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Biomedical subjects

P I Lelkes

Publications and source records attributed to P I Lelkes.

At least 19 recordsLinked to original sources
PubMed2005

Dynamic culture in a rotating-wall vessel bioreactor differentially inhibits murine T-lymphocyte activation by mitogenic stimuli upon return to static conditions in a time-dependent manner.

Depressed immune function is a well-documented effect of spaceflight. Both in-flight studies and ground-based studies using microgravity analogs, such as rotating wall vessel (RWV) bioreactors, have demonstrated that mitogen-stimulated T lymphocytes exhibit decreased proliferation, IL-2 secretion, and activation marker expression in true microgravity and the dynamic RWV-culture environment. This study investigates the kinetics of RWV-induced T lymphocyte inhibition by monitoring the ability of Balb/c mouse splenocytes to become activated under static culture conditions after concanavalin A (Con A) stimulation in an RWV. Splenocytes were stimulated with Con A and cultured for up to 24 h in the RWV before being allowed to "recover" under static culture conditions in the continued presence of Con A. The T-lymphocyte fraction of splenocytes was assayed during the recovery period for IL-2 secretion, expansion of the T-lymphocyte population, and expression of the activation marker CD25. Our results indicate that CD25 expression was not affected by any duration of RWV exposure. In contrast, proliferation and IL-2 secretion were inhibited by >8 and 12 h of exposure, respectively. Culture in the RWV for 24 h resulted in a near-complete loss of cellular viability during the recovery period, which was not seen in cells maintained in the RWV for 16 h or less. Taken together, these results indicate that for up to 8 h of RWV culture activation is not significantly impaired upon return to static conditions; longer duration RWV culture results in a gradual loss of activation during the recovery period most likely because of decreased T-cell viability and/or IL-2 production.

Animals↗
PubMed2001

Reactive oxygen species, apoptosis and altered NGF-induced signaling in PC12 pheochromocytoma cells cultured in elevated glucose: an in vitro cellular model for diabetic neuropathy.

Diabetic neuropathies, affecting the autonomic, sensory, and motor peripheral nervous system, are among the most frequent complications of diabetes. The symptoms of diabetic polyneuropathies are multi-faceted; the etiology and the underlying mechanisms are as yet unclear. Clinical studies established a significant correlation between the control of the patients' blood glucose level and the severity of the damage to the peripheral nervous system. Recent in vitro studies suggest that elevated glucose levels induced dysfunction and apoptosis in cultured cells of neuronal origin, possibly through the formation of reactive oxygen species (ROS). Based on these results, we hypothesized that elevated glucose levels impair neuronal survival and function via ROS dependent intracellular signaling pathways. In order to test this hypothesis, we cultured neural crest-derived PC12 pheochromocytoma cells under euglycemic (5 mM) and hyperglycemic (25 mM) conditions. Continuous exposure of undifferentiated PC12 cells for up to 72 h to elevated glucose induced the enhanced generation of ROS, as assessed from the increase in the cell-associated fluorescence of the ROS-sensitive fluorogenic indicator, 2,7-dichlorodihydrofluorescein diacetate. In cells cultured in high glucose, both basal and secretagogue-stimulated catecholamine release were enhanced. Furthermore, high glucose, reduced (by ca. 30%) the rate of cell proliferation and enhanced the occurrence of apoptosis, as assessed by DNA fragmentation, TUNEL assay and the activation of an apoptosis-specific protease, caspase CCP32. Elevated glucose levels significantly attenuated nerve growth factor (NGF)-induced neurite extension, as quantitated by computer-aided image analysis. Culturing PC12 cells in high glucose resulted in alterations in basal and NGF-stimulated mitogen-activated protein kinase (MAPK) signaling pathways, specifically in a switch from the neuronal survival/differentiation-associated MAPK ERK to that of apoptosis/stress-associated MAPK p38 and JNK. Based on our results we present a model in which the prolonged, excess formation of ROS represents a common mechanism for hyperglycemia-induced damage to neuronal cells. We propose that this simple in vitro system might serve as an appropriate model for evaluating some of the effects of elevated glucose on cultured cells of neuronal origin.

Journal Article↗
PubMed2001

Expression of VACM-1 protein in cultured rat adrenal endothelial cells is linked to the cell cycle.

