaired NO-cGKI signaling.     

Nitric oxide sensitivity in pulmonary artery and airway smooth muscle: a possible role for cGMP responsiveness

We aimed to assess intrinsic smooth muscle mechanisms contributing to greater nitric oxide (NO) responsiveness in pulmonary vascular vs. airway smooth muscle. Porcine pulmonary artery smooth muscle (PASM) and tracheal smooth muscle (TSM) strips were used in concentration-response studies to the NO donor (Z)-1-[N-2-aminoethyl-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO). PASM consistently exhibited greater relaxation at a given DETA-NO concentration (NO responsiveness) than TSM NO responsiveness, with DETA-NO log EC(50) being -6.55 +/- 0.11 and -5.37 +/- 0.13 for PASM and TSM, respectively (P < 0.01). We determined relationships between tissue cGMP concentration ([cGMP](i)) and relaxation using the particulate guanylyl cyclase agonist atrial natriuretic peptide. Atrial natriuretic peptide resulted in nearly complete relaxation, with no detectable increase in [cGMP](i) in PASM and only 20% relaxation (10-fold increase in [cGMP](i)) in TSM, indicating that TSM is less cGMP responsive than PASM. Total cGMP-dependent protein kinase I (cGKI) mRNA expression was greater in PASM than in TSM (2.23 +/- 0.36 vs. 0.93 +/- 0.31 amol mRNA/mug total RNA, respectively; P < 0.01), but total cGKI protein expression was not significantly different (0.56 +/- 0.07 and 0.49 +/- 0.04 ng cGKI/mug protein, respectively). The phosphotransferase assay for the soluble fraction of tissue homogenates demonstrated no difference in the cGMP EC(50) between PASM and TSM. The maximal phosphotransferase activity indexed to the amount of total cGKI in the homogenate differed significantly between PASM and TSM (1.61 +/- 0.15 and 1.04 +/- pmol.min(-1).ng cGKI(-1), respectively; P < 0.05), suggesting that cGKI may be regulated differently in the two tissues. A novel intrinsic smooth muscle mechanism accounting for greater NO responsiveness in PASM vs. TSM is thus greater cGMP responsiveness from increased cGKI-specific activity in PASM.     

Cysteine-rich protein 2, a novel substrate for cGMP kinase I in enteric neurons and intestinal smooth muscle

Nitric oxide/cGMP/cGMP kinase I (cGKI) signaling causes relaxation of intestinal smooth muscle. In the gastrointestinal tract substrates of cGKI have not been identified yet. In the present study a protein interacting with cGKIbeta has been isolated from a rat intestinal cDNA library using the yeast two-hybrid system. The protein was identified as cysteine-rich protein 2 (CRP2), recently cloned from rat brain (Okano, I., Yamamoto, T., Kaji, A., Kimura, T., Mizuno, K., and Nakamura, T. (1993) FEBS Lett. 333, 51-55). Recombinant CRP2 is specifically phosphorylated by cGKs but not by cAMP kinase in vitro. Co-transfection of CRP2 and cGKIbeta into COS cells confirmed the phosphorylation of CRP2 in vivo. Cyclic GMP kinase I phosphorylated CRP2 at Ser-104, because the mutation to Ala completely prevented the in vivo phosphorylation. Immunohistochemical analysis using confocal laser scan microscopy showed a co-localization of CRP2 and cGKI in the inner part of the circular muscle layer, in the muscularis mucosae, and in specific neurons of the myenteric and submucosal plexus. The co-localization together with the specific phosphorylation of CRP2 by cGKI in vitro and in vivo suggests that CRP2 is a novel substrate of cGKI in neurons and smooth muscle of the small intestine.     

