Programmed Cell Death and Functional Activity of Platelets in Case of Oncohematologic Diseases.

Programmed cell death of non-nucleated blood cells - platelets - could be associated with pathophysiology of oncologic and oncohematologic diseases. It contributes to both bleedings (caused by the thrombocytopenia, which is induced by elimination of the platelets) and thrombosis (caused by the processes of blood coagulation on the surface of phosphatidylserine exposing platelets). Here we characterized functional responses of platelets from the patients with various oncological disorders undergoing chemotherapy and compared them to the platelets from the healthy donors and platelets pre-incubated with apoptosis inducer ABT-737. Some patients exhibited diminished capability of platelets to aggregate. Immunophenotyping of these platelets revealed their pre-activation in comparison to the platelets from the healthy donors. Calcium signaling analysis revealed that in the patient-derived platelets, as well as in the apoptotic platelets, intracellular calcium levels were increased in resting cells. However, moderate level of this increase together with weak expression of phosphatidylserine allows us to assume that apoptotic processes in the circulating platelets from the patients are limited.

Hemoglobin deoxygenation and methemoglobinemia prevent regulatory volume decrease in crucian carp (Carassius carassius) red blood cells.

Fish red blood cells (RBCs) exhibit an oxygen-dependent regulatory volume decrease (RVD) in hypoosmotic environment. In higher vertebrates, membrane-associated hemoglobin is involved in the regulation of osmotic ion movements across the cellular membrane. However, whether the hemoglobin conformational state plays a role in the regulation of osmotic responses in fish red blood cells is still not fully understood. We found that changes in hemoglobin conformation influence the pattern of the regulatory volume decrease in Carassius carassius red blood cells. In oxygenated cells (96.4 ± 3.7% oxygenated hemoglobin), the volume recovery was completed within 125 min. Deoxygenation of hemoglobin (96.5 ± 2.7% of deoxygenated hemoglobin) inhibited the volume decrease in hyposmotically swollen red blood cells. Reoxygenation restored regulatory volume decrease in cells within 5 min. Induced methemoglobinemia (48.4 ± 1.8% of methemoglobin and 41.3 ± 2.3% of deoxygenated hemoglobin) blocked the process of volume recovery and significantly decreased osmotic stability of red blood cells.

The sGC stimulator riociguat inhibits platelet function in washed platelets but not in whole blood.

BACKGROUND AND PURPOSE: Stimulation of soluble guanylyl cyclase (sGC) is a valuable therapeutic strategy for the treatment of several cardiovascular diseases. The sGC stimulator riociguat has been approved for the treatment of two forms of pulmonary hypertension. Platelets contain large amounts of sGC and play a key role in the regulation of haemostasis. Therefore, we investigated the effects of riociguat on platelet function. EXPERIMENTAL APPROACH: The effect of riociguat treatment on human platelet activation and aggregation was investigated. The sGC-specific effects of riociguat were determined by comparing wild-type and platelet-specific sGC-knockout mice. KEY RESULTS: Riociguat induced cGMP synthesis and subsequent PKG activation in human platelets, suggesting that the inhibitory effects are mediated by cGMP signalling. This finding was confirmed when sGC-knockout platelets were not inhibited by riociguat. In washed human platelets, 100 nM riociguat reduced ADP-induced GPIIb/IIIa activation, while a 10-fold higher concentration was required to reduce convulxin-stimulated GPIIb/IIIa activation. Riociguat inhibited ADP-induced platelet shape change and aggregation, while ATP-induced shape change remained unaffected. However, in PRP and whole blood, 50-100 µM riociguat was required to inhibit platelet activation and aggregation. Riociguat in combination with iloprost significantly inhibited platelet aggregation, even in whole blood. CONCLUSIONS AND IMPLICATIONS: Riociguat inhibits platelet activation in whole blood only at concentrations above 50 µM, while the plasma concentrations in riociguat-treated patients are 150 to 500 nM. This finding indicates that riociguat treatment does not affect platelet function in patients. Nevertheless, the possibility that riociguat acts synergistically with iloprost to inhibit platelet activation should be considered.

