Dermatological surgery: an update on suture materials and techniques. Part 2.

This is the second part of a two-part series summarizing the latest evidence related to suture materials and wound closure techniques in dermatological surgery. We critically appraised evidence focusing on the following consequences of suture choice: scar/cosmesis, pain, patient satisfaction, cost, infection and wound complications. We searched the databases MEDLINE, PubMed and Embase using the keywords 'skin surgery', 'dermatological surgery', 'sutures', 'braided sutures', 'monofilament sutures' and 'antibacterial sutures' to identify relevant English-language articles. This part of the review assesses the evidence for different types of buried sutures, including braided vs. monofilament sutures, longer-absorbing sutures and antibacterial sutures. The majority of trials were noted to be of poor quality, single-centre (thus lacking external validity) and underpowered, which presents challenges in comparing suture techniques in skin surgery. Future large-scale, multicentre, randomized trials are needed, with both surgeon and patient-assessed validated outcomes.

Dermatological surgery: an update on suture materials and techniques. Part 1.

Significant variation exists in the surgical suture materials and techniques used for dermatological surgery. Many wound-closure techniques are now practised, including use of sutures, staples and topical adhesives. The focus of our review article is to summarize the latest evidence relating to suture materials and wound-closure techniques, considering the following areas: scar/cosmesis, pain, patient satisfaction, cost, infection and wound complications. We searched the databases Medline, PubMed and Embase using the keywords 'skin surgery', 'dermatologic surgery', 'sutures', 'suture techniques', 'suturing techniques' and 'surgical techniques' to identify relevant English-language articles. Absorbable superficial sutures may be a preferred alternative to nonabsorbable sutures by both patients and surgeons. Subcuticular sutures may be preferable to simple interrupted sutures for superficial wound closure, and there may also be a role for skin staples in dermatological surgery, particularly on the scalp. However, there remains limited evidence specific to dermatological surgery supporting the use of particular suture materials and suturing techniques. Further high-quality research is required, including multicentre randomized trials with larger cohorts.

Comparative proteomics reveals unexpected quantitative phosphorylation differences linked to platelet activation state.

There is a need to assess platelet activation in patients with thrombotic disorders. P-selectin and activated integrin αIIbß3 are usually quantified by flow cytometry to measure platelet activation. Monitoring changes in vasodilator-stimulated phosphoprotein (VASP) phosphorylation is an established method to determine the platelet-reactivity status. To study disruptions of platelet reactivity more comprehensively, we compared the human non-secretory platelet proteome after in-vitro -activation and -inhibition with their respective untreated controls using unbiased fluorescence two-dimensional differential in-gel electrophoresis. The non-secretory platelet proteome was more severely affected during inhibition than during activation. Strikingly, while VASP reached a 1.3-fold increase in phosphorylation levels in inhibited platelets, other protein kinase A targets showed several-fold stronger inhibition-induced phosphorylation levels, including LIM and SH3 domain protein 1 (6.7-fold), Src kinase-associated phosphoprotein 2 (4.6-fold), and Ras-related protein Rap1b (4.1-fold). Moreover, phosphorylation of integrin-linked protein kinase (ILK) and pleckstrin (PLEK) species was associated with P-selectin surface expression. The discrimination power between activation and inhibition was more pronounced for dephosphorylated ILK (3.79 Cohen's d effect size) and phosphorylated PLEK (3.77) species than for P-selectin (2.35). These data reveal new insights into the quantitative changes of the platelet reactivity proteome and suggest powerful alternatives to characterise their activation and inactivation potential.

The cytoskeletal protein LASP-1 differentially regulates migratory activities of choriocarcinoma cells.