The structural determinations of the leucine zipper coiled-coil domains of the cGMP-dependent protein kinase Iα and its interaction with the myosin binding subunit of the myosin light chains phosphase

Physiologic relaxation of vascular smooth muscle is induced by the cyclic guanosine monophosphate (cGMP)- dependent protein kinase Iα enzyme (cGKIα), which activates myosin phosphatase (MLCP). This activation process is thought to occur through the interaction involving both N- and C-terminal leucine zipper coiled-coil (LZCC) domains of the kinase enzyme (cGKIα) with the myosin binding subunit (MBS) of MLCP. In this review, I summarize how to define the LZCC domains in both N-terminal cGKIα(1-59) and C-terminal MBS proteins using predictive and experimental methods, how to make a rapid and accurate structure determination of a cGKIα(1-59) molecule using NMR's residual dipolar coupling (RDC) measurements, and how to indentify the existence of a weak protein interaction between N-terminal LZCC domain (cGKIα(1-59)) and a LZCC domain (MBSCT42) within the C-terminal MBS. In addition, the location and orientation of the residues in LZCC proteins can be readily visualized using a novel diagram, the so-called "wenxiang diagram", which is more advantageous than traditional helical wheel diagrams in analyzing LZCC protein structures and their action mechanisms. Using the composed wenxiang diagrams, we have characterized the interaction between cGKIα(1- 59) and another LZCC molecule (MBSCT42), and deduced that the most affected residues of these two LZCC molecules might be at the positions d, a, e and g. These studies and findings are also covered in this review. It is intriguing to see that the successful incorporation of wenxiang diagrams and NMR spectroscopy in the LZCC structural and functional studies may provide some insights into protein-protein interaction mechanisms.     

Prolonged treatment of porcine pulmonary artery with nitric oxide decreases cGMP sensitivity and cGMP-dependent protein kinase specific activity

A cultured porcine pulmonary artery (PA) model was used to examine the effects of prolonged nitric oxide (NO) treatment on the response to acutely applied NO, cGMP analog, or atrial natriuretic peptide (ANP). Twenty-four-hour treatment with the NO donor (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO) resulted in >10-fold decrease in the response to acutely applied DETA-NO. In parallel with this, the relaxant response to acutely applied cGMP analog, beta-phenyl-1,N(2)-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothioate, Sp isomer (Sp-8-Br-PET-cGMPS), and ANP decreased. The reduction in ANP responsiveness in PA was not associated with a reduction in cGMP levels evoked by 10(-6) M ANP. Twenty-four hours in culture and treatment with DETA-NO decreased total cGMP-dependent protein kinase (cGKI) mRNA level compared with that in freshly prepared PA (1.05 +/- 0.12, 0.42 +/- 0.08, and 0.11 +/- 0.01 amol/mug, respectively). Total cGKI protein levels were decreased to a lesser extent by 24 h in culture and further decreased by 24-h DETA-NO treatment compared with that in freshly prepared PA (361 +/- 33, 272 +/- 20, and 238 +/- 25 ng/mg total protein, respectively). Maximal cGMP-stimulated phosphotransferase activity was reduced in 24-h cultured and DETA-NO-treated PA (986 +/- 84, 815 +/- 81, and 549 +/- 78 pmol P(i).min(-1).mg soluble protein(-1)), but the cGMP concentration resulting in 50% of maximal phosphotransferase activity was not. cGKI specific activity (maximal cGMP-activated phosphotransferase activity/ng cGKI) was significantly reduced in PA treated with DETA-NO for 24 h compared with freshly prepared and 24-h cultured PA (1.95 +/- 0.22, 2.64 +/- 0.25, and 2.85 +/- 0.28 pmol P(i).min(-1).ng cGKI(-1), respectively). We conclude that prolonged NO treatment induces decreased acute NO responsiveness in PA in part by decreasing cGMP sensitivity. It does so by decreasing both cGKI expression and cGKI specific activity.     

