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T M Lincoln

Publications and source records attributed to T M Lincoln.

At least 19 recordsLinked to original sources
PubMed1999

Activation of mitogen-activated protein kinase pathways by cyclic GMP and cyclic GMP-dependent protein kinase in contractile vascular smooth muscle cells.

Vascular smooth muscle cells (VSMC) exist in either a contractile or a synthetic phenotype in vitro and in vivo. The molecular mechanisms regulating phenotypic modulation are unknown. Previous studies have suggested that the serine/threonine protein kinase mediator of nitric oxide (NO) and cyclic GMP (cGMP) signaling, the cGMP-dependent protein kinase (PKG) promotes modulation to the contractile phenotype in cultured rat aortic smooth muscle cells (RASMC). Because of the potential importance of the mitogen-activated protein kinase (MAP kinase) pathways in VSMC proliferation and phenotypic modulation, the effects of PKG expression in PKG-deficient and PKG-expressing adult RASMC on MAP kinases were examined. In PKG-expressing adult RASMC, 8-para-chlorophenylthio-cGMP activated extracellular signal- regulated kinases (ERK1/2) and c-Jun N-terminal kinase (JNK). The major effect of PKG activation was increased activation by MAP kinase kinase (MEK). The cAMP analog, 8-Br-cAMP inhibited ERK1/2 activation in PKG-deficient and PKG-expressing RASMC but had no effect on JNK activity. The effects of PKG on ERK and JNK activity were additive with those of platelet-derived growth factor (PDGF), suggesting that PKG activates MEK through a pathway not used by PDGF. The stimulatory effects of cGMP on ERK and JNK activation were also observed in low-passaged, contractile RASMC still expressing endogenous PKG, suggesting that the effects of PKG expression were not artifacts of cell transfections. These results suggest that in contractile adult RASMC, NO-cGMP signaling increases MAP kinase activity. Increased activation of these MAP kinase pathways may be one mechanism by which cGMP and PKG activation mediate c-fos induction and increased proliferation of contractile adult RASMC.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1999

Regulation of myosin phosphatase by a specific interaction with cGMP- dependent protein kinase Ialpha.

Contraction and relaxation of smooth muscle are regulated by myosin light-chain kinase and myosin phosphatase through phosphorylation and dephosphorylation of myosin light chains. Cyclic guanosine monophosphate (cGMP)-dependent protein kinase Ialpha (cGKIalpha) mediates physiologic relaxation of vascular smooth muscle in response to nitric oxide and cGMP. It is shown here that cGKIalpha is targeted to the smooth muscle cell contractile apparatus by a leucine zipper interaction with the myosin-binding subunit (MBS) of myosin phosphatase. Uncoupling of the cGKIalpha-MBS interaction prevents cGMP-dependent dephosphorylation of myosin light chain, demonstrating that this interaction is essential to the regulation of vascular smooth muscle cell tone.

Amino Acid Motifs↗
PubMed1999

Analysis of expression of cGMP-dependent protein kinase in rabbit heart cells.

We previously showed that stimulation of cGMP-dependent protein kinase (PKG) stimulates L-type calcium current in newborn but not in adult rabbit ventricular myocytes. We have now isolated rabbit PKG type Ialpha cDNA (+1 to 2095), determined the sequence, and analyzed specific expression of PKG in adult and newborn rabbit heart by Western and Northern analyses to elucidate the developmental decline in the significance of PKG in cardiac function. We obtained full-length cDNA of PKG Ialpha from newborn rabbit heart mRNA with reverse transcription-polymerase chain reaction. The coding region of rabbit PKG Ialpha showed 94% homology to sequences of human and bovine PKG Ialpha. The deduced amino acid sequence of 671 amino acids showed seven substitutions between rabbit and either human or bovine PKG Ialpha. The major substitutions were found in the cGMP-binding domain. The cloned PKG 1alpha cDNA was expressed in COS cells. Expressed PKG showed cGMP stimulated PKG activity and immunoreactivity. Northern blot analysis of cardiac tissue demonstrated PKG Ialpha mRNA of 6.8 kb, with much higher levels in newborn than in adult cells. Western analysis in homogenates from ventricular tissues and isolated ventricular myocytes of rabbit heart showed much higher expression of PKG type I protein in newborn compared with adult cells. These findings suggest that PKG is developmentally regulated in rabbit heart and is expressed at a much higher level in newborn than in adult cells. The greater expression of PKG in newborn cells could be responsible for differences in the significance of cGMP in adult and newborn rabbit cells.