PURPOSE: During healthy pregnancy, a distinct but limited invasion of trophoblast cells into the uterus occurs. In contrast, excessive trophoblast invasion is associated with placental choriocarcinoma (CC). Overexpression of the cytoskeletal protein LASP-1 was shown to contribute to cancer aggressiveness. Here, the yet unknown role of LASP-1 in CC cells is analysed. METHODS: Expression of LASP-1 in human primary carcinoma was assessed by immunohistochemistry and confirmed in CC-derived cell lines by immunocytochemistry, RT-PCR and Western blot. After down-regulation of LASP-1 expression with specific si-RNA in CC-derived cell lines, migratory and proliferative activities were analysed by matrigel migration assay and WST-8 test. RESULTS: LASP-1 expression was detected in human primary choriocarcinoma and in JEG-3, JAR and BeWo cells. Knock down of LASP-1 resulted in a decreased expression of LASP-1 protein in JEG-3 and JAR cells accompanied by a diminished migration and a decreased proliferative activity of these two cell lines. Knockdown of LASP-1 in BeWo cells failed. In consequence, migratory function and proliferation was unaffected. CONCLUSION: This is the first study describing LASP-1 expression in CC cells. Detecting an affection of migratory processes after LASP-1 silencing, we propose that LASP-1 could impact on metastasis of CC cells.

Nuclear import of LASP-1 is regulated by phosphorylation and dynamic protein-protein interactions.

LASP-1 is a multidomain protein predominantly localized at focal contacts, where it regulates cytoskeleton dynamics and cell migration. However, in different tumor entities, a nuclear LASP-1 accumulation is observed, thought to have an important role in cancer progression. Until now, the molecular mechanisms that control LASP-1 nuclear import were not elucidated. Here, we identified a novel LASP-1-binding partner, zona occludens protein 2 (ZO-2), and established its role in the signal transduction pathway of LASP-1 nucleo-cytoplasmatic shuttling. Phosphorylation of LASP-1 by PKA at serine 146 induces translocation of the LASP-1/ZO-2 complex from the cytoplasm to the nucleus. Interaction occurs within the carboxyterminal proline-rich motif of ZO-2 and the SH3 domain in LASP-1. In situ proximity ligation assay confirmed the direct binding between LASP-1 and ZO-2 and visualized the shuttling. Nuclear export is mediated by Crm-1 and a newly identified nuclear export signal in LASP-1. Finally, dephosphorylation of LASP-1 by phosphatase PP2B is suggested to relocalize the protein back to focal contacts. In summary, we define a new pathway for LASP-1 in tumor progression.

Nuclear localisation of LASP-1 correlates with poor long-term survival in female breast cancer.

BACKGROUND: LIM and SH3 protein 1 (LASP-1) is a nucleo-cytoplasmatic signalling protein involved in cell proliferation and migration and is upregulated in breast cancer in vitro studies have shown that LASP-1 might be regulated by prostate-derived ETS factor (PDEF), p53 and/or LASP1 gene amplification. This current study analysed the prognostic significance of LASP-1 on overall survival (OS) in 177 breast cancer patients and addressed the suggested mechanisms of LASP-1-regulation. METHODS: Nucleo-cytoplasmatic LASP-1-positivity of breast carcinoma samples was correlated with long-term survival, clinicopathological parameters, Ki67-positivity and PDEF expression. Rate of LASP1 amplification was determined in micro-dissected primary breast cancer cells using quantitative RT-PCR. Cell-phase dependency of nuclear LASP-1-localisation was studied in synchronised cells. In addition, LASP-1, PDEF and p53 expression was compared in cell lines of different tumour entities to define principles for LASP-1-regulation. RESULTS: We showed that LASP-1 overexpression is not due to LASP1 gene amplification. Moreover, no correlation between p53-mutations or PDEF-expression and LASP-1-status was observed. However, nuclear LASP-1-localisation in breast carcinomas is increased during proliferation with peak in G2/M-phase and correlated significantly with Ki67-positivity and poor OS. CONCLUSION: Our results provide evidence that nuclear LASP-1-positivity may serve as a negative prognostic indicator for long-term survival of breast cancer patients.

Overexpression of LASP-1 mediates migration and proliferation of human ovarian cancer cells and influences zyxin localisation.

