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1.
Biochemistry (Mosc) ; 81(7): 691-9, 2016 Jul.
Article in English | MEDLINE | ID: mdl-27449615

ABSTRACT

The intracellular PI3K-AKT-mTOR pathway is involved in regulation of numerous important cell processes including cell growth, differentiation, and metabolism. The PI3Kα isoform has received particular attention as a novel molecular target in gene therapy, since this isoform plays critical roles in tumor progression and tumor blood flow and angiogenesis. However, the role of PI3Kα and other class I isoforms, i.e. PI3Kß, γ, δ, in the regulation of vascular tone and regional blood flow are largely unknown. We used novel isoform-specific PI3K inhibitors and mice deficient in both PI3Kγ and PI3Kδ (Pik3cg(-/-)/Pik3cd(-/-)) to define the putative contribution of PI3K isoform(s) to arterial vasoconstriction. Wire myography was used to measure isometric contractions of isolated murine mesenteric arterial rings. Phenylephrine-dependent contractions were inhibited by the pan PI3K inhibitors wortmannin (100 nM) and LY294002 (10 µM). These vasoconstrictions were also inhibited by the PI3Kα isoform inhibitors A66 (10 µM) and PI-103 (1 µM), but not by the PI3Kß isoform inhibitor TGX 221 (100 nM). Pik3cg(-/-)/Pik3cd(-/-)-arteries showed normal vasoconstriction. We conclude that PI3Kα is an important downstream element in vasoconstrictor GPCR signaling, which contributes to arterial vasocontraction via α1-adrenergic receptors. Our results highlight a regulatory role of PI3Kα in the cardiovascular system, which widens the spectrum of gene therapy approaches targeting PI3Kα in cancer cells and tumor angiogenesis and regional blood flow.


Subject(s)
Phosphatidylinositol 3-Kinases/metabolism , Androstadienes/pharmacology , Animals , Chromones/pharmacology , Class I Phosphatidylinositol 3-Kinases , Class Ib Phosphatidylinositol 3-Kinase/deficiency , Class Ib Phosphatidylinositol 3-Kinase/genetics , Furans/pharmacology , Mesenteric Arteries/physiology , Mice , Mice, Knockout , Morpholines/pharmacology , Neoplasms/blood supply , Neoplasms/pathology , Neoplasms/therapy , Neovascularization, Pathologic , Phenylephrine/pharmacology , Phosphatidylinositol 3-Kinases/deficiency , Phosphatidylinositol 3-Kinases/genetics , Phosphoinositide-3 Kinase Inhibitors , Pyridines/pharmacology , Pyrimidines/pharmacology , Pyrimidinones/pharmacology , Receptors, Adrenergic, alpha-1/metabolism , Signal Transduction/drug effects , Vasoconstriction/drug effects , Wortmannin
2.
Photodiagnosis Photodyn Ther ; 14: 178-84, 2016 Jun.
Article in English | MEDLINE | ID: mdl-27109055

ABSTRACT

BACKGROUND: Basal cell carcinoma (BCC) is a locally destructive form of skin cancer, mainly affecting Caucasians. In the last few years non-surgical treatments of BCC have become widely used and non-invasive methods for treatment monitoring and follow-up are therefore becoming increasingly warranted. The objective of this study was to investigate the utility of adjunct use of non-invasive optical coherence tomography (OCT) imaging for the detection of recurrent BCC over clinical and dermoscopic examination alone, in a real-world setting. METHODS: A total of 58 patients, previously treated with curettage and/or MAL-PDT for BCC, were included in the study. The patients were examined clinically and dermoscopically for recurrence by a dermatologist before joining the study. The included patients were then OCT scanned and if the OCT images raised suspicion of recurrent BCC the area was biopsied. RESULTS: In 6 cases the clinical examination revealed suspicions recurrent lesions and OCT correctly identified all of these (6/6). In 49 cases the follow-up examinations showed no clinical or dermoscopic signs of recurrence, but in 12.2% (6/49) of these cases the subsequent OCT examination revealed a subclinical recurrent BCC lesion. These were all confirmed by histology. In 3 cases both the clinical and the OCT diagnosis was unclear and recurrent BCC could not be ruled out, but histology showed no sign of malignancy. CONCLUSIONS: These results suggest that the adjunct use of OCT increases the detection rate of recurrent BCC over clinical/dermoscopy examination alone.


Subject(s)
Carcinoma, Basal Cell/diagnosis , Photochemotherapy , Skin Neoplasms/diagnosis , Tomography, Optical Coherence , Adult , Aged , Aged, 80 and over , Female , Follow-Up Studies , Humans , Male , Middle Aged , Photosensitizing Agents/pharmacology , Tomography, Optical Coherence/methods
3.
Acta Physiol (Oxf) ; 207(3): 546-64, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23253200

ABSTRACT

Recent preclinical data indicate that activators of transient receptor potential channels of the vanilloid receptor subtype 1 (TRPV1) may improve the outcome of ischaemic acute kidney injury (AKI). The underlying mechanisms are unclear, but may involve TRPV1 channels in dorsal root ganglion neurones that innervate the kidney. Recent data identified TRPV4, together with TRPV1, to serve as major calcium influx channels in endothelial cells. In these cells, gating of individual TRPV4 channels within a four-channel cluster provides elementary calcium influx (calcium sparklets) to open calcium-activated potassium channels and promote vasodilation. The TRPV receptors can also form heteromers that exhibit unique conductance and gating properties, further increasing their spatio-functional diversity. This review summarizes data on electrophysiological properties of TRPV1/4 and their modulation by endogenous channel agonists such as 20-HETE, phospholipase C and phosphatidylinositide 3-kinase (PI3 kinase). We review important roles of TRPV1 and TRPV4 in kidney physiology and renal ischaemia reperfusion injury; further studies are warranted to address renoprotective mechanism of vanilloid receptors in ischaemic AKI including the role of the capsaicin receptor TRPV1 in primary sensory nerves and/or endothelium. Particular attention should be paid to understand the kidneys' ability to respond to ischaemic stimuli after catheter-based renal denervation therapy in man, whereas the discovery of novel pharmacological TRPV modulators may be a successful strategy for better treatment of acute or chronic kidney failure.