Amino Acid Sequence↗
PubMed1998

Mechanism of platelet inhibition by nitric oxide: in vivo phosphorylation of thromboxane receptor by cyclic GMP-dependent protein kinase.

Nitric oxide (NO) is a potent vasodilator and inhibitor of platelet activation. NO stimulates production of cGMP and activates cGMP-dependent protein kinase (G kinase), which by an unknown mechanism leads to inhibition of Galphaq-phospholipase C-inositol 1, 4,5-triphosphate signaling and intracellular calcium mobilization for several important agonists, including thromboxane A2 (TXA2). To explore the mechanism of platelet inhibition by NO, activation of platelet TXA2 receptors in the presence of cGMP was studied. The nonhydrolyzable analog 8-bromo-cyclic GMP (8-Br-cGMP) potently inhibited activation of the TXA2-specific GTPase in platelet membranes in a concentration-dependent fashion, suggesting that G kinase catalyzes the phosphorylation of some proximal component of the receptor-G protein signaling pathway. Nanomolar concentrations of G kinase were found to catalyze the phosphorylation of platelet TXA2 receptors in vitro, but not Galphaq copurifying with the TXA2 receptors in these experiments. Using immunoaffinity methods, in vivo phosphorylation of TXA2 receptors by cyclic GMP was demonstrated from 32P-labeled cells treated with 8-Br-cGMP. Peptide mapping studies of in vivo phosphorylated TXA2 receptors demonstrated cGMP mediates phosphorylation of the carboxyl terminus of the TXA2 receptor. G kinase also catalyzed the phosphorylation of peptides corresponding to the cytoplasmic tails of both alpha and beta forms of the receptor but not control peptide or a peptide corresponding to the third intracytoplasmic loop of the TXA2 receptor. These data identify TXA2 receptors as cGMP-dependent protein kinase substrates and support a novel mechanism for the inhibition of cell function by NO in which activation of G kinase inhibits signaling by G protein-coupled receptors by catalyzing their phosphorylation.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1998

Cyclic GMP-dependent protein kinase inhibits osteopontin and thrombospondin production in rat aortic smooth muscle cells.

Vascular lesions resulting from injury are characterized by a thickening of the intima brought about in part through the production of increased amounts of extracellular matrix proteins by the vascular smooth muscle cells (VSMCs). In this study, we tested the hypothesis that cGMP-dependent protein kinase (PKG), an important mediator of NO and cGMP signaling in VSMCs, inhibits the production of two extracellular matrix proteins, osteopontin and thrombospondin, which are involved in the formation of the neointima. VSMCs deficient in PKG were stably transfected with cDNAs encoding either the holoenzyme PKG-Ialpha or the constitutively active catalytic domain of PKG-I in order to directly examine the effects of PKG on osteopontin and thrombospondin production. Cells expressing either of the PKG constructs had dramatically reduced levels of osteopontin and thrombospondin-1 protein compared with control-transfected PKG-deficient cells. PKG transfection also altered the morphology of the VSMCs. These results indicate that PKG may be involved in suppressing extracellular matrix protein expression, which is one important characteristic of synthetic secretory VSMCs. Suppression of these matrix proteins may underlie the effects of NO-cGMP signaling to inhibit VSMC migration and phenotypic modulation.