LIM and SH3 protein 1 (LASP-1), initially identified from human breast cancer, is a specific focal adhesion protein involved in cell proliferation and migration. In the present work, we analysed the effect of LASP-1 on biology and function of human ovarian cancer cell line SKOV-3 using small interfering RNA technique (siRNA). Transfection with LASP-1-specific siRNA resulted in a reduced protein level of LASP-1 in SKOV-3 cells. The siRNA-treated cells were arrested in G(2)/M phase of the cell cycle and proliferation of the tumour cells was suppressed by 60-90% corresponding to around 70% of the cells being transfected successfully as seen by immunofluorescence. Moreover, transfected tumour cells showed a 40% reduced migration. LASP-1 silencing is accompanied by a reduced binding of the LASP-1-binding partner zyxin to focal contacts without changes in actin stress fibre and microtubule organisation or focal adhesion morphology as observed by immunofluorescence. In contrast, silencing of zyxin is not influencing cell migration and had neither influence on LASP-1 expression nor actin cytoskeleton and focal contact morphology suggesting that LASP-1 is necessary and sufficient for recruiting zyxin to focal contacts. The data provide evidence for an essential role of LASP-1 in tumour cell growth and migration, possibly through influencing zyxin localization.

Heat shock protein 27 is a substrate of cGMP-dependent protein kinase in intact human platelets: phosphorylation-induced actin polymerization caused by HSP27 mutants.

Phosphorylation of heat shock protein 27 (Hsp27) in human platelets by mitogen-activated protein kinase-activated protein kinase (MAPKAP) 2 is associated with signaling events involved in platelet aggregation and regulation of microfilament organization. We now show that Hsp27 is also phosphorylated by cGMP-dependent protein kinase (cGK), a signaling system important for the inhibition of platelet aggregation. Stimulation of washed platelets with 8-para-chlorophenylthio-cGMP, a cGK specific activator, resulted in a time-dependent phosphorylation of Hsp27. This is supported by the ability of cGK to phosphorylate Hsp27 in vitro to an extent comparable with the cGK-mediated phosphorylation of its established substrate vasodilator-stimulated phosphoprotein. Studies with Hsp27 mutants identified threonine 143 as a yet uncharacterized phosphorylation site in Hsp27 specifically targeted by cGK. To test the hypothesis that cGK could inhibit platelet aggregation by phosphorylating Hsp27 and interfering with the MAPKAP kinase phosphorylation of Hsp27, the known MAPKAP kinase 2-phosphorylation sites (Ser15, Ser78, and Ser82) as well as Thr143 were replaced by negatively charged amino acids, which are considered to mimic phosphate groups, and tested in actin polymerization experiments. Mimicry at the MAPKAP kinase 2 phosphorylation sites led to mutants with a stimulating effect on actin polymerization. Mutation of the cGK-specific site Thr143 alone had no effect on actin polymerization, but in the MAPKAP kinase 2 phosphorylation-mimicking mutant, this mutation reduced the stimulation of actin polymerization significantly. These data suggest that phosphorylation of Hsp27 and Hsp27-dependent regulation of actin microfilaments contribute to the inhibitory effects of cGK on platelet function.

Activation of cGMP-dependent protein kinase Ibeta inhibits interleukin 2 release and proliferation of T cell receptor-stimulated human peripheral T cells.