Subject(s)
Kidney Diseases/metabolism , Kidney/metabolism , TRPV Cation Channels/metabolism , Acute Kidney Injury/metabolism , Acute Kidney Injury/physiopathology , Animals , Humans , Ion Channel Gating , Kidney/blood supply , Kidney/drug effects , Kidney/innervation , Kidney/physiopathology , Kidney Diseases/drug therapy , Kidney Diseases/etiology , Kidney Diseases/physiopathology , Ligands , Membrane Potentials , Renal Insufficiency/metabolism , Renal Insufficiency/physiopathology , Reperfusion Injury/metabolism , Reperfusion Injury/physiopathology , Signal Transduction , TRPV Cation Channels/drug effects
4.
Oncogene ; 29(46): 6184-92, 2010 Nov 18.
Article in English | MEDLINE | ID: mdl-20729911

ABSTRACT

Microtubule-interfering cancer drugs such as paclitaxel (PTX) often cause chemoresistance and severe side effects, including neurotoxicity. To explore potentially novel antineoplastic molecular targets, we investigated the cellular response of breast carcinoma cells to short hairpin(sh)RNA-mediated depletion of the centrosomal protein transforming acidic coiled coil (TACC) 3, an Aurora A kinase target expressed during mitosis. Unlike PTX, knockdown of TACC3 did not trigger a cell death response, but instead resulted in a progressive loss of the pro-apoptotic Bcl-2 protein Bim that links microtubule integrity to spindle poison-induced cell death. Interestingly, TACC3-depleted cells arrested in G1 through a cellular senescence program characterized by the upregulation of nuclear p21(WAF), downregulation of the retinoblastoma protein and extracellular signal-regulated kinase 1/2, formation of HP1γ (phospho-Ser83)-positive senescence-associated heterochromatic foci and increased senescence-associated ß-galactosidase activity. Remarkably, the onset of senescence following TACC3 knockdown was strongly accelerated in the presence of non-toxic PTX concentrations. Thus, we conclude that mitotic spindle stress is a major trigger of premature senescence and propose that the combined targeting of the centrosomal Aurora A-TACC3 axis together with drugs interfering with microtubule dynamics may efficiently improve the chemosensitivity of cancer cells.


Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Cellular Senescence/drug effects , Microtubule-Associated Proteins/physiology , Paclitaxel/pharmacology , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p21/physiology , Doxorubicin/pharmacology , Extracellular Signal-Regulated MAP Kinases/physiology , Humans , Microtubules/drug effects
5.
Anticancer Res ; 29(9): 3669-74, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19667163

ABSTRACT

BACKGROUND: Reduced serum 25-hydroxyvitamin D3 (25(OH)D) levels are associated with an increased incidence and an unfavorable outcome of various types of cancer. However, the influence of serum 25(OH)D on the incidence and outcome of patients with malignant melanoma is unknown. PATIENTS AND METHODS: The association between serum 25(OH)D levels and clinical and histopathological data among 205 patients with malignant melanoma was examined. Additionally, 141 healthy controls were investigated. All the blood samples were taken between October and April to minimize seasonal variations; basal serum 25(OH)D levels were analyzed using the LIAISON 25-OH Vitamin D-Assay (DiaSorin, Dietzenbach, Germany). The study started in 1997. The patients were observed until death or March 2007, whichever came first. RESULTS: Serum 25(OH)D levels were significantly reduced in stage IV melanoma patients as compared to stage I melanoma patients (p=0.006). A trend toward a greater tumor thickness of the primary cutaneous melanomas was seen in the patients with low (<10 ng/ml) serum 25(OH)D levels (median: 2.55 mm) as compared to those with 25(OH)D serum levels >20 ng/ml (median: 1.5 mm), although this difference was not statistically significant (p=0.078). The patients with low 25(OH)D serum levels (<10 ng/ml) had earlier distant metastatic disease (median: 24.37 months) as compared to those with 25(OH)D serum levels >20 ng/ml (median: 29.47 months), although this difference was also not statistically significant (p=0.641). CONCLUSION: Among the patients with malignant melanoma, significantly reduced serum 25(OH)D levels were found in the stage IV patients as compared to stage I patients, and those with low 25(OH)D serum levels (<10 ng/ml) may develop earlier distant metastatic disease compared to those with higher 25(OH)D serum levels (>20 ng/ml). Further study of the vitamin D pathway and its influence on pathogenesis and progression of malignant melanoma is warranted.