The vasopressin-activated calcium-mobilizing (VACM-1) protein is a unique arginine vasopressin (AVP) receptor which shares sequence homology with the cullins, genes involved in the regulation of cell cycle transitions. Unlike either cullins or AVP receptors, however, VACM-1 is expressed exclusively in the vascular endothelial cells and in the renal collecting tubule cells. In order to test the hypothesis that the expression of VACM-1 might be correlated with the cell cycle, and to establish an endothelial cell model for the VACM-1 receptor, we examined VACM-1 expression in rat adrenal medulla endothelial cells (RAMEC). Northern and Western blot analyses of mRNA and protein from RAMEC identified presence of 6.4 kb mRNA and a Mr 81 kDa protein, respectively. Immunostaining of RAMEC with anti-VACM-1 antibodies and Western blot analyses indicated that in RAMEC, VACM-1 protein expression is dependent on the cell cycle. VACM-1 protein virtually disappears during the S phase and localizes to the cytosol during cell division and to the cell membrane at the completion of cytokinesis. Furthermore, pretreatment of RAMEC with anti-VACM-1 specific antibodies increased basal levels of Ca2+and attenuated the AVP-dependent increase in cytosolic Ca2+. In summary, these results indicate that VACM-1 protein expression in RAMEC membrane is linked to the cell cycle, and consequently, VACM-1 may be involved in the regulation of cell division.

Adrenal Medulla↗
PubMed2001

Regulation of extracellular matrix remodeling and MMP-2 activation in cultured rat adrenal medullary endothelial cells.

We previously reported that short-term exposure of cultured rat adrenal medullary endothelial cells (RAMEC) to thrombin enhances the subendothelial deposition of extracellular matrix (ECM) proteins fibronectin, laminin, and collagen types I (C-I) and IV (C-IV) (Papadimitriou et al. 1997). In this work, we extended our previous studies on factors that effect ECM protein deposition to include agents that activate or inhibit some of the most common intracellular signals such as cAMP, protein kinase C (PKC), and calcium. Furthermore, we investigated the possible link between the observed alterations in ECM protein deposition and the secretion of matrix metalloproteinase-2 (MMP-2). Forskolin (adenylyl cyclase activator) caused a dose-dependent increase in the deposition of all four ECM proteins studied. Isoproterenol beta-adrenergic receptor agonist) and the membrane permeant cAMP analogue dibutyryl-cAMP significantly increased the deposited amounts of ECM proteins at low concentrations, and this increase was reversed at higher concentrations of both agents. All these agents had the opposite effect on MMP-2 secretion, increasing it at doses where they decreased ECM protein deposition and vice versa. However, elevation of cAMP by the phosphodiesterase inhibitor IBMX had no effect either on the deposited amounts of any of the ECM proteins studied or on MMP-2 secretion. Activation of PKC by phorbol ester (PMA) resulted in a decrease in ECM protein deposition and an increase in MMP-2 secretion. Finally, chelation of intercellular calcium with BAPTA-AM resulted in an increased ECM deposition and a decrease in MMP-2 secretion. Our results show a complex pattern of regulation of ECM protein deposition by cAMP-mobilizing agents and also indicate an inverse correlation between ECM protein deposition and secretion of MMP-2. The concerted regulation of both of these processes is essential in the formation of new blood vessels, and for the integrity of the vascular wall.

Adrenal Medulla↗
PubMed2000

Inhibition of angiogenesis by blockers of volume-regulated anion channels.

Osmotic cell swelling activates an outwardly rectifying Cl(-) current in endothelial cells that is mediated by volume-regulated anion channels (VRACs). In the past, we have shown that serum-induced proliferation of endothelial cells is arrested in the presence of compounds that potently block the endothelial VRACs. Here we report on the effects of four chemically distinct VRAC blockers [5-nitro-2-(3-phenylpropylamino)benzoic acid] (NPPB), mibefradil, tamoxifen, and clomiphene-on several models of experimental angiogenesis. Mibefradil (20 microM), NPPB (100 microM), tamoxifen (20 microM), and clomiphene (20 microM) inhibited tube formation by rat microvascular endothelial cells plated on matrigel by 42.9 +/- 8.8%, 25.3 +/- 10.4%, 32.2 +/- 4.5%, and 20 +/- 5.8%, respectively (p < 0.05). Additionally, NPPB (50-100 microM) and mibefradil (10-30 microM) significantly inhibited bFGF (10 ng/ml) + TNFalpha (2.5 ng/ml)-stimulated microvessel formation by human microvascular endothelial cells plated on fibrin by 30-70%. Furthermore, NPPB, mibefradil, and clomiphene concentration dependently inhibited spontaneous microvessel formation in the rat aorta-ring assay and vessel development in the chick chorioallantoic membrane assay. These results suggest that VRAC blockers are potent inhibitors of angiogenesis and thus might serve as therapeutic tools in tumor growth and other angiogenesis-dependent diseases.