The fibrate gemfibrozil is a NO- and haem-independent activator of soluble guanylyl cyclase: in vitro studies.

BACKGROUND AND PURPOSE: Fibrates are a class of drugs widely used to treat dyslipidaemias. They regulate lipid metabolism and act as PPARα agonists. Clinical trials demonstrate that besides changes in lipid profiles, fibrates decrease the incidence of cardiovascular events, with gemfibrozil exhibiting the most pronounced benefit. This study aims to characterize the effect of gemfibrozil on the activity and function of soluble guanylyl cyclase (sGC), the key mediator of NO signalling. EXPERIMENTAL APPROACH: High-throughput screening of a drug library identified gemfibrozil as a direct sGC activator. Activation of sGC is unique to gemfibrozil and is not shared by other fibrates. KEY RESULTS: Gemfibrozil activated purified sGC, induced endothelium-independent relaxation of aortic rings and inhibited platelet aggregation. Gemfibrozil-dependent activation was absent when the sGC haem domain was deleted, but was significantly enhanced when sGC haem was lacking or oxidized. Oxidation of sGC haem enhanced the vasoactive and anti-platelet effects of gemfibrozil. Gemfibrozil competed with the haem-independent sGC activators ataciguat and cinaciguat. Computational modelling predicted that gemfibrozil occupies the space of the haem group and interacts with residues crucial for haem stabilization. This is consistent with structure-activity data which revealed an absolute requirement for gemfibrozil's carboxyl group. CONCLUSIONS AND IMPLICATIONS: These data suggest that in addition to altered lipid and lipoprotein state, the cardiovascular preventive benefits of gemfibrozil may derive from direct activation and protection of sGC function. A sGC-directed action may explain the more pronounced cardiovascular benefit of gemfibrozil observed over other fibrates and some of the described side effects of gemfibrozil.

Orai1-induced store-operated Ca(2+) entry enhances phospholipase activity and modulates canonical transient receptor potential channel 6 function in murine platelets.

BACKGROUND: Orai1, the major store-operated Ca(2+) entry (SOCE) channel in platelets, is not only critical for enhancing diverse signaling pathways, but may also regulate receptor-operated Ca(2+) entry (ROCE). Dynamic coupling of the Orai1 signalosome to canonical transient receptor potential channels (TRPCs) has been suggested as an essential step in the activation of SOCE and ROCE. However, the functional significance of the biochemical interaction between Orai and TRPC isoforms remains controversial. OBJECTIVE: We aimed to elucidate the role of Orai1 in diacylglycerol (DAG)-mediated ROCE. METHODS: Trpc6(-/-) , Orai1(-/-) and Orai1(-/-) /Trpc6(-/-) mice were generated, and their platelets were analyzed. RESULTS: Thapsigargin (TG)-induced SOCE was further reduced in Orai1(-/-) /Trpc6(-/-) platelets as compared with Orai1(-/-) platelets, thus revealing that TG-induced signaling pathways can activate TRPC6. Thapsigargin-induced SOCE leads to enhanced phospholipase C and D activity in wild-type platelets. The activity of both enzymes was significantly reduced in Orai1(-/-) platelets upon TG stimulation, whereas receptor-induced phospholipase activity was not affected. Furthermore, TG-induced and glycoprotein VI-mediated thromboxane A2 release was strongly dependent on Orai1-mediated SOCE. CONCLUSION: The regulation of TRPC6 activity can occur independently of the physical interaction with Orai1. TRPC6 operates in crosstalk with Orai1 through Orai1-induced DAG production via phospholipase activation. Orai1-induced DAG production and thromboxane release amplify the second phase of Ca(2+) signaling in platelets.