Subject(s)
Melanoma/blood , Skin Neoplasms/blood , Vitamin D/analogs & derivatives , Adolescent , Adult , Aged , Female , Humans , Male , Melanoma/pathology , Middle Aged , Neoplasm Metastasis , Seasons , Skin Neoplasms/pathology , Sunlight , Vitamin D/blood , Young Adult
6.
Br J Dermatol ; 160(5): 1026-33, 2009 May.
Article in English | MEDLINE | ID: mdl-19183171

ABSTRACT

BACKGROUND: Accurate assessment of tumour size is important when planning treatment of nonmelanoma skin cancer (NMSC). Imaging with optical coherence tomography (OCT) has the potential to diagnose and measure depth of NMSC. OBJECTIVES: To compare accuracy of mean tumour thickness measurement in NMSC tumours < 2 mm of depth using OCT and 20-MHz high-frequency ultrasound (HFUS). In addition, OCT morphology of NMSC was studied in OCT images and the influence of histological and colorimetric values on the quality and penetration depth in OCT images was estimated. METHODS: In total, 93 patients were scanned and 34 lesions [23 basal cell carcinoma (BCC) and 11 actinic keratosis (AK) lesions] < 2 mm thick and easily identified in OCT images were studied. OCT and HFUS were compared with biopsies. The influence of skin pigmentation and infiltration analgesia on OCT image quality was studied. Skin colour was measured with a colorimeter. RESULTS: OCT presented narrower limits of agreement than HFUS. Both methods overestimated thickness but OCT was significantly less biased (0.392 mm vs. 0.713 mm). No relation between OCT penetration depth and skin colour was found. CONCLUSIONS: OCT appears more precise and less biased than HFUS for thickness measurement in AK and BCC lesions < 2 mm, but both OCT and especially HFUS tended to overestimate tumour thickness.


Subject(s)
Carcinoma, Basal Cell/pathology , Keratosis, Actinic/pathology , Skin Neoplasms/pathology , Adult , Aged , Aged, 80 and over , Carcinoma, Basal Cell/diagnostic imaging , Female , Humans , Image Enhancement/instrumentation , Keratosis, Actinic/diagnostic imaging , Male , Middle Aged , Sensitivity and Specificity , Skin Neoplasms/diagnostic imaging , Tomography, Optical Coherence/instrumentation , Tumor Burden , Ultrasonography
7.
Eur J Nucl Med Mol Imaging ; 36(6): 928-37, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19153733

ABSTRACT

PURPOSE: We compared the outcome of a 1-day and a 2-day sentinel node (SN) biopsy procedure, evaluated in terms of lymphoscintigraphic, surgical and pathological findings. METHODS: We studied 476 patients with melanoma from two melanoma centres using static scintigraphy and blue dye. A proportional odds model was used for statistical analysis. RESULTS: The number of SNs visualized at scintigraphy increased significantly with time from injection to scintigraphy and activity left in the patient at scintigraphy, and depended on the melanoma location. The number of SNs removed at surgery increased with the number of SNs visualized at scintigraphy and time from injection to surgery. The frequency of nodal metastasis increased with increasing thickness and Clark level of the melanoma, and was highest for two SNs visualized at scintigraphy. CONCLUSION: This study showed that early vs. late imaging and surgery do make a difference on the outcome of the SN procedure and confirmed the importance of the scintigraphic visualization of all true SNs.


Subject(s)
Melanoma/diagnosis , Melanoma/surgery , Sentinel Lymph Node Biopsy/methods , Adolescent , Adult , Aged , Aged, 80 and over , Female , Humans , Male , Melanoma/diagnostic imaging , Melanoma/pathology , Middle Aged , Radionuclide Imaging , Time Factors
8.
J Eur Acad Dermatol Venereol ; 22(12): 1458-64, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18811603

ABSTRACT

BACKGROUND: Optical coherence tomography (OCT) is a non-invasive optical imaging technique with a micrometer resolution that may potentially offer real-time bedside imaging of sufficient detail to allow for morphological discrimination between different types of bullae. OBJECTIVE: To explore the potential of OCT in bullous skin disorders by looking at a set of patients with skin blisters of known origin and study the OCT images for possible hallmarks of the blistering level. MATERIALS AND METHODS: OCT provides cross-sectional, tomographic images of the skin. A consecutive series of patients were recruited and their lesions imaged by OCT: 3 patients with bullous pemphigoid (BP), 1 patient with extensive bullae following burns, 1 patient with pemphigus, 1 patient with subcorneal pustular dermatosis, and a patient with Dariers disease. The latter two were included due to similarity to pemphigus with respect to the level of defect cell adhesion. RESULTS: In OCT images, BP bullae are easily depicted as dark, ovoid to round well-demarquated areas, and BP bulla morphology is clearly different from the burn blisters and the pemphigus-like disease with respect to the blistering level. DISCUSSION: Differentiation of epidermal and subepidermal blisters is demonstrated using OCT. The variation within pemphigoid lesions and pemphigus-like diseases is however too subtle to allow for differential diagnosis; this may be ascribed to limited resolution. Enhanced resolution of OCT may overcome this obstacle.


Subject(s)
Skin Diseases, Vesiculobullous/pathology , Aged , Cross-Sectional Studies , Female , Humans , Male , Middle Aged , Skin Diseases, Vesiculobullous/diagnosis , Tomography, Optical Coherence
9.
Oncogene ; 27(1): 116-25, 2008 Jan 03.
Article in English | MEDLINE | ID: mdl-17599038

ABSTRACT

Regulators of the mitotic spindle apparatus are attractive cellular targets for antitumor therapy. The centrosomal protein transforming acidic coiled coil (TACC) 3 is required for spindle assembly and proper chromosome segregation. In this study, we employed an inducible RNA interference approach to downregulate TACC3 expression. We show that TACC3 knock-down in NIH3T3 fibroblasts caused aneuploidy, but failed to overtly impair mitotic progression. TACC3 depletion rather triggered a postmitotic p53-p21(WAF) pathway and led to a reversible cell cycle arrest. Similar effects were induced by low concentrations of paclitaxel, a spindle poison used in antitumor therapy. Interestingly, however, and unlike in TACC3-proficient cells, paclitaxel was able to induce strong polyploidy and subsequent apoptosis in TACC3-depleted cells. Even though paclitaxel treatment was associated with the activation of the survival kinase Akt and an antiapoptotic expression of cytoplasmic p21(WAF) and cyclin D1, this inhibition of cell death was abrogated by depletion of TACC3. Thus, our data identify TACC3 as a potential target to overcome p21(WAF)-associated protection of transformed cells against paclitaxel-induced cell death.


Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Carrier Proteins/genetics , Cell Cycle/genetics , Cyclin-Dependent Kinase Inhibitor p21/physiology , Fetal Proteins/deficiency , Fetal Proteins/genetics , Paclitaxel/pharmacology , Animals , Apoptosis/drug effects , Apoptosis/physiology , Carrier Proteins/physiology , Cell Death/drug effects , Cell Death/genetics , Down-Regulation/genetics , Fetal Proteins/physiology , Humans , Mice , Microtubule-Associated Proteins , NIH 3T3 Cells , Tumor Suppressor Protein p53/physiology
10.
Biochem J ; 380(Pt 3): 831-6, 2004 Jun 15.
Article in English | MEDLINE | ID: mdl-15025562

ABSTRACT

Previously, we have shown that the human insulin receptor (IR) interacts with G(i)2, independent of tyrosine kinase activity and stimulates NADPH oxidase via the Galpha subunit of G(i)2. We have now investigated the regulatory role of G(i)2-proteins in IR function. For the experiments, isolated IRs from plasma membranes of human fat cells were used. The activation of IR autophosphorylation by insulin was blocked by G-protein inactivation through GDPbetaS (guanosine 5'-[beta-thio]disphosphate). Consistently, activation of G-proteins by micromolar concentrations of GTPgammaS (guanosine 5'-[gamma-thio]triphosphate) induced receptor autophosphorylation 5-fold over baseline and increased insulin-induced autophosphorylation by 3-fold. In the presence of 10 microM GTPgammaS, insulin was active at picomolar concentrations, indicating that insulin acted via its cognate receptor. Pretreatment of the plasma membranes with pertussis toxin prevented insulin- and GTPgammaS-induced autophosphorylation, but did not disrupt the IR-G(i)2 complex. The functional nature of the IR-G(i)2 complex was made evident by insulin's ability to increase association of G(i)2 with the IR. This leads to an augmentation of maximal receptor autophosphorylation induced by insulin and GTPgammaS. The specificity of this mechanism was further demonstrated by the use of isolated preactivated G-proteins. Addition of G(i)2alpha and Gbetagamma mimicked maximal response of insulin, whereas Galphas or Galphao had no stimulatory effect. These results define a novel mechanism by which insulin signalling mediates tyrosine kinase activity and autophosphorylation of the IR through recruitment of G(i)-proteins.


Subject(s)
GTP-Binding Protein alpha Subunits, Gi-Go/physiology , Receptor, Insulin/metabolism , Adipocytes/chemistry , Adipocytes/enzymology , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , GTP-Binding Protein beta Subunits/metabolism , GTP-Binding Protein gamma Subunits/metabolism , Guanine Nucleotides/pharmacology , Humans , Ligands , Pertussis Toxin/pharmacology , Phosphorylation/drug effects , Receptor Protein-Tyrosine Kinases/metabolism , Recombinant Proteins/metabolism , Signal Transduction/drug effects , Signal Transduction/physiology
11.
FASEB J ; 17(1): 38-40, 2003 Jan.
Article in English | MEDLINE | ID: mdl-12424219

ABSTRACT

Recent findings indicate that platelet-derived growth factor (PDGF) plays a role in the generation of reactive oxygen species (ROS) as second messengers in smooth muscle cells (SMC). To identify the source and signal transduction pathway of ROS formation in SMC, we investigated PDGF-induced ROS formation. Stimulation of SMC with PDGF resulted in a rapid increase of ROS production. Using an inactivating antibody, we identified the increase to be dependent on p22phox, a NAD(P)H-oxidase subunit. ROS release was completely inhibited by the Gi protein inhibitor PTX as well as an antibody against Galphai1,2, however, not by antibodies against Galphai3/0, Gas, and Gbeta1beta2. The effect of PDGF on ROS production in SMC membranes could likewise be mimicked by the use of a recombinant Galphai2 subunit but not by Galphai3, Galphai0, Gas, and Gbetagamma subunits. Immunoaffinity chromatography demonstrated coupling of Galphai1,2 to the PDGF a-receptor, which, after preincubation of the SMC membranes with PDGF, was increased in the absence of GTPgammaS but decreased in the presence of GTPgammaS and prevented by PTX treatment. These data define a novel G protein-dependent mechanism by which PDGF signaling is transduced through direct coupling of the Gai1,2 subunit of the trimeric G proteins to the PDGF tyrosine kinase receptor.