Allantois↗
PubMed2000

Biodesign of a skeletal muscle flap as a model for cardiac assistance.

In using autologous muscles for cardiac assistance, it is crucial to reduce ischemia-reperfusion injury in the surgically traumatized skeletal muscle. In adult sheep, we developed a simple model of surgically designed 2 latissimus dorsi muscle leaflets by modifying the vascular supply to these leaflets. Three pockets with graded injury were established, and muscle morphology and vascular remodeling were monitored in 3 experimental groups: muscle leaflets without any treatment (Group 1, n = 6) that served as controls; muscle leaflets integrated with a fibrin interlayer (Group 2, n = 6); and leaflets integrated with fibrin and entrapped pyrrolostatin (Group 3, n = 6). We applied the fibrinogen and thrombin solutions, which polymerize to form a three-dimensional meshwork joining the tissues, creating a provisional matrix for angiogenesis, and acting as a delivery depot for agents aimed at minimizing ischemia-reperfusion lesion formation. After 2 months, the muscle leaflets biointegrated with the fibrin interface showed none of the signs of necrosis or ischemia-reperfusion lesions seen in the controls. Although no angiogenic factors were incorporated, the fibrin interlayer rapidly (<2 weeks) became a densely vascularized tissue replete with a voluminous capillary network. In contrast, controls showed poor bonding between the tissues, muscle fiber deterioration, and a compromised vascular network. Muscle structure was best preserved and angiogenesis was greatest when pyrrolostatin, a free radical scavenger, was added to the fibrin meshwork to reduce damage caused by overproduction of free radicals. This newly designed model will be useful to study many current approaches in cardiovascular biology, from pharmaceuticals to gene therapy, which might prove advantageous in muscle-designed cardiac assistance.

Animals↗
PubMed1999

Tissue-specific alternative mRNA splicing of phenylethanolamine N-methyltransferase (PNMT) during development by intron retention.

The expression of phenylethanolamine N-methyl transferase (EC 2. 1.1.2.8, PNMT), the final enzyme in the cascade of catecholamine synthesis, is differentially regulated in adrenergic neurons in the brain and in adrenal chromaffin cells. Using reverse transcription-polymerase chain reaction-based techniques, we detected in the prenatal developing rat brainstem, two species of PNMT mRNA which were produced by a rare alternative splicing mechanism known as intron retention. The spliced, intronless message was downregulated postnatally, while the intron-retained mRNA species continued to be constitutively expressed through adulthood. By contrast in the adrenals, at all stages of development examined, only the intronless message was expressed. In line with previous reports on the failure of glucocorticoids to induce PNMT expression in the brain, the pattern of PNMT splicing in brainstem explants was not affected by the presence of the synthetic glucocorticoid dexamethasone. Undifferentiated sympathoadrenal PC12 pheochromocytoma cells expressed very low basal levels of both mRNA variants, accompanied by a very low basal PNMT enzymatic activity. Exposure of PC12 cells to dexamethasone resulted in the upregulation of only the spliced mRNA variant concomitant with a 3-fold increase in PNMT enzymatic activity. In contrast, treatment of PC 12 cells with nerve growth factor (NGF) enhanced the expression of both the intron-retained and the intronless mRNA species without changes in the basal enzyme activity. This latter result suggests that the translation of the intronless mRNA species may be regulated by the intron-retained mRNA species, which by itself may yield a truncated, yet enzymatically functional translational product. Our data suggest that the tissue-specific regulation of PNMT expression is based on a rare alternative splicing mechanism termed intron retention, and that in the adrenal, but not in the brain, this mechanism is sensitive to regulation by glucocorticoids. Thus, this system is uniquely suited for studying the hormonal control of tissue-specific splicing in the nervous system.