Animals↗
PubMed1998

Evidence that additional mechanisms to cyclic GMP mediate the decrease in intracellular calcium and relaxation of rabbit aortic smooth muscle to nitric oxide.

1. The role of cyclic GMP in the ability of nitric oxide (NO) to decrease intracellular free calcium concentration [Ca2+]i and divalent cation influx was studied in rabbit aortic smooth muscle cells in primary culture. In cells stimulated with angiotensin II (AII, 10(-1) M), NO (10(-10) - 10(-6) M) increased cyclic GMP levels measured by radioimmunoassay and decreased [Ca2+]i and cation influx as indicated by fura-2 fluorimetry. 2. Zaprinast (10(-4) M), increased NO-stimulated levels of cyclic GMP by 3-20 fold. Although the phosphodiesterase inhibitor lowered the level of [Ca2+]i reached after administration of NO, the initial decreases in [Ca2+]i initiated by NO were not significantly different in magnitude or duration from those that occurred in the absence of zaprinast. 3. The guanylyl cyclase inhibitor, H-(1,2,4) oxadiazolo(4,3-a) quinoxallin-1-one (ODQ, 10(-5) M), blocked cyclic GMP accumulation and activation of protein kinase G, as measured by back phosphorylation of the inositol trisphosphate receptor. ODQ and Rp-8-Br-cyclic GMPS, a protein kinase G inhibitor, decreased the effects of NO, 10(-10) - 10(-8) M, but the decrease in [Ca2+]i or cation influx caused by higher concentrations of NO (10(-7) - 10(-6) M) were unaffected. Relaxation of intact rabbit aorta rings to NO (10(-7) - 10(-5) M) also persisted in the presence of ODQ without a significant increase in cyclic GMP. Rp-8-Br-cyclic GMPS blocked the decreases in cation influx caused by a cell permeable cyclic GMP analog, but ODQ and/or the protein kinase G inhibitor had no significant effect on the decrease caused by NO. 4. Although inhibitors of cyclic GMP, protein kinase G and phosphodiesterase can be shown to affect the decrease in [Ca2+]i and cation influx via protein kinase G, these studies indicate that when these mechanisms are blocked, cyclic GMP-independent mechanisms also contribute significantly to the decrease in [Ca2+]i and smooth muscle relaxation to NO.

1-Methyl-3-isobutylxanthine↗
PubMed1998

Nitric oxide--cyclic GMP pathway regulates vascular smooth muscle cell phenotypic modulation: implications in vascular diseases.

The role of cGMP-dependent protein kinase (PKG) in the regulation of rat aortic vascular smooth muscle cells (VSMC) phenotype was examined using a transfected cell culture system. Repetitively passaged VSMC do not express PKG and exist in the synthetic phenotype. Transfection of PKG-l alpha cDNA, or the active catalytic domain of PKG-l alpha, resulted in the appearance of VSMC having a morphology consistent with the contractile phenotype. PKG-expressing cells also contained markers for the contractile phenotype (for example, smooth muscle specific myosin heavy chain, calponin, alpha-actin) and reduced levels of synthetic phenotype markers (osteopontin, thrombospondin). PKG-transfected VSMC have also reduced the levels of fibroblast growth factor receptors 1 and 2, consistent with the establishment of a more contractile phenotype. The regulation of PKG expression in VSMC is largely undefined; however, continuous exposure of cultured bovine aortic smooth muscle cells with nitric oxide (NO)-donor drugs or cyclic nucleotide analogues reduced the expression of PKG. These results suggest that PKG occupies a critical role in VSMC phenotype and that suppression of PKG expression during inflammation or injury promotes a more synthetic state of the VSMC.

Animals↗
PubMed1998

Protein kinase G is not essential to NO-cGMP modulation of basal tone in rat pulmonary circulation.