Subject(s)
GTP-Binding Protein alpha Subunits, Gi-Go/physiology , Membrane Transport Proteins , Muscle, Smooth, Vascular/enzymology , NADH, NADPH Oxidoreductases/metabolism , Platelet-Derived Growth Factor/pharmacology , Proto-Oncogene Proteins/physiology , Reactive Oxygen Species/metabolism , Animals , Enzyme Activation , GTP-Binding Protein alpha Subunit, Gi2 , Models, Biological , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/metabolism , NADPH Dehydrogenase/physiology , NADPH Oxidases , Phosphoproteins/physiology , Receptor, Platelet-Derived Growth Factor alpha/metabolism , Signal Transduction
12.
Mol Cell Biol ; 21(24): 8452-60, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11713280

ABSTRACT

Costimulation of G protein-coupled receptors (GPCRs) may result in cross talk interactions between their downstream signaling pathways. Stimulation of GPCRs may also lead to cross talk regulation of receptor tyrosine kinase signaling and thereby to activation of mitogen-activated protein kinase (MAPK). In COS-7 cells, we investigated the interactions between two particular mitogenic receptor pathways, the endogenously expressed beta-adrenergic receptor (beta-AR) and the transiently transfected human bradykinin (BK) B(2) receptor (B(2)R). When beta-AR and B(2)R are costimulated, we found two different cross talk mechanisms. First, the predominantly G(q) protein-coupled B(2)R is enabled to activate a G(i) protein and, subsequently, type II adenylate cyclase. This results in augmentation of beta-AR-mediated cyclic AMP (cAMP) accumulation by BK, which alone is unable to increase the cAMP level. Second, independently of BK-induced superactivation of the cAMP system, costimulation of beta-AR leads to protein kinase A-mediated blockade of phospholipase C activation by BK. Thereby, the pathway from B(2)R to MAPK, which essentially involves protein kinase C activation, is selectively switched off. The MAPK activation in response to isoproterenol was not affected due to costimulation. Furthermore, in the presence of isoproterenol, BK lost its ability to stimulate DNA synthesis in COS-7 cells. Thus, our findings might establish a novel paradigm: cooperation between simultaneously activated mitogenic pathways may prevent multiple stimulation of MAPK activity and increased cell growth.


Subject(s)
Guanosine Triphosphate/analogs & derivatives , Receptors, Adrenergic, beta/metabolism , Receptors, Bradykinin/metabolism , Affinity Labels/pharmacology , Animals , Azides/pharmacology , COS Cells , Cyclic AMP/metabolism , DNA/metabolism , Dose-Response Relationship, Drug , Enzyme Activation , ErbB Receptors/metabolism , Guanosine Triphosphate/pharmacology , Immunohistochemistry , Isoproterenol/pharmacology , MAP Kinase Signaling System , Models, Biological , Phosphatidylinositols/metabolism , Phosphorylation , Protein Binding , Time Factors , Transcriptional Activation , Transfection , Tyrosine/metabolism
13.
Circ Res ; 89(8): 692-9, 2001 Oct 12.
Article in English | MEDLINE | ID: mdl-11597992

ABSTRACT

Heterodimeric class I phosphoinositide 3-kinase (PI3K) has been shown to be involved in the stimulation of voltage-gated Ca(2+) channels by various mediators. In this study, we bring evidences that vascular L-type Ca(2+) channels can be modulated by both tyrosine kinase-regulated class Ia and G protein-regulated class Ib PI3Ks. Purified recombinant PI3Ks increased the peak Ca(2+) channel current density when applied intracellularly. Furthermore, PI3Kalpha-, beta-, and delta-mediated stimulations of Ca(2+) channel currents were increased by preactivation by a phosphotyrosyl peptide, whereas PI3Kgamma- and beta-mediated effects were increased by Gbetagamma. In freshly isolated and cultured vascular myocytes, angiotensin II and Gbetagamma stimulated L-type Ca(2+) channel current. In contrast, platelet-derived growth factor (PDGF)-BB and the phosphotyrosyl peptide did not stimulate Ca(2+) channel current in freshly isolated cells despite the presence of endogenous PDGF receptors and PI3Kalpha and PI3Kgamma. Interestingly, when endogenous PI3Kbeta expression arose in cultured myocytes, both PDGF and phosphotyrosyl peptide stimulated Ca(2+) channels through PI3Kbeta, as revealed by the inhibitory effect of an anti-PI3Kbeta antibody. These results suggest that endogenous PI3Kbeta but not PI3Kalpha is specifically involved in PDGF receptor-induced stimulation of Ca(2+) channels and that different isoforms of PI3K regulate physiological increases of Ca(2+) influx in vascular myocytes stimulated by vasoconstrictor or growth factor.


Subject(s)
Calcium Channels, L-Type/metabolism , Muscle, Smooth, Vascular/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Angiotensin II/pharmacology , Animals , Barium/metabolism , Barium/pharmacology , Becaplermin , Calcium/metabolism , Calcium Channels, L-Type/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Gene Products, env/metabolism , Heterotrimeric GTP-Binding Proteins/metabolism , Ion Transport/drug effects , Iontophoresis , Isoenzymes/genetics , Isoenzymes/metabolism , Isoenzymes/pharmacology , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/drug effects , Patch-Clamp Techniques , Peptide Fragments/metabolism , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/pharmacology , Platelet-Derived Growth Factor/pharmacology , Proto-Oncogene Proteins c-sis , Rats , Rats, Wistar , Receptors, Platelet-Derived Growth Factor/metabolism , Transfection , Vasoconstrictor Agents/pharmacology
14.
J Biol Chem ; 276(35): 32545-51, 2001 Aug 31.
Article in English | MEDLINE | ID: mdl-11443116