Adrenal Medulla↗
PubMed1999

Angiogenesis, angiogenic growth factors, and cell adhesion molecules are upregulated in chronic pancreatic diseases: angiogenesis in chronic pancreatitis and in pancreatic cancer.

Recent epidemiologic evidence suggests that patients with chronic pancreatitis (CP) have an increased risk of developing pancreatic carcinoma (PCA). In spite of numerous similarities in both diseases, mechanisms for progression from CP to PCA are poorly understood. We hypothesized that enhanced angiogenesis might play a pivotal role in the etiology and histopathology of both CP and PCA, and thus form a possible link between precancer and carcinoma. In surgical specimens of 18 patients with CP, 10 with PCA, and 18 controls, absolute numbers of blood vessels and relative blood vessel density were assessed after immunostaining of endothelial cells for von Willebrand factor and PECAM-1 (platelet/ endothelial cell adhesion molecule-1). Furthermore, the expression of cell adhesion molecules ICAM-1 (intercellular adhesion molecule-1) and VCAM-1 (vascular cell adhesion molecule-1) and of VEGF (vascular endothelial growth factor) was investigated in all specimens. Both CP and PCA exhibited areas of high vascular density ("hot spots"). The mean number of blood vessels in these areas in PCA was 132.2+/-16.8 per mm2, and in CP, 99.2+/-7.4 per mm2. The mean vessel count in controls was 25.1+/-5.1. Relative vessel density was increased in both PCA (41.3+/-3.5%) and CP (30.6+/-2.6%) versus controls (8.0+/-0.8%). Both absolute vessel count and relative vessel density were significantly higher (p<0.05) in PCA than in CP. Enhanced expression of ICAM-1 in CP and PCA was seen in ductal cells in CP and cancer cells. In controls, ICAM-1 and VCAM-1 were expressed only at low levels in endothelial cells. VCAM-1 was strongly expressed in acinar cells as well as in ductal cells. In CP and PCA, VEGF was strongly expressed in ductal cells in CP as well as in cancer cells. We show for the first time that angiogenic activity is increased in both CP and PCA. Based on this study, we suggest that antiangiogenesis might be a novel target for prevention or therapy in chronic pancreatic diseases.

Adenocarcinoma↗
PubMed1999

Tissue factor activity is increased in human endothelial cells cultured under elevated static pressure.

We tested the hypothesis that elevated blood pressure, a known stimulus for vascular remodeling and an independent risk factor for the development of atherosclerotic disease, can modulate basal and cytokine-induced tissue factor (TF; CD 142) expression in cultured human endothelial cells (EC). Using a chromogenic enzymatic assay, we measured basal and tumor necrosis factor-alpha (TNF-alpha; 10 ng/ml, 5 h)-induced TF activities in human aortic EC (HAEC) and vena cava EC (HVCEC) cultured at atmospheric pressure and at 170 mmHg imposed pressure for up to 48 h. Basal TF activities were 22 +/- 10 U/mg protein for HAEC and 14 +/- 9 U/mg protein for HVCEC and were upregulated in both cell types >10-fold by TNF-alpha. Exposure to pressure for 5 h induced additional elevation of basal TF activity by 47 +/- 16% (P < 0.05, n = 6) for HAEC and 17 +/- 5% (P < 0.05, n = 3) for HVCEC. Pressurization also enhanced TF activity in TNF-alpha-treated cells from 240 +/- 28 to 319 +/- 32 U/mg protein in HAEC (P < 0.05, n = 4) and from 148 +/- 25 to 179 +/- 0.8 U/mg protein (P < 0.05, n = 3) in HVCEC. Cytokine stimulation caused an approximately 100-fold increase in steady-state TF mRNA levels in HAEC, whereas pressurization did not alter either TF mRNA or cell surface antigen expression, as determined by quantitative RT-PCR methodology and ELISA. Elevated pressure, however, modulated the EC plasma membrane organization and/or permeability as inferred from the increased cellular uptake of the fluorescent amphipathic dye merocyanine 540 (33 +/- 7%, P < 0.05). Our data suggest that elevated static pressure modulates the hemostatic potential of vascular cells by modifying the molecular organization of the plasma membrane.

Aorta↗
PubMed1999

Characterization of pardaxin-induced dopamine release from pheochromocytoma cells: role of calcium and eicosanoids.