Several major functions of type I cGMP-dependent protein kinase (cGK I) have been established in smooth muscle cells, platelets, endothelial cells, and cardiac myocytes. Here we demonstrate that cGK Ibeta is endogenously expressed in freshly purified human peripheral blood T lymphocytes and inhibits their proliferation and interleukin 2 release. Incubation of human T cells with the NO donor, sodium nitroprusside, or the membrane-permeant cGMP analogs PET-cGMP and 8-pCPT-cGMP, activated cGK I and produced (i) a distinct pattern of phosphorylation of vasodilator-stimulated phosphoprotein, (ii) stimulation of the mitogen-activated protein kinases ERK1/2 and p38 kinase, and, upon anti-CD3 stimulation, (iii) inhibition of interleukin 2 release and (iv) inhibition of cell proliferation. cGK I was lost during in vitro culturing of primary T cells and was not detectable in transformed T cell lines. The proliferation of these cGK I-deficient cells was not inhibited by even high cGMP concentrations indicating that cGK I, but not cGMP-regulated phosphodiesterases or channels, cAMP-dependent protein kinase, or other potential cGMP mediators, was responsible for inhibition of T cell proliferation. Consistent with this, overexpression of cGK Ibeta, but not an inactive cGK Ibeta mutant, restored cGMP-dependent inhibition of cell proliferation of Jurkat cells. Thus, the NO/cGMP/cGK signaling system is a negative regulator of T cell activation and proliferation and of potential significance for counteracting inflammatory or lymphoproliferative processes.

Endothelial nitric-oxide synthase (type III) is activated and becomes calcium independent upon phosphorylation by cyclic nucleotide-dependent protein kinases.

Endothelial nitric-oxide synthase (NOS-III) is defined as being strictly dependent on Ca(2+)/calmodulin (CaM) for activity, although NO release from endothelial cells has been reported to also occur at intracellular free Ca(2+) levels that are substimulatory for the purified enzyme. We demonstrate here that NOS-III, but neither NOS-I nor -II, is rapidly and strongly activated and phosphorylated on both Ser and Thr in the presence of cGMP-dependent protein kinase II (cGK II) and the catalytic subunit of cAMP-dependent protein kinase (cAK) in vitro. Phosphopeptide analysis by mass spectrometry identified Ser(1177), as well as Ser(633) which is situated in a recently defined CaM autoinhibitory domain within the flavin-binding region of human NOS-III. Phosphoamino acid analysis identified a putative phosphorylation site at Thr(495) in the CaM-binding domain. Importantly, both cAK and cGK phosphorylation of NOS-III in vitro caused a highly reproducible partial (10-20%) NOS-III activation which was independent of Ca(2+)/CaM, and as much as a 4-fold increase in V(max) in the presence of Ca(2+)/CaM. cAK stimulation in intact endothelial cells also increased both Ca(2+/)CaM-independent and -dependent activation of NOS-III. These data collectively provide new evidence for cAK and cGK stimulation of both Ca(2+)/CaM-independent and -dependent NOS-III activity, and suggest possible cross-talk between the NO and prostaglandin I(2) pathways and a positive feedback mechanism for NO/cGMP signaling.

Inhibition of cyclic GMP-dependent protein kinase-mediated effects by (Rp)-8-bromo-PET-cyclic GMPS.