Nitric oxide (NO) is important in modulating increased pulmonary vascular tone. Whereas in other systems it is believed that the action of NO is mediated through guanosine 3',5'-cyclic monophosphate (cGMP) and protein kinase G (PKG), the validity of this pathway in the pulmonary circulation has not been established. Using isolated salt-perfused normotensive and hypertensive rat lungs, we studied the effects of the soluble guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), and the PKG inhibitors, KT5823, Rp-8-pCPT-cGMPS, and (N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide) (H-8), on pulmonary vascular resistance. In isolated normotensive lungs, ODQ-mediated inhibition of soluble guanylyl cyclase augmented hypoxic pulmonary vasoconstriction, whereas the PKG inhibitors had no effect. Despite the marked differences in the physiological effect, ODQ and Rp-8-pCPT-cGMPS inhibited PKG activity to a similar degree as determined by a back-phosphorylation assay showing decreased PKG-mediated phosphorylation of serine 1755 on the D-myo-inositol 1,4,5-trisphosphate receptor. In hypertensive lungs, inhibition of soluble guanylyl cyclase by ODQ increased perfusion pressure by 101 +/- 20% (P < 0.05), an increase similar to that seen with inhibition of NO synthase (NOS), confirming an essential role for cGMP. In contrast, KT5823, Rp-8-pCPT-cGMPS, and H-8 (used in doses 5- to 100-fold in excess of their reported inhibitory concentrations for PKG) caused only a small increase in baseline perfusion pressure (14 +/- 2%, P = not significant from vehicle control). Effectiveness of PKG inhibition in the hypertensive lungs was also confirmed with the back-phosphorylation assay. These studies suggest that whereas NO-mediated modulation of vascular tone in the normotensive and hypertensive pulmonary circulation is dependent on cGMP formation, activation of PKG may not be essential.

Alkaloids↗
PubMed1997

Smooth muscle cell expression of type I cyclic GMP-dependent protein kinase is suppressed by continuous exposure to nitrovasodilators, theophylline, cyclic GMP, and cyclic AMP.

A key component of the nitric oxide-cyclic guanosine monophosphate (cGMP) pathway in smooth muscle cells (SMC) is the type I GMP-dependent protein kinase (PK-G I). Activation of PK-G I mediates the reduction of cytoplasmic calcium concentrations and vasorelaxation. In this manuscript, we demonstrate that continuous exposure of SMC in culture to the nitrovasodilators S-nitroso-N-acetylpenicillamine (SNAP) or sodium nitroprusside (SNP) results in approximately 75% suppression of PK-G I mRNA by 48 h. PK-G I mRNA and protein were also suppressed by continuous exposure to cGMP analogues 8-bromo- and 8-(4-chlorophenylthio) guanosine-3,5-monophosphate or the cAMP analogue dibutyryl cAMP. These results suggest that activation of one or both of the cyclic nucleotide-dependent protein kinases mediates PK-G I mRNA suppression. Using isoform-specific cDNA probes, only the PK-G I alpha was detected in SMC, either at baseline or after suppression, while PK-G I beta was not detected, indicating that isoform switch was not contributing to the gene regulation. Using the transcription inhibitor actinomycin D, the PK-G I mRNA half-life in bovine SMC was observed to be 5 h. The half-life was not affected by the addition of SNAP to actinomycin D, indicating no effect on PK-G I mRNA stability. Nuclear runoff studies indicated a suppression of PK-G I gene transcription by SNAP. PK-G I suppression was also observed in vivo in rats given isosorbide dinitrate in the drinking water, with a dose-dependent suppression of PK-G I protein in the aorta. PK-G I antigen in whole rat lung extract was also suppressed by administration of isosorbide or theophylline in the drinking water. These data may contribute to our understanding of nitrovasodilator resistance, a phenomenon resulting from continuous exposure to nitroglycerin or other nitrovasodilators.

Animals↗
PubMed1997

Nitric oxide synthase and cyclic GMP-dependent protein kinase concentrated at the neuromuscular endplate.