Dual role of the p38 MAPK/cPLA2 pathway in the regulation of platelet apoptosis induced by ABT-737 and strong platelet agonists.

p38 Mitogen-activated protein (MAP) kinase is involved in the apoptosis of nucleated cells. Although platelets are anucleated cells, apoptotic proteins have been shown to regulate platelet lifespan. However, the involvement of p38 MAP kinase in platelet apoptosis is not yet clearly defined. Therefore, we investigated the role of p38 MAP kinase in apoptosis induced by a mimetic of BH3-only proteins, ABT-737, and in apoptosis-like events induced by such strong platelet agonists as thrombin in combination with convulxin (Thr/Cvx), both of which result in p38 MAP kinase phosphorylation and activation. A p38 inhibitor (SB202190) inhibited the apoptotic events induced by ABT-737 but did not influence those induced by Thr/Cvx. The inhibitor also reduced the phosphorylation of cytosolic phospholipase A2 (cPLA2), an established p38 substrate, induced by ABT-737 or Thr/Cvx. ABT-737, but not Thr/Cvx, induced the caspase 3-dependent cleavage and inactivation of cPLA2. Thus, p38 MAPK promotes ABT-737-induced apoptosis by inhibiting the cPLA2/arachidonate pathway. We also show that arachidonic acid (AA) itself and in combination with Thr/Cvx or ABT-737 at low concentrations prevented apoptotic events, whereas at high concentrations it enhanced such events. Our data support the hypothesis that the p38 MAPK-triggered arachidonate pathway serves as a defense mechanism against apoptosis under physiological conditions.

Phosphorylation of CalDAG-GEFI by protein kinase A prevents Rap1b activation.

BACKGROUND: Signaling via protein kinase A (PKA) and protein kinase G (PKG) is critical for maintaining platelets in the resting state. Both kinases down-regulate the activity of the small GTPase Rap1b, a critical signaling switch for integrin activation and platelet aggregation. However, the mechanism of Rap1b regulation by PKA and PKG is largely unknown. OBJECTIVE: To identify the PKA phosphorylation sites in calcium and diacylglycerol-regulated guanine nucleotide exchange factor I (CalDAG-GEFI), the main GEF for Rap1b in platelets, and the effect of CalDAG-GEFI phosphorylation in Rap1b activation. METHODS: The phosphorylation sites in CalDAG-GEFI were identified by radio-active phosphate incorporation assay and mass spectrometry. Phospho-antibody was developed to detect CalDAG-GEFI phosphorylation in Western blots. Rap1b activation was detected by Rap1-GTP pull-down assay. RESULTS: S587 was identified as the major PKA phosphorylation site in CalDAG-GEFI, while S116/117 was weakly phosphorylated. Phosphorylation of S587 correlated with the inhibitory effect of PKA on Rap1b activation in platelets. In HEK293 cells, expression of a phospho-mimetic mutant of CalDAG-GEFI (S587D) abolished agonist-induced Rap1b activation. Mutation of S587 to alanine partially reversed the inhibitory effect of PKA signaling on Rap1b activation, while mutation of S116, S117 and S587 to alanine completely abolished the inhibitory effect of PKA on Rap1b activation. CONCLUSION: Our study strongly suggests that phosphorylation of CalDAG-GEFI is a critical mechanism by which PKA controls Rap1b-dependent platelet aggregation.

Downregulation of AKT reverses platinum resistance of human ovarian cancers in vitro.

Platinum resistance is the most crucial problem for treatment of ovarian cancer. Increasing evidence points towards AKT overexpression as a mechanistic reason for this clinical condition. The present study evaluates the effect of overexpression and downregulation of AKT on the sensitivity to cisplatin in a platinum-resistant human ovarian cancer cell line and the corresponding platinum-sensitive parental cell line. A2780 and A2780cis ovarian cancer cell lines were stably transfected with an AKT-sense and AKT-antisense plasmid. Successful transfection was evaluated by western blot analysis. Cytotoxic effects of cisplatin were evaluated by metabolic (MTT) and clonogenicity assays as well as by FACS analysis. AKT overexpression (confirmed by western blotting) converted platinum-sensitive A2780 into platinum-resistant cells as shown by MTT assay. Importantly, platinum resistance of A2780cis cells could be reversed by downregulation of AKT, as demonstrated by MTT and clonogenicity assays and FACS analysis. Our data provide strong evidence that cisplatin resistance in ovarian cancer is mediated by AKT overexpression and can be overcome by AKT downregulation, thus, providing a rationale for clinical phase II/III studies combining AKT inhibitors with cisplatin.