1. The modulation of the guanosine 3':5'-cyclic monophosphate (cyclic GMP)- and adenosine 3':5'-cyclic monophosphate (cyclic AMP)-dependent protein kinase activities by the diastereomers of 8-bromo-beta phenyl-1, N2-ethenoguanosine 3':5'-cyclic monophosphorothioate, ((Rp)- and (Sp)-8-bromo-PET-cyclic GMPS) was investigated by use of purified protein kinases. In addition, the effects of (Rp)-8-bromo-PET-cyclic GMPS on protein phosphorylation in intact human platelets and on [3H]-noradrenaline release and neurogenic vasoconstriction in electrical field stimulated rat tail arteries were also studied. 2. Kinetic analysis with purified cyclic GMP-dependent protein kinase (PKG) type I alpha and I beta, which are expressed in the rat tail artery, revealed that (Rp)-8-bromo-PET-cyclic GMPS is a competitive inhibitor with an apparent Ki of 0.03 microM. The activation of purified cyclic AMP-dependent protein kinase (PKA) type II was antagonized with an apparent Ki of 10 microM. 3. In human platelets, (Rp)-8-bromo-PET-cyclic GMPS (0.1 mM) antagonized the activation of the PKG by the selective activator 8-(4-chlorophenylthio)-guanosine 3':5'-cyclic monophosphate (8-pCPT-cyclic GMP; 0.2 mM) without affecting the activation of PKA by (Sp)-5, 6-dichloro-1-beta-D-ribofurano-sylbenzimidazole- 3':5'-cyclic monophosphorothioate ((Sp)-5,6-DCl-cyclic BiMPS; 0.1 mM). 4. (Rp)-8-bromo-PET-cyclic GMPS was not hydrolysed by the cyclic GMP specific phosphodiesterase (PDE) type V from bovine aorta but potently inhibited this PDE. 5. The corresponding sulphur free cyclic nucleotide of the two studied phosphorothioate derivatives, 8-bromo-beta-phenyl-1, N2-ethenoguanosine-3':5'-cyclic monophosphate (8-bromo-PET-cyclic GMP), had no effect on electrically-induced [3H]-noradrenaline release but concentration-dependently decreased the stimulation-induced vasoconstriction. (Rp)-8-bromo-PET-cyclic GMPS (3 microM) shifted the vasoconstriction response to the right without affecting stimulation evoked tritium overflow. 6. The NO donor, 3-morpholinosydnonimine (SIN-1) relaxed rat tail arteries precontracted with phenylephrine (1 microM). The SIN-1 concentration-relaxation curve was shifted in a parallel manner to the right by (Rp)-8-bromo-PET-cyclic GMPS, suggesting that the relaxation was mediated by a cyclic GMP/PKG-dependent mechanism. 7. The [3H]-noradrenaline release-enhancing effect and stimulation-induced decrease in vasoconstriction of forskolin were unaffected by (Rp)-8-bromo-PET-cyclic GMPS. Moreover, the forskolin concentration-relaxation curve was not changed in the presence of the PKG inhibitor, suggesting a high selectivity in intact cells for PKG- over PKA-mediated effects. 8. The results obtained indicate that (Rp)-8-bromo-PET-cyclic GMPS presently is the most potent and selective inhibitor of PKG and is helpful in distinguishing between cyclic GMP and cyclic AMP messenger pathways activation. Therefore, this phosphorothioate stereomer may be a useful tool for studying the role of cyclic GMP in vitro.

Characterization of cyclic nucleotide phosphodiesterases with cyclic AMP analogs: topology of the catalytic sites and comparison with other cyclic AMP-binding proteins.

To define essential interactions of cAMP with the catalytic sites of cyclic nucleotide phosphodiesterases (PDEs) and to begin to map the topology of the sites, we have tested a series of cAMP analogs as competitive inhibitors of the PDEs that hydrolyze cAMP with high efficiency (PDE1, PDE2, PDE3, and PDE4). Comparisons of IC50 values, relative to cAMP, were used to predict which functional groups on cAMP interact with each isozyme. Common to all PDEs tested, except for the calcium/calmodulin-dependent PDE (CaM-PDE, PDE1), is an interaction at the N1-position of cAMP and a distinct lack of binding to the 2'-hydroxyl group of the ribose moiety. Only the cGMP-stimulated (PDE2) and cAMP-specific (PDE4) PDEs appear to interact strongly at the N7-position. The cGMP-inhibited PDE (cGI-PDE, PDE3) may interact less strongly with this nitrogen. The PDE4 and PDE3 both interact with cAMP through the 6-amino group, which most likely serves as a hydrogen bond donor. PDE4 and PDE3 appear to be able to bind to the anti-conformer of cAMP, whereas the PDE1 and PDE2 bind the syn-conformer. The CaM-PDE exhibits no appreciable specificity for any of the analogs tested, showing little or no interaction with the 6-amino group or with any of the ring nitrogens. Large differences exist in the nucleotide-binding requirements for the PDE catalytic sites, compared with the regulatory sites of cAMP-dependent protein kinase and the catabolite activator protein.

(Rp)-8-pCPT-cGMPS, a novel cGMP-dependent protein kinase inhibitor.