Nitric oxide mediates diverse functions in development and physiology of vertebrate skeletal muscle. Neuronal type nitric oxide synthase-mu is enriched in fast-twitch fibers and binds to syntrophin, a component of the sarcolemmal dystrophin glycoprotein complex. Here, we show that cyclic GMP-dependent protein kinase type I, a primary effector for nitric oxide, occurs selectively at the neuromuscular junction, in mice and rats, and both neuronal type nitric oxide synthase-mu and cyclic GMP-dependent protein kinase type I remain at skeletal muscle endplates at least two weeks following muscle denervation. Expression of neuronal type nitric oxide synthase-mu and cyclic GMP-dependent protein kinase type I are up-regulated following fusion of cultured primary myotubes. Interestingly, the highest levels of neuronal type nitric oxide synthase-mu in muscle are found complexed with dystrophin at the sarcolemma of intrafusal fibers in muscle spindles. Localization of neuronal type nitric oxide synthase-mu and cyclic GMP-dependent protein kinase type I at the neuromuscular junction suggests functions for nitric oxide and cyclic GMP in the regulation of synaptic actions of intra- and extrafusal muscle fibers.

Animals↗
PubMed1997

Expression of guanylyl cyclase-A/atrial natriuretic peptide receptor blocks the activation of protein kinase C in vascular smooth muscle cells. Role of cGMP and cGMP-dependent protein kinase.

To understand the molecular mechanisms of cellular signaling of atrial natriuretic peptide (ANP), we have studied its effect on the enzymatic activity of endogenous and overexpressed protein kinase C (PKC) in rat thoracic aortic vascular smooth muscle (RTASM) cells. Angiotensin II (ANG II), endothelin-1 (ET-1), and 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulated fourfold to fivefold PKC activity in PKC-alpha cDNA-transfected RTASM cells. However, pretreatment of these cells with ANP significantly inhibited the agonist-stimulated PKC activity in a dose-dependent manner. The inhibitory effect of ANP was more effective if cells were transfected with both PKC-alpha and guanylyl cyclase-A/atrial natriuretic peptide receptor (Npra) cDNAs. The agonist-stimulated PKC activity was also inhibited if RTASM cells were pretreated with cGMP analog 8-bromo-cGMP; however, the treatment of cells with a cAMP analog, dibutyryl-cAMP, did not show any discernible effect. The pretreatment of cells with Npra antagonist A-71915, significantly blocked the production of cGMP as well as the inhibitory effect of ANP on PKC activity. To further examine whether the antagonistic action of ANP and 8-bromo-cGMP on agonist-stimulated PKC activity were mediated through cGMP-dependent protein kinase (PKG), cells were treated with ANP or 8-bromo-cGMP and activators of PKC in the presence of KT-5823, a specific inhibitor of PKG. The treatment of cells with KT-5823 significantly attenuated the inhibitory effects of both ANP and 8-bromo-cGMP on agonist-stimulated PKC activity. The results from these studies provide strong evidence that ANP antagonizes the activation of PKC in RTASM cells, involving guanylyl cyclase-A receptor Npra and second messenger cGMP. Our data further support the notion that ANP acts as a negative mediator of signaling cross-talks between Npra and PKC in a cGMP-dependent manner, probably involving cGMP-dependent protein kinase in this process.

Angiotensin II↗
PubMed1996

Phosphorylation of the inositol 1,4,5-trisphosphate receptor. Cyclic GMP-dependent protein kinase mediates cAMP and cGMP dependent phosphorylation in the intact rat aorta.