Pardaxin, an excitatory neurotoxin, induced dopamine release from pheochromocytoma (PC12) cells both in the presence and absence of extracellular calcium ([Ca]o). In the presence of extracellular calcium, nifedipine, an L-type calcium channel blocker, did not affect dopamine release, whereas 1,2-bis (2-aminophenoxy) ethane N,N, N'N'-tetra-acetic acid (BAPTA), a chelator of cytosolic calcium, and dantrolene, a blocker of calcium release from intracellular stores, inhibited only partially (30-40%) pardaxin-induced dopamine release. In the absence of [Ca]o, BAPTA and dantrolene were ineffective. Pardaxin stimulated the arachidonic acid (AA) cascade in PC12 cells independently of [Ca]o. The phospholipase inhibitors mepacrine and bromophenacyl bromide inhibited both pardaxin-induced AA release and pardaxin-induced dopamine release. Dopamine release induced by pardaxin also was blocked by the lipoxygenase inhibitors nordihydroguaiaretic acid, esculetin, and 2-(12-hydroxydodeca-5, 10-diynyl)-3,5,6-trimethyl-1,4-benzoquinone. Under these conditions, a parallel reduction in 5-hydroxyeicosatetranoic acid release also was observed. Suppression of pardaxin-induced dopamine release by inhibitors of phospholipase A2 and lipoxygenase was more pronounced in calcium-free medium. These results indicate the involvement of the lipoxygenase pathway in pardaxin-induced dopamine release and suggest the use of this toxin as a novel pharmacological tool for investigating the mechanism of calcium-independent neurotransmitter release.

Animals↗
PubMed1998

On the possible role of reactive oxygen species in angiogenesis.

Human microvascular endothelial cells grown on a 3-D reconstituted extracellular matrix (Matrigel) spontaneously and rapidly form a capillary network of tubular structures, thus modeling part of the angiogenic cascade. Exposure of the cells at the time of plating onto Matrigel to a brief episode of hypoxia (40-60) min and subsequent reoxygenation, significantly accelerated (up to 3-fold) the rate of tubular morphogenesis, as determined by computer-aided morphometry. This effect was not dependent on activation of PKC or upregulation/release of angiogenic growth factors. Rather, hypoxia/reoxygenation (H/R), but not hypoxia alone, caused the formation of reactive oxygen species (ROS) and the activation of the nuclear transcription factor NF kappa B, both of which were inhibited by ROS-scavengers, such as pyrollidine dithiocarbamate. Tube formation was inhibited, also under normoxic conditions, by diverse ROS antagonists in a dose-dependent fashion. Our results indicate that angiogenesis is accompanied by and/or requires generation of ROS. We hypothesize that in the clinical setting of hypoxia/reoxygenation during ischemic pre-conditioning, enhanced activation of ROS-dependent intracellular signaling may accelerate the rate of neovascularization also in vivo, thus contributing to the alleviation of certain ischemic lesions.

Cell Hypoxia↗
PubMed1998

Simulated microgravity conditions enhance differentiation of cultured PC12 cells towards the neuroendocrine phenotype.

We are studying microenvironmental cues which contribute to neuroendocrine organ assembly and tissue-specific differentiation. As our in vitro model, we cultured rat adrenal medullary PC12 pheochromocytoma cells in a novel cell culture system, the NASA rotating wall vessel (RWV) bioreactors. This "simulated microgravity" environment in RWV bioreactors, characterized by randomizing gravitational vectors and minimizing shear stress, has been shown to favor macroscopic tissue assembly and to induce tissue-specific differentiation. We hypothesized that the unique culture conditions in the RWV bioreactors might enhance the in vitro formation of neuroendocrine organoids. To test our hypothesis, we evaluated the expression of several markers of neuroendocrine differentiation in cultures of PC12 cells maintained for up to 20 d in the slow turning lateral vessel (STLV) type RWV. PC12 cell differentiation was assessed by morphological, immunological, biochemical and molecular techniques. PC12 cells, cultured under "simulated microgravity" conditions, formed macroscopic, tissue-like organoids several millimeters in diameter. Concomitantly, the expression of phenylethanolamine-N-methyl transferase (PNMT), but not of other catecholamine synthesizing enzymes, was enhanced. Increased PNMT expression, as verified on both the gene and protein level, was accompanied by an increase in the specific activity of the enzyme. Furthermore, after 20 d in culture in the STLV, we observed altered patterns of protein tyrosine phosphorylation and prolonged activation of c-fos, a member of the AP-1 nuclear transcription factor complex. We conclude that culture conditions in the RWV appear to selectively activate signal transduction pathways leading to enhanced neuroendocrine differentiation of PC12 cells.