Interaction of cCMP with the cGK,cAK and MAPK Kinases in Murine Tissues

cAMP and cGMP are well established second messengers that are essential for numerous (patho)physiological processes. These purine cyclic nucleotides activate cAK and cGK, respectively. Recently, the existence of cCMP was described, and a possible function for this cyclic nucleotide was investigated. It was postulated that cCMP plays a role as a second messenger. However, the functions regulated by cCMP are mostly unknown. To elucidate probable functions, cCMP-binding and -activated proteins were identified using different methods. We investigated the effect of cCMP on purified cyclic nucleotide-dependent protein kinases and lung and jejunum tissues of wild type (WT), cGKI-knockout (cGKI KO) and cGKII-knockout (cGKII KO) mice. The catalytic activity of protein kinases was measured by a (γ-³²P) ATP kinase assay. Cyclic nucleotide-dependent protein kinases (cAK, cGKI and cGKII) in WT tissue lysates were stimulated by cCMP. In contrast, there was no stimulation of phosphorylation in KO tissue lysates. Competitive binding assays identified cAK, cGKI, and cGKII as cCMP-binding proteins. An interaction between cCMP/MAPK and a protein-protein complex of MAPK/cGK were detected via cCMP affinity chromatography and co-immunoprecipitation, respectively. These complexes were abolished or reduced in jejunum tissues from cGKI KO or cGKII KO mice. In contrast, these complexes were observed in the lung tissues from WT, cGKI KO and cGKII KO mice. Moreover, cCMP was also able to stimulate the phosphorylation of MAPK. These results suggest that MAPK signaling is regulated by cGMP-dependent protein kinases upon activation by cCMP. Based on these results, we propose that additional cCMP-dependent protein kinases that are capable of modulating MAPK signaling could exist. Hence, cCMP could potentially act as a second messenger in the cAK/cGK and MAPK signaling pathways and play an important role in physiological processes of the jejunum and lung.     

The amino-terminal cyclic nucleotide binding site of the type II cGMP-dependent protein kinase is essential for full cyclic nucleotide-dependent activation

For the type I cGMP-dependent protein kinases (cGKIalpha and cGKIbeta), a high affinity interaction exists between the C2 amino group of cGMP and the hydroxyl side chain of a threonine conserved in most cGMP binding sites. To examine the effect of this interaction on ligand binding and kinase activation in the type II isozyme of cGMP-dependent protein kinase (cGKII), alanine was substituted for the conserved threonine or serine. cGKII was found to require the C2 amino group of cGMP and its cognate serine or threonine hydroxyl for efficient cGMP activation. Of the two binding sites, disruption of cGMP-specific binding in the NH(2)-terminal binding site had the greatest effect on cGMP-dependent kinase activation, like cGKI. However, ligand dissociation studies showed that the location of the rapid and slow dissociation sites of cGKII was reversed relative to cGKI. Another set of mutations that prevented cyclic nucleotide binding demonstrated the necessity of the NH(2)-terminal, rapid dissociation binding site for cyclic nucleotide-dependent activation of cGKII. These findings suggest distinct mechanisms of activation for cGKII and cGKI isoforms. Because cGKII mediates the effects of heat-stable enterotoxins via the cystic fibrosis transmembrane regulator Cl(-) channel, these findings define a structural target for drug design.     

Regulation of Sensory Neuron Precursor Proliferation by Cyclic GMP-Dependent Protein Kinase

Abstract: Cyclic GMP (cGMP) is a molecular messenger involved in diverse cellular processes. Recently, cGMP-dependent protein kinase (cGK) type II was determined to be a regulator of endochondral ossification and bone growth, identifying a role for cGMP in the regulation of cellular proliferation. Here, we demonstrate the presence of cGK type I (cGKI) in cells of the developing trigeminal ganglia. cGKI occurs in some proliferating precursors as evidenced by double labeling with an antibody to cGKI and 5-bromo-2'-deoxyuridine(BrdU) incorporation. Inhibition of cGKI with KT5823 or Rp -8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphorothioate ( Rp -8-pCPT-cGMPS) in chick embryos results in a 30–40% decrease in trigeminal ganglia cell number, and this effect is independent of nitric oxide synthase (NOS). In addition, inhibition of cGKI with Rp -8-pCPT-cGMPS results in a 60% decrease in BrdU incorporation in the trigeminal ganglia of embryonic day 5 chicks. We find that PC12 cells expressing cGKI proliferate more rapidly and incorporate more BrdU than do control cells. The cGKI inhibitor Rp -8-pCPT-cGMPS decreases proliferation and BrdU incorporation in transfected PC12 cells but has no effect on control cells. The PC12 cells do not express NOS, indicating that this effect is also independent of NOS. Thus, cGKI regulates the proliferation of sensory neurons as a result of activation of a NOS-independent pathway, representing a novel pathway by which the number of sensory neurons is regulated.     