ABSTRACT

Previous results have shown that in rat portal vein myocytes the betagamma dimer of the G(13) protein transduces the angiotensin II-induced stimulation of calcium channels and increase in intracellular Ca(2+) concentration through activation of phosphoinositide 3-kinase (PI3K). In the present work we determined which class I PI3K isoforms were involved in this regulation. Western blot analysis indicated that rat portal vein myocytes expressed only PI3Kalpha and PI3Kgamma and no other class I PI3K isoforms. In the intracellular presence of an anti-p110gamma antibody infused by the patch clamp pipette, both angiotensin II- and Gbetagamma-mediated stimulation of Ca(2+) channel current were inhibited, whereas intracellular application of an anti-p110alpha antibody had no effect. The anti-PI3Kgamma antibody also inhibited the angiotensin II- and Gbetagamma-induced production of phosphatidylinositol 3,4,5-trisphosphate. In Indo-1 loaded cells, the angiotensin II-induced increase in [Ca(2+)](i) was inhibited by intracellular application of the anti-PI3Kgamma antibody, whereas the anti-PI3Kalpha antibody had no effect. The specificity of the anti-PI3Kgamma antibody used in functional experiments was ascertained by showing that this antibody did not recognize recombinant PI3Kalpha in Western blot experiments. Moreover, anti-PI3Kgamma antibody inhibited the stimulatory effect of intracellularly infused recombinant PI3Kgamma on Ca(2+) channel current without altering the effect of recombinant PI3Kalpha. Our results show that, although both PI3Kgamma and PI3Kalpha are expressed in vascular myocytes, the angiotensin II-induced stimulation of vascular L-type calcium channel and increase of [Ca(2+)](i) involves only the PI3Kgamma isoform.


Subject(s)
Angiotensin II/pharmacology , Calcium Channels, L-Type/physiology , Isoenzymes/metabolism , Muscle, Smooth, Vascular/physiology , Phosphatidylinositol 3-Kinases/metabolism , Animals , Antibodies/pharmacology , Barium/pharmacology , Blotting, Western , Calcium/metabolism , Calcium Channels, L-Type/drug effects , Cell Membrane/drug effects , Cell Membrane/enzymology , Cell Membrane/physiology , Class Ib Phosphatidylinositol 3-Kinase , In Vitro Techniques , Isoenzymes/isolation & purification , Kinetics , Membrane Potentials/drug effects , Membrane Potentials/physiology , Microsomes/enzymology , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/drug effects , Patch-Clamp Techniques , Phorbol 12,13-Dibutyrate/pharmacology , Phosphatidylinositol 3-Kinases/isolation & purification , Portal Vein/physiology , Protein Subunits , Rats , Recombinant Proteins/metabolism
15.
Mol Endocrinol ; 15(7): 1211-21, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11435619

ABSTRACT

L-type calcium channels mediate depolarization-induced calcium influx in insulin-secreting cells and are thought to be modulated by G protein-coupled receptors (GPCRs). The major fraction of L-type alpha1-subunits in pancreatic beta-cells is of the neuroendocrine subtype (CaV1.3 or alpha1D). Here we studied the biophysical properties and receptor regulation of a CaV1.3 subunit previously cloned from HIT-T15 cells. In doing so, we compared this neuroendocrine CaV1.3 channel with the cardiac L-type channel CaV1.2a (or alpha1C-a) after expression together with alpha2delta- and beta3-subunits in Xenopus oocytes. Both the current voltage relation and voltage dependence of inactivation for the neuroendocrine CaV1.3 channel were shifted to more negative potentials compared with the cardiac CaV1.2 channel. In addition, the CaV1.3 channel activated and inactivated more rapidly than the CaV1.2a channel. Both subtypes showed a similar sensitivity to the dihydropyridine (+)isradipine. More interestingly, the CaV1.3 channels were found to be stimulated by ligand-bound G(i)/G(o)-coupled GPCRs whereas a neuronal CaV2.2 (or alpha1B) channel was inhibited. The observed receptor-induced stimulation of CaV1.3 channels could be mimicked by phorbol-12-myristate-13-acetate and was sensitive to inhibitors of protein kinases, but not to the phosphoinositol-3-kinase-inhibitor wortmannin, pointing to serine/threonine kinase-dependent regulation. Taken together, we describe a neuroendocrine L-type CaV1.3 calcium channel that is stimulated by G(i)/G(o)-coupled GPCRs and differs significantly in distinct biophysical characteristics from the cardiac subtype (CaV1.2a), suggesting that the channels have different roles in native cells.


Subject(s)
Calcium Channels, L-Type/genetics , Calcium Channels/genetics , Ion Channel Gating/physiology , Androstadienes/pharmacology , Animals , Barium/metabolism , Calcium Channels/drug effects , Calcium Channels/physiology , Calcium Channels, L-Type/drug effects , Calcium Channels, L-Type/physiology , Dihydropyridines/pharmacology , Electric Conductivity , Enkephalin, Ala(2)-MePhe(4)-Gly(5)-/pharmacology , Enzyme Inhibitors/pharmacology , Female , GTP-Binding Proteins/physiology , Gene Expression , Isradipine/pharmacology , Myocardium/chemistry , Neurons/chemistry , Oocytes/metabolism , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase Inhibitors , Receptors, Opioid, mu/agonists , Tetradecanoylphorbol Acetate/pharmacology , Wortmannin , Xenopus laevis
16.
J Neurophysiol ; 85(2): 816-27, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11160515