In the present study, the inhibitory effect of the cGMP analog (Rp)-8-(para-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate ((Rp)-8-pCPT-cGMPS) on the cGMP-dependent protein kinase-mediated protein phosphorylation in intact human platelets was investigated. In vitro phosphorylation experiments with the substrate kemptide demonstrated an inhibition of the cGMP-dependent protein kinase by (Rp)-8-pCPT-cGMPS with a Ki of 0.5 microM. In intact human platelets, (Rp)-8-pCPT-cGMPS antagonized the activation of the cGMP-dependent protein kinase by 8-pCPT-cGMP without affecting cAMP-dependent protein kinase or cGMP-regulated phosphodiesterases. The data obtained suggest that (Rp)-8-pCPT-cGMPS may be a useful tool for studying the role of cGMP in vitro and in intact cells.

cAMP- and cGMP-dependent protein kinase phosphorylation sites of the focal adhesion vasodilator-stimulated phosphoprotein (VASP) in vitro and in intact human platelets.

The vasodilator-stimulated phosphoprotein (VASP) is a major substrate for cAMP-dependent- (cAK) and cGMP-dependent protein kinase (cGK) in human platelets and other cardiovascular cells. To identify the VASP phosphorylation sites, purified VASP was phosphorylated by either protein kinase and subjected to trypsin, V8 and Lys-C proteolysis. The phosphorylated proteolytic fragments obtained were separated by reversed phase high performance liquid chromatography. Sequence analysis of the phosphorylated peptides and 32P measurement of the released 32P-labeled amino acids revealed three phosphorylation sites: a serine 1-containing site (LRKVSKQEEA), a serine 2-containing site (HIERRVSNAG), and a threonine-containing site (MNAVLARRRKATQVGE). Additional experiments with purified VASP demonstrated that both cAK and cGK phosphorylated serine 2 rapidly and the threonine residue slowly, whereas cGK phosphorylated the serine 1 residue more rapidly than the cAK. These differences in the phosphorylation rates of VASP by the two protein kinases were also observed with synthetic peptides corresponding to the sequences of the three identified phosphorylation sites. These experiments also established the synthetic peptide serine 1 as one of the best in vitro cGK substrates and the serine 2-containing site as the site responsible for the phosphorylation-induced mobility shift of VASP in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Experiments with 32P-labeled platelets provided evidence that VASP is phosphorylated at the same three identified sites also in intact cells and that selective activation of cAK or cGK primarily increased the phosphorylation of both serine 2 and serine 1 but not threonine. Our results demonstrated overlapping substrate specificities of cAK and cGK in vitro and in intact cells. However, important quantitative and qualitative differences between cAK- and cGK-mediated phosphorylation of the focal adhesion protein VASP in human platelets were also observed, suggesting distinct functions of the two types of cyclic nucleotide-mediated VASP phosphorylation.

Derivatives of 1-beta-D-ribofuranosylbenzimidazole 3',5'-phosphate that mimic the actions of adenosine 3',5'-phosphate (cAMP) and guanosine 3',5'-phosphate (cGMP).

A series of new analogues of 1-beta-D-ribofuranosylbenzimidazole 3',5'-phosphate (cBIMP) has been designed according to the properties predicted by the MNDO method, and synthesised from substituted benzimidazoles. Dipole vectors and HOMO and LUMO energies for each benzimidazole base were calculated by the MNDO method and the lipophilicities of the cBIMP derivatives were determined. In general, the cBIMP derivatives activate cAMP-dependent protein kinases I and II and preferentially bind to site B, especially for the type II kinase, with 2-trifluoromethyl-cBIMP and 5,6-difluoro-cBIMP exhibiting the highest site selectivity. Each cBIMP derivative can stimulate cGMP-stimulated cyclic phosphodiesterase (cGS-PDE), with 5,6-dimethyl-cBIMP being as potent as cGMP, and also inhibit cGMP-inhibited phosphodiesterase (cGI-PDE). Only the 2-trifluoromethyl-cBIMP and the Rp-phosphorothioates (cBIMPS) (equatorial P = S) were resistant to hydrolysis by cPDE. The Sp-phosphorothioates were hydrolysed slowly, if at all. In addition to exhibiting a high lipophilicity, the most active compounds for the induction of apoptosis and inhibition of proliferation were also resistant to cPDE (Sp-5,6-dichloro-cBIMPS) and/or were potent activators of cAMP-dependent protein kinase (5,6-dichloro-cBIMP).