Hypertrophy and altered activity of the adrenal cortex in Homer 1 knockout mice.

Homer 1 gene products are involved in synaptic transmission and plasticity, and hence, distinct behavioral abnormalities, including anxiety- and depression-like behaviors, have been observed in Homer 1 knockout (KO) mice. Here we report that Homer 1 KO mice additionally exhibit a pronounced endocrine phenotype, displaying a profoundly increased adrenal gland weight and increased adrenal/body weight ratio. Histological examinations of Homer 1 deficient adrenal glands revealed an increased size of the adrenal cortex, especially the sizes of the zona fasciculata and zona glomerulosa. Moreover, the plasma corticosterone and aldosterone were higher in Homer 1 KO than wild-type (WT) mice while the plasma ACTH levels were not different between the genotypes. The in vivo ACTH test revealed that corticosterone and aldosterone plasma levels were higher in saline injected Homer 1 KO mice than in WT mice (saline injected mice served as controls for the respective groups of ACTH-injected animals), but the magnitude of steroid responses to ACTH was similar in both genotypes. In contrast, an in vitro experiment performed on isolated cells of adrenal cortex clearly showed increased production of both steroids in response to ACTH in Homer 1 KO cells, which is in line with an ~8-fold increase in the expression of ACTH receptor mRNA in the adrenal cortex of these mutants. These results, together with the detection of Homer 1 mRNA and protein in the adrenal cortex of WT mice, indicate that Homer 1 directly affects the steroidogenic function of the adrenal glands.

Differentiation of cGMP-dependent and -independent nitric oxide effects on platelet apoptosis and reactive oxygen species production using platelets lacking soluble guanylyl cyclase.

Platelet activation is an irreversible process resulting in platelet apoptosis and necrosis, and circulating platelets contain many components of the apoptotic machinery. Cyclic guanosine monophosphate (cGMP) generated by nitric oxide (NO) activated soluble guanylyl cyclase (sGC) plays a crucial role in preventing platelet activation. However, in addition to activation of sGC, cGMP-independent NO effects in platelets have been described. To differentiate between cGMP-dependent and -independent NO effects on platelet apoptosis and reactive oxygen species (ROS) production, we generated platelet-specific sGC-deficient mice (PS-GCKO). Platelet apoptosis was induced by a combination of thrombin/convulxin (Thr/Cvx) and assessed by phosphatidylserine (PS) surface exposure, and loss of the mitochondrial membrane potential. NO-induced inhibition of PS externalisation was mediated only by cGMP-dependent mechanisms. Inhibition of the mitochondrial membrane potential decrease at low NO concentration was also cGMP-dependent but became cGMP-independent at high NO concentrations. In contrast, inhibition of ROS formation at any NO concentration was mediated by cGMP-independent mechanisms, very likely due to direct radical scavenging. NO inhibits platelet apoptosis by cGMP-dependent mechanisms and ROS production by cGMP-independent mechanisms. The PS-GCKO mouse model is an important tool for the differentiation of cGMP-dependent and -independent NO effects on platelets.

NO-synthase-/NO-independent regulation of human and murine platelet soluble guanylyl cyclase activity.