ABSTRACT

Voltage-dependent calcium channels (VDCCs) are multimeric complexes composed of a pore-forming alpha(1) subunit together with several accessory subunits, including alpha(2)delta, beta, and, in some cases, gamma subunits. A family of VDCCs known as the L-type channels are formed specifically from alpha(1S) (skeletal muscle), alpha(1C) (in heart and brain), alpha(1D) (mainly in brain, heart, and endocrine tissue), and alpha(1F) (retina). Neuroendocrine L-type currents have a significant role in the control of neurosecretion and can be inhibited by GTP-binding (G-) proteins. However, the subunit composition of the VDCCs underlying these G-protein-regulated neuroendocrine L-type currents is unknown. To investigate the biophysical and pharmacological properties and role of G-protein modulation of alpha(1D) calcium channels, we have examined calcium channel currents formed by the human neuronal L-type alpha(1D) subunit, co-expressed with alpha(2)delta-1 and beta(3a), stably expressed in a human embryonic kidney (HEK) 293 cell line, using whole cell and perforated patch-clamp techniques. The alpha(1D)-expressing cell line exhibited L-type currents with typical characteristics. The currents were high-voltage activated (peak at +20 mV in 20 mM Ba2+) and showed little inactivation in external Ba2+, while displaying rapid inactivation kinetics in external Ca2+. The L-type currents were inhibited by the 1,4 dihydropyridine (DHP) antagonists nifedipine and nicardipine and were enhanced by the DHP agonist BayK S-(-)8644. However, alpha(1D) L-type currents were not modulated by activation of a number of G-protein pathways. Activation of endogenous somatostatin receptor subtype 2 (sst2) by somatostatin-14 or activation of transiently transfected rat D2 dopamine receptors (rD2(long)) by quinpirole had no effect. Direct activation of G-proteins by the nonhydrolyzable GTP analogue, guanosine 5'-0-(3-thiotriphospate) also had no effect on the alpha(1D) currents. In contrast, in the same system, N-type currents, formed from transiently transfected alpha(1B)/alpha(2)delta-1/beta(3), showed strong G-protein-mediated inhibition. Furthermore, the I-II loop from the alpha(1D) clone, expressed as a glutathione-S-transferase (GST) fusion protein, did not bind Gbetagamma, unlike the alpha(1B) I-II loop fusion protein. These data show that the biophysical and pharmacological properties of recombinant human alpha(1D) L-type currents are similar to alpha(1C) currents, and these currents are also resistant to modulation by G(i/o)-linked G-protein-coupled receptors.


Subject(s)
Calcium Channels, L-Type/physiology , Neurons/metabolism , 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester/pharmacology , Calcium Channel Agonists/pharmacology , Calcium Channels/metabolism , Calcium Channels, L-Type/drug effects , Cell Line , Dihydropyridines/agonists , Dihydropyridines/antagonists & inhibitors , Dihydropyridines/pharmacology , Electric Conductivity , GTP-Binding Proteins/physiology , Glutathione Transferase/metabolism , Humans , Protein Isoforms/metabolism , Recombinant Fusion Proteins/metabolism
17.
Eur J Biochem ; 267(24): 7102-8, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11106421

ABSTRACT

Human platelets contain a Na+/H+ exchanger (NHE) that regulates the cytosolic pH. The role of trimeric G-proteins in NHE control was investigated in plasma membrane vesicles by measuring exchange of intravesicular protons for extravesicular Na+. Exchange was saturable, independent of membrane potential and inhibited by ethylisopropyl amiloride (Ki 0.05 micromol.L-1), demonstrating the involvement of NHE-1. The G-protein activators AlF4- and GMP-P(NH)P reduced exchange by increasing the Km for Na+ from 11.3 +/- 2.1 mM to 21.6 +/- 1.4 mM (AlF4-) and 19.8 +/- 1.1 mM (GMP-P(NH)P), leaving Vmax and the Hill coefficient unchanged. This effect was abolished by inhibitors of Gi-proteins (N-ethylmaleimide, holoenzyme- and A-protomer of pertussis toxin) and by an anti-Galpha Ig and GDP(beta)S. Activation of Gi-proteins by mastoparan and its synthetic analogue Mas7 also strongly reduced NHE activity. These data show that in platelets NHE-1 is under negative control of the Gi-family of trimeric G-proteins.


Subject(s)
GTP-Binding Proteins/metabolism , Sodium-Hydrogen Exchangers/metabolism , Acridine Orange , Cell Membrane/metabolism , Spectrometry, Fluorescence
18.
J Biol Chem ; 275(18): 13746-54, 2000 May 05.
Article in English | MEDLINE | ID: mdl-10788495

ABSTRACT

In this study, Gbeta specificity in the regulation of Gbetagamma-sensitive phosphoinositide 3-kinases (PI3Ks) and phospholipase Cbeta (PLCbeta) isozymes was examined. Recombinant mammalian Gbeta(1-3)gamma(2) complexes purified from Sf9 membranes stimulated PI3Kgamma lipid kinase activity with similar potency (10-30 nm) and efficacy, whereas transducin Gbetagamma was less potent. Functionally active Gbeta(5)gamma(2) dimers were purified from Sf9 cell membranes following coexpression of Gbeta(5) and Ggamma(2-His). This preparation as well as Gbeta(1)gamma(2-His) supported pertussis toxin-mediated ADP-ribosylation of Galpha(i1). Gbeta(1)gamma(2-His) stimulated PI3Kgamma lipid and protein kinase activities at nanomolar concentrations, whereas Gbeta(5)gamma(2-His) had no effect. Accordingly, Gbeta(1)gamma(2-His), but not Gbeta(5)gamma(2-His), significantly stimulated the lipid kinase activity of PI3Kbeta in the presence or absence of tyrosine-phosphorylated peptides derived from the p85-binding domain of the platelet derived-growth factor receptor. Conversely, both preparations were able to stimulate PLCbeta(2) and PLCbeta(1). However, Gbeta(1)gamma(2-His), but not Gbeta(5)gamma(2-His), activated PLCbeta(3). Experimental evidence suggests that the mechanism of Gbeta(5)-dependent effector selectivity may differ between PI3K and PLCbeta. In conclusion, these data indicate that Gbeta subunits are able to discriminate among effectors independently of Galpha due to selective protein-protein interaction.