Mapping of the epitope/paratope interactions of a monoclonal antibody directed against adenosine 3',5'-monophosphate.

A series of systematically modified cyclic AMP (cAMP) analogues, including newly synthesized benzimidazole ribofuranosyl 3',5'-monophosphates was used to map the essential molecular interactions between cAMP and the monoclonal antibody 4/2C2 (mab 4/2C2) directed against 2'-O-succinoyl cAMP [Colling, Gilles, Nass, Moka & Jaenicke (1988) Second Messengers Phosphoproteins 12, 123-133]. Its paratope binds the purine base in syn conformation by dipole-dipole interactions and hydrophobic forces and/or stacking interactions. The ribose phosphate moiety is recognized by a combination of charge interactions and H-bonds to the exocyclic and the 5'-oxygen atoms and a hydrophobic interaction at the 2'-position. There is no regioselectivity for the exocyclic oxygen atoms. Compared with the known types of binding, mab 4/2C2 thus shows a new combination of molecular interactions which may be the basis of its strikingly specific recognition and binding of the cyclic adenylates. On this account mab 4/2C2 may become an important tool in studies on cAMP metabolism.

Role of cGMP and cGMP-dependent protein kinase in nitrovasodilator inhibition of agonist-evoked calcium elevation in human platelets.

Most platelet agonists activate and elevate the cytosolic free calcium concentration in human platelets through receptor-dependent mechanisms that are antagonized by cAMP- and cGMP-elevating agents. Nitrovasodilators such as nitroprusside and endothelium-derived relaxing factor are potent cGMP-elevating platelet inhibitors. In the present study, the role of cGMP and cGMP-dependent protein kinase in nitrovasodilator inhibition of ADP- and thrombin-evoked calcium elevation and activation of human platelets was investigated. Preincubation of platelets with 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate (8-pCPT-cGMP; a membrane-permeant selective activator of the cGMP-dependent protein kinase that does not significantly affect cGMP-regulated phosphodiesterases) inhibited the thrombin-induced phosphorylation mediated by myosin light chain kinase and protein kinase C. Nitrovasodilator-induced protein phosphorylation in human platelets was distinct from that induced by cAMP-elevating prostaglandins and could be mimicked by 8-pCPT-cGMP. Preincubation of human platelets with nitrovasodilators or 8-pCPT-cGMP inhibited the ADP- and thrombin-evoked calcium elevation in the presence and absence of external calcium. Nitrovasodilators and 8-pCPT-cGMP also inhibited the agonist-induced Mn2+ influx, but stopped-flow experiments indicated that the ADP receptor-operated cation channel was not significantly inhibited. These results suggest that in human platelets nitrovasodilators inhibit the agonist-induced calcium mobilization from intracellular stores and the secondary store-related calcium influx but not the ADP receptor-operated cation channel. The results also suggest that these nitrovasodilator effects are mediated by cGMP and the cGMP-dependent protein kinase.

Characterization of Sp-5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole- 3',5'-monophosphorothioate (Sp-5,6-DCl-cBiMPS) as a potent and specific activator of cyclic-AMP-dependent protein kinase in cell extracts and intact cells.