OBJECTIVES: Platelets, specialized adhesive cells, play key roles in normal and pathological hemostasis through their ability to rapidly adhere to subendothelial matrix proteins (adhesion) and to other activated platelets (aggregation), functions which are inhibited by nitric oxide (NO). Platelets have been reported to be regulated not only by exogenous endothelium-derived NO, but also by two isoforms of NO synthase, endothelial (eNOS) and inducible (iNOS), endogenously expressed in platelets. however, data concerning expression, regulation and function of eNOS AND iNOS in platelets remain controversial. METHODS AND RESULTS: Using important positive (endothelial cells, stimulated macrophages) and negative (eNOS/iNOS knock-out mouse) controls, as well as human platelets highly purified by a newly developed protocol, we now demonstrate that human and mouse platelets do not contain eNOS/iNOS proteins or mRNA. NOS substrate (L-arginine), NOS inhibitors (L-NAME, L-NMMA), and eNOS/iNOS deficiency did not produce detectable functional effects on human and mouse platelets. von Willebrand factor (VWF)/ristocetin treatment of platelets increased cGMP by NO-independent activation of soluble guanylyl cyclase (sGC) which correlated with Src kinase-dependent phosphorylation of sGC beta(1)-subunit-Tyr(192). CONCLUSIONS: Human and mouse platelets do not express eNOS/iNOS. VWF/ristocetin-mediated activation of the sGC/cGMP signaling pathway may contribute to feedback platelet inhibition.

Insulin inhibition of platelet-endothelial interaction is mediated by insulin effects on endothelial cells without direct effects on platelets.

OBJECTIVES: Platelet hyperreactivity contributes to adverse vascular events in diabetes mellitus. It is unclear whether platelet hyperreactivity is due to impaired insulin effects directly on platelets and /or originates from endothelial cells. Here, acute effects of insulin on platelet activation and platelet-endothelial cell interactions were analyzed. METHODS AND RESULTS: Washed human platelets were treated with insulin alone or in combinations with thrombin, collagen and ADP. Insulin signaling was analyzed by intracellular phosphorylation markers of platelet activation (ERK, p38 MAPK, PKB) or inhibition (VASP), platelet aggregation, intracellular Ca(2+) levels, and platelet adhesion to collagen coated surfaces and endothelial cells under flow. Insulin up to 100 nm for 5 min did not change phosphorylation status of VASP, p38, ERK or PKB in platelets. Integrin alpha(IIb)beta(3) activation, P-selectin expression, aggregation, and platelet adhesion to collagen coated surfaces and endothelial cells under flow were not altered by insulin. An insulin receptor was detected on endothelial cells but not on human platelets. Insulin treatment decreased platelet adhesion to endothelial cells through insulin stimulation of endothelial NO production and NOS inhibition interfered with this process. CONCLUSIONS: Insulin exerts no direct acute effects on platelet function but inhibits platelet-endothelial interaction by insulin stimulation of endothelial NO production.

Localization of cGMP-dependent protein kinase type II in rat brain.

In brain, signaling pathways initiated by atrial natriuretic peptide, or transmitters which stimulate nitric oxide synthesis, increase cGMP as their second messenger. One important class of target molecules for cGMP is cGMP-dependent protein kinases, and in the present study, biochemical and immunocytochemical analyses demonstrate the widespread distribution of type II cGMP-dependent protein kinase in rat brain, from the cerebral cortex to the brainstem and cerebellum. Also, colocalization of cGMP-dependent protein kinase type II with its activator, cGMP, was found in several brain regions examined after in vitro stimulation of brain slices with sodium nitroprusside. In western blots, cGMP-dependent protein kinase type II was observed in all brain regions examined, although cerebellar cortex and pituitary contained comparatively less of the kinase. Immunocytochemistry revealed cGMP-dependent protein kinase type II in certain neurons, and occasionally in putative oligodendrocytes and astrocytes, however, its most striking and predominant localization was in neuropil. Electron microscopy examination of neuropil in the medial habenula showed localization of the kinase in both axon terminals and dendrites. As a membrane-associated protein, cGMP-dependent protein kinase type II often appeared to be transported to cell processes to a greater extent than being retained in the cell body. Thus, immunocytochemical labeling of cGMP-dependent protein kinase type II often did not coincide with the localization of kinase mRNA previously observed by others using in situ hybridization. We conclude that in contrast to cGMP-dependent protein kinase type I, which has a very restricted localization to cerebellar Purkinje cells and a few other sites, cGMP-dependent protein kinase type II is a very ubiquitous brain protein kinase and thus a more likely candidate for relaying myriad cGMP effects in brain requiring protein phosphorylation.