The effects of cyclic GMP (cGMP) and activation of cGMP-dependent protein kinase (PKG) on the phosphorylation of the inositol 1,4, 5-trisphosphate (IP3) receptor were examined in intact rat aorta using the technique of back phosphorylation. Aorta treated with the nitric oxide donors, S-nitroso-N-acetylpenicillamine and sodium nitroprusside, or the selective PKG activator, 8-(4-para-chlorophenylthio)-cGMP (8-CPT-cGMP), demonstrated increased IP3 receptor phosphorylation in situ, which was both time- and concentration-dependent with a stoichiometry of 0.5 mol of phosphate/mol of receptor above control. Treatment of aorta with the adenyl cyclase activator, forskolin, also demonstrated increased phosphorylation of the IP3 receptor on the PKG site, although the selective cAMP-dependent protein kinase activator, 8-(4-para-chlorophenylthio)-cAMP (8-CPT-cAMP), did not increase the phosphorylation of the IP3 receptor. Moreover, the PKG selective inhibitor, KT 5823, inhibited both sodium nitroprusside and forskolin-induced IP3 receptor phosphorylation more potently than the selective cAMP-dependent protein kinase inhibitor, KT 5720, suggesting that PKG mediates the increase in IP3 receptor phosphorylation by both cyclic nucleotides in intact aorta. These results provide further support for the notion that PKG is activated by both cAMP and cGMP in intact vascular smooth muscle and that PKG performs a critical role in cyclic nucleotide-dependent relaxation of blood vessels.

Alkaloids↗
PubMed1996

cGMP-dependent protein kinase in dorsal root ganglion: relationship with nitric oxide synthase and nociceptive neurons.

Nitric oxide and cGMP influence plasticity of nociceptive processing in spinal cord. However, effectors for cGMP have not been identified in sensory pathways. We now demonstrate that cGMP-dependent protein kinase I (cGKl) occurs in the DRGs at levels comparable to that in cerebellum, the richest source of cGKl in the body. Immunohistochemical studies reveal that cGKl is concentrated in a subpopulation of small- and medium-diameter DRG neurons that partially overlap with substance P and calcitonin gene-related polypeptide containing cells. During development, cGKl expression throughout the embryo is essentially restricted to sensory neurons and to the spinal floor and roof plates. Neuronal nitric oxide synthase (nNOS) is coexpressed with cGKl in sensory neurons during embryonic development and after peripheral nerve axotomy. The primary target for cGKl in cerebellum, G-substrate, is not present in developing, mature, or regenerating sensory neurons, indicating that other proteins serve as effectors for cGKl in sensory processing. These data establish sensory neurons as a primary locus for cGMP actions during development and suggest a role for cGKl in plasticity of nociception.

Animals↗
PubMed1996

Cyclic GMP-dependent protein kinase is required for thrombospondin and tenascin mediated focal adhesion disassembly.

Focal adhesions are specialized regions of cell membranes that are foci for the transmission of signals between the outside and the inside of the cell. Intracellular signaling events are important in the organization and stability of these structures. In previous work, we showed that the counter-adhesive extracellular matrix proteins, thrombospondin, tenascin, and SPARC, induce the disassembly of focal adhesion plaques and we identified the active regions of these proteins. In order to determine the mechanisms whereby the anti-adhesive matrix proteins modulate cytoskeletal organization and focal adhesion integrity, we examined the role of protein kinases in mediating the loss of focal adhesions by these proteins. Data from these studies show that cGMP-dependent protein kinase is necessary to mediate focal adhesion disassembly triggered by either thrombospondin or tenascin, but not by SPARC. In experiments using various protein kinase inhibitors, we observed that selective inhibitors of cyclic GMP-dependent protein kinase, KT5823 and Rp-8-Br-cGMPS, blocked the effects of both the active sequence of thrombospondin 1 (hep I) and the alternatively-spliced segment (TNfnA-D) of tenascin-C on focal adhesion disassembly. Moreover, early passage rat aortic smooth muscle cells which have high levels of cGMP-dependent protein kinase were sensitive to hep I treatment, in contrast to passaged cGMP-dependent protein kinase deficient cells which were refractory to hep I or TNfnA-D treatment, but were sensitive to SPARC. Transfection of passaged smooth muscle cells with the catalytic domain of PKG I alpha restored responsiveness to hep I and TNfnA-D. While these studies show that cGMP-dependent protein kinase activity is necessary for thrombospondin and tenascin-mediated focal adhesion disassembly, kinase activity alone is not sufficient to induce disassembly as transfection of the catalytic domain of the kinase in the absence of additional stimuli does not result in loss of focal adhesions.