A newly designed cyclic AMP (cAMP) analogue, Sp-5,6-dichloro-1-beta-D- ribofuranosylbenzimidazole-3',5'-monophosphorothioate (Sp-5,6-DCl-cBiMPS), and 8-(p-chlorophenylthio)-cAMP (8-pCPT-cAMP) were compared with respect to their chemical and biological properties in order to assess their potential as activators of the cAMP-dependent protein kinases (cAMP-PK) in intact cells. Sp-5,6-DCl-cBiMPS was shown to be both a potent and specific activator of purified cAMP-PK and of cAMP-PK in platelet membranes, whereas 8-pCPT-cAMP proved to be a potent activator of cAMP-PK and cyclic-GMP-dependent protein kinase (cGMP-PK) both as purified enzymes and in platelet membranes. Sp-5,6-DCl-cBiMPS was not significantly hydrolysed by three types of cyclic nucleotide phosphodiesterases, whereas 8-pCPT-cAMP (and 8-bromo-cAMP) was hydrolysed to a significant extent by the Ca2+/calmodulin-dependent phosphodiesterase and by the cGMP-inhibited phosphodiesterase. The apparent lipophilicity, a measure of potential cell-membrane permeability, of Sp-5,6-DCl-cBiMPS was higher than that of 8-pCPT-cAMP. Extracellular application of Sp-5,6-DCl-cBiMPS to intact human platelets reproduced the pattern of protein phosphorylation induced by prostaglandin E1, a cAMP-increasing inhibitor of platelet activation. In intact platelets, Sp-5,6- DCl-cBiMPS was also more effective than 8-pCPT-cAMP in inducing quantitative phosphorylation of the 46/50 kDa vasodilator-stimulated phosphoprotein (VASP), a major substrate of cAMP-PK in platelets. As observed with prostaglandin E1, pretreatment of human platelets with Sp-5,6-DCl-cBiMPS prevented the aggregation induced by thrombin. The results suggest that Sp-5,6-DCl-cBiMPS is a very potent and specific activator of cAMP-PK in cell extracts and intact cells and, in this respect, is superior to any other cAMP analogue used for intact-cell studies. In contrast with 8-pCPT-cAMP, Sp-5,6-DCl-cBiMPS can be used to distinguish the signal-transduction pathways mediated by cAMP-PK and cGMP-PK.

Starfish oocyte maturation: evidence for a cyclic AMP-dependent inhibitory pathway.

Oocyte maturation (meiosis reinitiation) in starfish is induced by the natural hormone 1-methyladenine (1-MeAde). Cyclic AMP seems to play a negative role in maturation since 1-MeAde triggers a decrease of the oocyte cAMP concentration and since intracellular microinjections of cAMP delay or inhibit maturation. Cyclic GMP is also inhibitory but other nucleotides such as cCMP, cIMP, and cUMP are inactive. The involvement of cAMP and cGMP in the control of oocyte maturation has been further investigated by the use of the stereoisomers of the phosphodiesterase-stable adenosine and guanosine 3',5'-phosphorothioates (cAMPS and cGMPS). The Sp isomers of cAMPS and cGMPS respectively activate cAMP-dependent protein kinase and cGMP-dependent kinase, while the Rp isomers inhibit the kinases. Extracellular addition of these cAMPS and cGMPS isomers has no effect on the oocytes. Intracellular microinjection of the kinase-activating (Sp)-cAMPS and (Sp)-cGMPS delays or inhibits 1-MeAde-induced maturation in a concentration-dependent manner (I50, 30 and 300 microM, respectively). Microinjections of (Rp)-cAMPS and (Rp)-cGMPS have no inhibitory effects and neither trigger nor facilitate maturation. Using various analogs, we found that the delaying or inhibiting effect is restricted to the compounds activating cAMP-dependent kinase, while the compounds inactive on or inhibiting the kinase have no effects on maturation. The inhibitory effect of (Sp)-cAMPS can be reversed by comicroinjection of the heat-stable inhibitor of cAMP-dependent protein kinase, by comicroinjection of the antagonist (Rp)-cAMPS, or by an increase in the 1-MeAde concentration. The negative effects of (Sp)-cAMPS or (Sp)-cGMPS are observed only when these isomers are microinjected during the hormone-dependent period. These results suggest that a cAMP-dependent inhibitory pathway participates in the maintenance of the prophase arrest of oocytes and that 1-MeAde acts both by inhibiting this negative pathway (dis-inhibitory pathway) and by stimulating a parallel activatory pathway leading to oocyte maturation. The generality of this mechanism is discussed.