Expression of cGMP-dependent protein kinase type I in mature white adipocytes

The presence of cGMP-dependent protein kinase I (cGKI) in murine adipocytes has been questioned, although cGKI was implicated in the thermogenic program of fat cells (FCs) and to exert anti-hypertrophic/-inflammatory effects in white adipose tissue. Herein, cGKI was detected in adipocytes from control mice, whereas FCs from global cGKI knockouts (cGKI^−/−) and cGKIα rescue (αRM) mice remained cGKI-negative. cGKI mutants exhibit decreased adipocyte size, plasma leptin levels and reduced body-weights as compared to litter-matched controls. Low abundance of adiponectin in WAT and plasma of αRM animals together with previously confirmed high IL-6 levels indicate a low-grade inflammation. However, αRMs were protected from streptozotocin-induced hyperglycemia. Our results suggest that cGMP/cGKI affects both glucose and FC homeostasis in more complex mode than previously thought.     

PCNA Up-Regulation in Cerebral Vessels with Angiotensin II-Hypertension: Abnormal Regulation of Ca2+ Channel and Nitrate Tolerance Associated with Alternative Splicing of cGKI

We studied the responses of the basilar arteries from control rats and from rats infused with angiotensin II (Ang; 240 μg/kg/h × 4 weeks), which were hypertensive (137 ± 13 vs 205 ± 10 mm Hg). Ang-hypertensive rats (AHR) showed significant up-regulation of the expression of proliferative cell nuclear antigen (PCNA) (PCNA index, 0.65) in vascular smooth muscle cell (VSMC) layers. Both hypertension and PCNA up-regulation were absent in animals co-treated with the hydrophilic dihydropyridine Ca²⁺ channel blocker amlodipine (100 mg/liter in the drinking water). Quantitative patch-clamp analysis of freshly isolated VSMC showed a significant increase in L-type Ca²⁺ channel currents in AHR that was attributed to an increase in the open channel probability, with no change in other biophysical properties, pharmacological characteristics, or in the channel expression. Compared with controls, regulation of Ca²⁺ channels in AHR was abnormal, with nitrate tolerance manifested as a reduction in down-regulation of the Ca²⁺ channel activity in response to the NO donor Na nitroprusside. A diminished sensitivity to 8-Br-cGMP was also observed, consistent with a mechanism downstream of soluble guanylyl cyclase. The nitrate tolerance was found to be attributable to alternative splicing of cGKI, with a decrease in the cGKIalpha expression and an increase in the cGKIbeta expression, with the latter known to be less sensitive to activation by cGMP and related analogs. We conclude that the increase in the proliferative response of VSMC in AHR is associated with an abnormal response to NO by VSMC due to alternative splicing of cGKI, resulting in an increase in the Ca²⁺ channel activation and up-regulation of the pro-proliferative Ca²⁺-sensitive gene for PCNA.     