Activation of cardiac c-Jun NH(2)-terminal kinases and p38-mitogen-activated protein kinases with abrupt changes in hemodynamic load.

The role of mitogen-activated protein kinase (MAPK) pathways as signal transduction intermediates of hemodynamic stress leading to cardiac hypertrophy in the adult heart is not fully established. In a rat model of pressure-overload hypertrophy, we examined whether activation of MAPK pathways, namely, the extracellular signal-regulated protein kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), and the p38-MAPK pathways, occurs during rapid changes in hemodynamic load in vivo. A slight activation of ERK2 and marked increases in JNK1 and p38-MAPK activities were observed 30 minutes after aortic banding. The increase in p38-MAPK activity was accompanied by an increase in the phosphorylation of the p38 substrate MAPK-activated protein kinases 2 and 3. Activation of these kinases was coincident with an increase in phosphorylation of c-Jun and activating transcription factor-2 (ATF-2) and enhanced DNA binding of activator protein-1 factors. Thus, hemodynamic stress of the adult rat heart in vivo results in rapid activation of several parallel MAPK kinase cascades, particularly stress-activated MAPK and p38-MAPK and their target transcription factors c-Jun and ATF-2.

Immunofluorescent imaging of beta 1- and beta 2-adrenergic receptors in rat kidney.

BACKGROUND: beta-Adrenergic receptors (beta-ARs) are known to participate in the regulation of glomerular filtration, NaCl reabsorption, acid-base balance, and renin secretion; however, the precise histologic localization of beta-AR at putative signaling sites involved in these processes remains an open issue. METHODS: We used a set of subtype-specific rabbit antibodies to visualize beta(1)- and beta(2)-AR in rat kidney by immunohistochemistry and specified cells and segments of the nephron thought to be regulated by catecholamines. In addition, the relative proportion of beta-AR subtypes in cortical and medullary portions of rat kidney was determined by Western blotting and by competing [(125)I]-cyanopindolol binding with the beta(1)- or beta(2)-selective antagonists bisoprolol or ICI 118,551, respectively. RESULTS: Immunoreactivity for beta(1)-AR was found in mesangial cells, juxtaglomerular granular cells, the macula densa epithelium, proximal and distal tubular segments, and acid-secreting type A intercalated cells of the cortical and medullary collecting ducts. Immunoreactivity for beta(2)-AR was predominantly localized in the apical and subapical compartment of proximal and, to a lesser extent, distal tubular epithelia (suggesting interactions with luminal fluid catecholamines). Both subtypes were dense in the membranes of smooth muscle cells from renal arteries. Concordant data were obtained by radioligand binding and immunoblotting of membranes prepared from cortical and medullary portions of the kidney. CONCLUSION: Our data provide an immunohistochemical basis for the cellular targets of beta-adrenergic regulation of renal function. Moreover, they could help to devise therapeutic strategies directed at renal beta-ARs.

Presynaptic role of cGMP-dependent protein kinase during long-lasting potentiation.

Previous research has suggested that cGMP-dependent protein kinases (cGKs) may play a role in long-term potentiation in hippocampus, but their site of action has been unknown. We examined this question at synapses between pairs of hippocampal neurons in dissociated cell culture. Injection of a specific peptide inhibitor of cGK into the presynaptic but not the postsynaptic neuron blocked long-lasting potentiation induced by tetanic stimulation of the presynaptic neuron. As controls, injection of a scrambled peptide or a peptide inhibitor of cAMP-dependent protein kinase into either neuron did not block potentiation. Conversely, injection of the alpha isozyme of cGK type I into the presynaptic but not the postsynaptic neuron produced activity-dependent potentiation that did not require NMDA receptor activation. Evidence from Western blots, reverse transcription-PCR, activity assays, and immunocytochemistry indicates that endogenous cGK type I is present in the neurons, including presynaptic terminals. These results support the idea that cGK plays an important presynaptic role during the induction of long-lasting potentiation in hippocampal neurons.