Subject(s)
GTP-Binding Protein beta Subunits , GTP-Binding Protein gamma Subunits , GTP-Binding Proteins/metabolism , Heterotrimeric GTP-Binding Proteins , Isoenzymes/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phospholipids/metabolism , Type C Phospholipases/metabolism , Enzyme Activation , Humans , Phospholipase C beta , Recombinant Proteins/metabolism , Substrate Specificity
19.
J Neurosci ; 20(6): 2131-41, 2000 Mar 15.
Article in English | MEDLINE | ID: mdl-10704487

ABSTRACT

Monoamines such as noradrenaline and serotonin are stored in secretory vesicles and released by exocytosis. Two related monoamine transporters, VMAT1 and VMAT2, mediate vesicular transmitter uptake. Previously we have reported that in the rat pheochromocytoma cell line PC 12 VMAT1, localized to peptide-containing secretory granules, is controlled by the heterotrimeric G-protein Go(2). We now show that in BON cells, a human serotonergic neuroendocrine cell line derived from a pancreatic tumor expressing both transporters on large, dense-core vesicles, VMAT2 is even more sensitive to G-protein regulation than VMAT1. The activity of both transporters is only downregulated by Galphao(2), whereas comparable concentrations of Galphao(1) are without effect. In serotonergic raphe neurons in primary culture VMAT2 is also downregulated by pertussis toxin-sensitive Go(2). By electron microscopic analysis from prefrontal cortex we show that VMAT2 and Galphao(2) associate preferentially to locally recycling small synaptic vesicles in serotonergic terminals. In addition, Go(2)-dependent modulation of VMAT2 also works when using a crude synaptic vesicle preparation from this brain area. We conclude that regulation of monoamine uptake by the heterotrimeric G proteins is a general feature of monoaminergic neurons that controls the content of both large, dense-core and small synaptic vesicles.


Subject(s)
Heterotrimeric GTP-Binding Proteins/metabolism , Membrane Glycoproteins/metabolism , Membrane Transport Proteins , Neurons/enzymology , Neuropeptides , Animals , Carcinoid Tumor , Cell Membrane Permeability/physiology , Down-Regulation/physiology , GTP-Binding Protein alpha Subunits , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Histamine/pharmacokinetics , Humans , Membrane Glycoproteins/analysis , Microscopy, Immunoelectron , Neuronal Plasticity/physiology , Neurons/chemistry , Neurons/ultrastructure , PC12 Cells , Pancreatic Neoplasms , Rabbits , Raphe Nuclei/cytology , Rats , Recombinant Fusion Proteins/metabolism , Serotonin/pharmacokinetics , Tritium , Tumor Cells, Cultured , Vesicular Biogenic Amine Transport Proteins , Vesicular Monoamine Transport Proteins
20.
J Biol Chem ; 274(41): 29311-7, 1999 Oct 08.
Article in English | MEDLINE | ID: mdl-10506190

ABSTRACT

By using purified preparations we show that nanomolar concentrations of Gbetagamma significantly stimulated lipid kinase activity of phosphatidylinositol 3-kinase (PI3K) beta and PI3Kgamma in the presence as well as in the absence of non-catalytic subunits such as p85alpha or p101. Concomitantly, Gbetagamma stimulated autophosphorylation of the catalytic subunit of PI3Kgamma (EC(50), 30 nM; stoichiometry >/=0.6 mol of P(i)/mol of p110gamma), which also occurred in the absence of p101. Surprisingly, we found that p101 affected the lipid substrate preference of PI3Kgamma in its Gbetagamma-stimulated state. With phosphatidylinositol as substrate, p110gamma but not p101/p110gamma was significantly stimulated by Gbetagamma to form PI-3-phosphate (EC(50), 20 nM). The opposite situation was found when PI-4,5-bisphosphate served as substrate. Gbetagamma efficiently and potently (EC(50), 5 nM) activated the p101/p110gamma heterodimer but negligibly stimulated the p110gamma monomer to form PI-3,4,5-trisphosphate. However, this weak stimulatory effect on p110gamma was overcome by excess concentrations of Gbetagamma (EC(50), 100 nM). This finding is in accordance with the in vivo situation, where activated PI3K catalyzes the formation of PI-3,4,5-trisphosphate but not PI-3-phosphate. We conclude that p101 is responsible for PI-4, 5-bisphosphate substrate selectivity of PI3Kgamma by sensitizing p110gamma toward Gbetagamma in the presence of PI-4,5-P(2).


Subject(s)
GTP-Binding Proteins/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Androstadienes/pharmacology , Animals , Cattle , Dimerization , Enzyme Activation , Phosphatidylinositol 3-Kinases/classification , Phosphatidylinositol 4,5-Diphosphate/metabolism , Phosphatidylinositol Phosphates/metabolism , Phosphorylation , Protein Binding , Protein Conformation , Receptor Protein-Tyrosine Kinases/metabolism , Recombinant Proteins/metabolism , Second Messenger Systems , Signal Transduction , Substrate Specificity , Wortmannin
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