cGMP signals mainly through cAMP kinase in permeabilized murine aorta

GMP affects vascular tone by multiple mechanisms, including inhibition of the Rho/Rho kinase-mediated Ca(2+) sensitization, a process identified as Ca(2+) desensitization. Ca(2+) desensitization is mediated probably by both cGMP- and cAMP-dependent protein kinases (cGKI and PKA). We investigate to which extent Ca(2+) desensitization is initiated by cGKI and PKA. cGMP/cAMP-induced relaxation was studied at constant [Ca(2+)] in permeabilized aortas from wild-type and cGKI-deficient mice. [Ca(2+)] increased aortic tone in the absence and presence of 50 microM GTPgammaS with EC(50) values of 160 and 30 nM, respectively. In the absence of GTPgammaS, the EC(50) for [Ca(2+)] was shifted rightward from 0.16 microM to 0.43 and 0.82 microM by 1 and 300 microM 8-bromo-cGMP (8-Br-cGMP), and to 8 microM by 10 microM Y-27632. Contractions induced by 300 nM [Ca(2+)] were relaxed by 8-Br-cGMP with an EC(50) of 2.6 microM. Surprisingly, [Ca(2+)]-induced contractions were also relaxed by 8-Br-cGMP in aortas from cGKI(-/-) mice (EC(50) of 19 microM). Western blot analysis of the vasodilator-stimulated phosphoprotein indicated "cross"-activation of PKA by 1 mM 8-Br-cGMP in aortic smooth muscle cells from cGKI(-/-) mice. Indeed, the PKA inhibitor peptide (PKI 5-24) completely abolished the relaxant effect of 8-Br-cGMP in muscles from cGKI(-/-) mice and to 65% in wild-type aortas. The thromboxane analogue U-46619 induced contraction at constant [Ca(2+)], which was only partially relaxed by 8-Br-cGMP but completely relaxed by Y-27632. The effect of 8-Br-cGMP on U-46619-induced contraction was attenuated by PKI 5-24. These results show that cGKI has only a small inhibitory effect on Ca(2+) sensitization in murine aortas.     

A catalytically inactive mutant of type I cGMP-dependent protein kinase prevents enhancement of large conductance, calcium-sensitive K+ channels by sodium nitroprusside and cGMP

The activation of large conductance, calcium-sensitive K(+) (BK(Ca)) channels by the nitric oxide (NO)/cyclic GMP (cGMP) signaling pathway appears to be an important cellular mechanism contributing to the relaxation of smooth muscle. In HEK 293 cells transiently transfected with BK(Ca) channels, we observed that the NO donor sodium nitroprusside and the membrane-permeable analog of cGMP, dibutyryl cGMP, were both able to enhance BK(Ca) channel activity 4-5-fold in cell-attached membrane patches. This enhancement correlated with an endogenous cGMP-dependent protein kinase activity and the presence of the alpha isoform of type I cGMP-dependent protein kinase (cGKI). We observed that co-transfection of cells with BK(Ca) channels and a catalytically inactive ("dead") mutant of human cGKIalpha prevented enhancement of BK(Ca) channel in response to either sodium nitroprusside or dibutyryl cGMP in a dominant negative fashion. In contrast, expression of wild-type cGKIalpha supported enhancement of channel activity by these two agents. Importantly, both endogenous and expressed forms of cGKIalpha were found to associate with BK(Ca) channel protein, as demonstrated by a reciprocal co-immunoprecipitation strategy. In vitro, cGKIalpha was able to directly phosphorylate immunoprecipitated BK(Ca) channels, suggesting that cGKIalpha-dependent phosphorylation of BK(Ca) channels in situ may be responsible for the observed enhancement of channel activity. In summary, our data demonstrate that cGKIalpha alone is sufficient to promote the enhancement of BK(Ca) channels in situ after activation of the NO/cGMP signaling pathway.     

Impairment of LTD and cerebellar learning by Purkinje cell-specific ablation of cGMP-dependent protein kinase I

The molecular basis for cerebellar plasticity and motor learning remains controversial. Cerebellar Purkinje cells (PCs) contain a high concentration of cGMP-dependent protein kinase type I (cGKI). To investigate the function of cGKI in long-term depression (LTD) and cerebellar learning, we have generated conditional knockout mice lacking cGKI selectively in PCs. These cGKI mutants had a normal cerebellar morphology and intact synaptic calcium signaling, but strongly reduced LTD. Interestingly, no defects in general behavior and motor performance could be detected in the LTD-deficient mice, but the mutants exhibited an impaired adaptation of the vestibulo-ocular reflex (VOR). These results indicate that cGKI in PCs is dispensable for general motor coordination, but that it is required for cerebellar LTD and specific forms of motor learning, namely the adaptation of the VOR.