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.

Distribution, cellular localization, and postnatal development of VASP and Mena expression in mouse tissues.

Vasodilator-stimulated phosphoprotein (VASP) and mammalian Enabled (Mena) are members of the proline-rich Ena/VASP protein family that links the cell membrane proteins, signal transduction pathways, and the actin cytoskeleton. VASP and Mena, substrates of cyclic nucleotide-dependent protein kinases, are associated in different cell types with microfilaments, focal adhesions, cell-cell contacts, and highly dynamic membrane regions. Here, the analysis of mRNA and protein expression, cellular localization, and postnatal development of VASP in different mouse tissues is reported and compared with that of Mena. The expression levels of VASP and Mena differ markedly among various tissues and cell types. The highest levels of VASP are observed in platelets, but stomach, intestine, spleen, lung, and blood vessels are also rich sources of VASP. Mena is abundantly expressed in brain, whereas it is not detectable in platelets and spleen. In intestine and stomach, prominent VASP and Mena immunoreactivity is detected in intestinal smooth muscle cells and blood vessels and cellular membranes of epithelial cells. In kidney, VASP and Mena are abundantly expressed in glomerular mesangial cells and in papilla. VASP and Mena immunoreactivity in heart is associated with blood vessels and with the intercalated discs of cardiac myocytes, where they colocalize with connexin-43. During postnatal development of heart, the level of VASP and Mena expression gradually decreases from neonatal to adult animals. The data demonstrate a clear colocalization of VASP and Mena in cells of stomach, intestine, kidney, and heart. These data and other recent developments suggest that proteins of the Ena/VASP family exert similar functions and may compensate for each other in these tissues.

KT5823 inhibits cGMP-dependent protein kinase activity in vitro but not in intact human platelets and rat mesangial cells.

Many signal transduction pathways are mediated by the second messengers cGMP and cAMP, cGMP- and cAMP-dependent protein kinases (cGK and PKA), phosphodiesterases, and ion channels. To distinguish among the different cGMP effectors, inhibitors of cGK and PKA have been developed including the K-252 compound KT5823 and the isoquinolinesulfonamide H89. KT5823, an in vitro inhibitor of cGK, has also been used in numerous studies with intact cells to implicate or rule out the involvement of this protein kinase in a given cellular response. However, the efficacy and specificity of KT5823 as cGK inhibitor in intact cells or tissues have never been demonstrated. Here, we analyzed the effects of both KT5823 and H89 on cyclic-nucleotide-mediated phosphorylation of vasodilator-stimulated phosphoprotein (VASP) in intact human platelets and rat mesangial cells. These two cell types both express high levels of cGK. KT5823 inhibited purified cGK. However, with both intact human platelets and rat mesangial cells, KT5823 failed to inhibit cGK-mediated serine 157 and serine 239 phosphorylation of VASP induced by nitric oxide, atrial natriuretic peptide, or the membrane-permeant cGMP analog, 8-pCPT-cGMP. KT5823 enhanced 8-pCPT-cGMP-stimulated VASP phosphorylation in platelets and did not inhibit forskolin-stimulated VASP phosphorylation in either platelets or mesangial cells. In contrast H89, an inhibitor of both PKA and cGK, clearly inhibited 8-pCPT-cGMP and forskolin-stimulated VASP phosphorylation in the two cell types. The data indicate that KT5823 inhibits purified cGK but does not affect a cGK-mediated response in the two different cell types expressing cGK I. These observations indicate that data that interpret the effects of KT5823 in intact cells as the major or only criteria supporting the involvement of cGK clearly need to be reconsidered.