Animals↗
PubMed1998

Comparison of ICAM-1 and VCAM-1 expression in various human endothelial cell types and smooth muscle cells.

The diversity in the local manifestation of inflammatory vascular lesions might be partially attributable to heterogenous cell adhesion molecule (CAM) expression among endothelial cells (EC) derived from different anatomical locations. We compared basal and tumor necrosis factor-alpha (TNFalpha, 0-100 ng/ml, 0-48 h)-induced intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression in cultured human aortic EC (HAEC), vena cava EC (HVCEC), dermal microvascular EC (HMVEC), and vena cava smooth muscle cells (HVCSM), using a fluorescent ELISA and the competitive quantitative RT-PCR. We found marked differences in basal ICAM-1 expression, both at the protein and mRNA levels, such that HAEC>HVCEC approximately equal to HMVEC>>HVCSM. Basal VCAM-1 mRNA levels were significantly lower in HVCEC than in HAEC and HVCSM, while protein levels were indistinguishable. TNFalpha-induced ICAM-1 and VCAM-1 levels in all EC were similar and significantly higher than in HVCSM (2.5- and 5-fold, respectively). Dissimilar levels of basal and TNFalpha-induced CAM expression in vascular cells may explain the varied predisposition of different blood vessels to developing certain vasculopathies.

Cells, Cultured↗
PubMed1998

Launch conditions might affect the formation of blood vessels in the quail chorioallantoic membrane.

As a part of the first joint USA-Russian MIR/Shuttle program, fertilized quail eggs were flown on the MIR 18 mission. Post-flight examination indicated impaired survival of both the embryos in space and also of control embryo exposed to vibrational and g-forces simulating the condition experienced during the launch of Progress 227. We hypothesized that excess mechanical forces and/or other conditions during the launch might cause abnormal development or the blood supply in the chorioallantoic membrane (CAM) leading to the impaired survival of the embryos. The CAM, a highly vascularized extraembryonic organ, provides for the oxygen exchange across the egg shell and is thus pivotal for proper embryonic development. To test our hypothesis, we compared angiogenesis in CAMs of eggs which were either exposed to the vibration and g-force profile simulating the conditions at launch of Progress 227 (synchronous controls), or kept under routine conditions in a laboratory incubator (laboratory controls). At various time points during incubation, the eggs were fixed in paraformaldehyde for subsequent dissection. At the time of dissection, the CAM was carefully lifted from the egg shell and examined as whole mounts by bright-field and fluorescent microscopy. The development of the vasculature (angiogenesis) was assessed from the density of blood vessels per viewing field and evaluated by computer aided image analysis. We observed a significant decrease in blood-vessel density in the synchronous controls versus "normal" laboratory controls beginning from day 10 of incubation. The decrease in vascular density was restricted to the smallest vessels only, suggesting that conditions during the launch and/or during the subsequent incubation of the eggs may affect the normal progress of angiogenesis in the CAM. Abnormal angiogensis in the CAM might contribute to the impaired survival of the embryos observed in synchronous controls as well as in space.

Allantois↗
PubMed1997

Expression of human p140trk receptors in p140trk-deficient, PC12/endothelial cells results in nerve growth factor-induced signal transduction and DNA synthesis.