Animals↗
PubMed1995

Cyclic guanosine monophosphate-dependent protein kinase is targeted to intermediate filaments and phosphorylates vimentin in A23187-stimulated human neutrophils.

The effects of the calcium ionophore, A23187, on human neutrophil activation were studied in relation to the signaling mechanism of cyclic guanosine monophosphate (cGMP)-dependent protein kinase (G-kinase). Immunocytochemistry demonstrated that G-kinase translocated from a diffuse localization in the cytoplasm to the cytoskeleton after stimulation with A23187. Over a period of 5 minutes, G-kinase was transiently colocalized with the intermediate filament protein, vimentin. At 3 minutes' stimulation with A23187, colocalization of G-kinase and vimentin was predominantly confined to filaments that extended into the uropod. The time of colocalization of G-kinase and vimentin was reduced in the A23187-stimulated cell from 3 minutes to 1 minute by 8-Br-cGMP. Coincident with colocalization was an increase in cGMP levels and transient phosphorylation of vimentin in adhered A23187-stimulated cells. Phosphorylation of vimentin was maximal after 3 minutes with A23187, and was essentially over at 5 minutes. The time of phosphorylation of vimentin was also reduced from 3 minutes to 1 minute when cells were preincubated with 8-Br-cGMP and then stimulated with A23187, which suggests that cyclic adenosine monophosphate (cAMP)-dependent protein kinase does not phosphorylate vimentin in A23187-treated neutrophils. Phosphorylation of vimentin was not observed in nonactivated cells treated only with 8-Br-cGMP. The presence of the protein kinase C inhibitors, staurosporine or H-7, did not inhibit vimentin phosphorylation in A23187-treated cells, which provides supportive data that protein kinase C is not the phosphorylating enzyme. These results suggest that vimentin and G-kinase are colocalized in a Ca(2+)-dependent manner in neutrophils, and that vimentin is transiently phosphorylated by G-kinase in response to the colocalization of the two proteins. The transient redistribution of compartmentalized G-kinase represents one type of neutrophil activation mechanism.

Calcimycin↗
PubMed1995

Localization of cyclic GMP-dependent protein kinase in human mononuclear phagocytes.

The presence and physiological role of cGMP-dependent protein kinase (G-kinase) was investigated in human mononuclear phagocytes. Western blots of monocyte extracts revealed a single polypeptide band that comigrated with purified bovine lung G-kinase. G-kinase was localized by immunofluorescence microscopy in freshly isolated adherent human monocytes, monocyte-derived macrophages cultured from 4 to 14 days, and alveolar macrophages. In monocytes, G-kinase was localized in granules or vesicles in the cytoplasm, at the microtubule organizing center, on filaments, and in the nucleus. In monocyte-derived macrophages, intense staining for G-kinase was found in the vicinity of the Golgi, in vesicles throughout the cytoplasm, and diffusely in the nucleus. Dual-label confocal laser scanning microscopy demonstrated that G-kinase was colocalized with the endoplasmic reticulum. For comparison, G-kinase was localized in alveolar macrophages that were adhered from 3 to 30 min. In these cells, G-kinase was prominent within the organelle-rich area pericortical to the nucleus. However, a well-defined area of intense staining was also observed at the cell periphery at early time points during adherence and spreading. Rhodamine-labeled phalloidin showed that this peripheral area was rich in F-actin. Cytochalasin D, but not nocodazole, inhibited G-kinase targeting to the cell margin. Furthermore, the guanylate cyclase inhibitor LY83583 inhibited alveolar macrophage spreading and staining for G-kinase at the cell periphery. These data suggest that G-kinase may play an important role in cGMP-mediated regulation involved in protein processing and cell motility.

Aminoquinolines↗