Nerve growth factor (NGF) regulates proliferation, differentiation, and survival of sympathetic and sensory neurons through the tyrosine kinase activity of its receptor, p140trk. These biological effects of NGF depend upon the signal-mediating function of p140trk substrates which are likely to differ from cell to cell. To define p140trk receptor substrates and the details of signalling by NGF in the hybrid cell PC12EN, we stably transfected cultures with a vector encoding a full-length human p140trk cDNA sequence. Two stably transfected clones, one expressing p140trk with higher affinity (PC12EN-trk3; Kd 57.4 pM, Bmax 9.7 pmole/mg) and one expressing p140trk with a lower affinity (PC12EN-trk1; Kd 392.4 pM, Bmax 5.7 pmole/mg) were generated. Radioreceptor assays indicate that transfected p140trk receptors show slow NGF-dissociation kinetics, are resistant to trypsin or Triton X-100 treatment, are specific for NGF compared to other neurotrophins, and are internalized or downregulated as are native PC12 p140trk receptors. NGF stimulates p140trk tyrosine phosphorylation in a dose- (0.01-10 ng/ml) and time- (5-120 min) dependent manner, and tyrosine phosphorylation was inhibited by 200-1,000 nM K-252a. NGF-induced Erk stimulation for 60 min was assessed using myelin basic protein as a substrate. NGF treatment also led to an increased phosphorylation of p70S6k, SNT, and phospholipase C gamma, demonstrating that the major NGF-stimulated signalling pathways found in other cells are activated in PC12EN-trk cells. Staurosporine (5-50 nM) rapidly and dBcAMP (1 mM) more slowly, but not NGF induced morphological differentiation in PC12EN-trk cells. Rather, NGF treatment in low-serum medium stimulated a 1.3- and 2.3-fold increase in DNA synthesis measured by [3H]thymidine incorporation in PC12EN-trk1 and PC12EN-trk3, respectively. These data highlight the functionality of the transfected p140trk receptors and indicate that these transfected cells may serve as a novel cellular model facilitating the study of the mitogenic properties of NGF signalling and the transducing role of the p140trk receptor substrates.

Animals↗
PubMed1997

The thrombin receptor in adrenal medullary microvascular endothelial cells is negatively coupled to adenylyl cyclase through a Gi protein.

The effects of thrombin on adenylyl cyclase activity were examined in rat adrenal medullary microvascular endothelial cells (RAMEC). Confluent RAMEC monolayers were stimulated for 5 min with cAMP-generating agents in the absence and presence of thrombin, and intracellular cAMP was measured with a radioligand binding assay. Thrombin (0.001-0.25 U/ml) dose-dependently inhibited IBMX-, isoproterenol- and forskolin-stimulated cAMP accumulation. A peptide agonist of the thrombin receptor, gamma-thrombin, and the serine proteases trypsin and plasmin, also inhibited agonist-stimulated cAMP levels, while proteolytically inactive PPACK- or DIP-alpha-thrombins were without effect. Moreover, the thrombin inhibitor hirudin abolished the inhibitory effect of thrombin but not of the peptide agonist. These results suggest that the inhibitory action of thrombin on cAMP accumulation is mediated by a proteolytically-activated thrombin receptor. The inhibitor of G(i)-proteins pertussis toxin abolished the inhibitory effect of thrombin on isoproterenol- or IBMX-stimulated cAMP production, while the phorbol ester PMA partly impaired it. The protein kinase C inhibitors staurosporine or H7 and the intracellular Ca2+ chelator BAPTA-AM were without effect. Collectively, our data suggest that the thrombin receptor in RAMEC is negatively coupled to adenylyl cyclase through a pertussis toxin-sensitive G(i)-protein.

Adenylate Cyclase Toxin↗
PubMed1997

GTSF-2: a new, versatile cell culture medium for diverse normal and transformed mammalian cells.

The aim of this study was to test the versatility of a new basal cell culture medium, GTSF-2. In addition to traditional growth-factors, GTSF-2 contains a blend of three sugars (glucose, galactose, and fructose) at their physiological levels. For these studies, we isolated normal endothelial cells from human, bovine, and rat large blood vessels and microvessels. In addition, GTSF-2 was also tested as a replacement for high-glucose-containing medium for PC12 pheochromocytoma cells and for other, transformed cell lines. The cell growth characteristics were assessed with a novel cell viability and proliferation assay, which is based on the bioreduction of the fluorescent dye, Alamar Blue. After appropriate calibration, the Alamar Blue assay was found to be equivalent to established cell proliferation assays. Alamar Blue offers the advantage that cell proliferation can be measured in the same wells over an extended period of time. For some of the cell types (e.g., endothelial cells isolated from the bovine aorta, the rat adrenal medulla, or the transformed cells), proliferation in unmodified GTSF-2 was equivalent to that in the original culture media. For others cell types (e.g., human umbilical vein endothelial cells and PC12 cells), GTSF-2 proved to be a superior growth medium, when supplemented with simple additives, such as endothelial cell growth supplement (bFGF) or horse serum. Our results suggest that GTSF-2 is a versatile basal medium that will be useful for studying organ-specific differentiation in heterotypic coculture studies.

Animals↗