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PubMed1992

Constitutive and inducible nitric oxide synthases in human megakaryoblastic cells.

Human megakaryoblastic cells (Meg-01) were found to possess constitutive and express inducible nitric oxide (NO) synthase activities. The constitutive NO synthase was Ca+(+)- and NADPH-dependent, as is the NO synthase found previously in human platelets. Stimulation of Meg-01 cells by the cytokines interleukin-1 beta (0.15-32.5 ng/ml) and tumor necrosis factor-alpha (0.15-10 ng/ml) resulted in expression of the inducible, Ca+(+)-independent, NO synthase. This activity was increased by the addition of NADPH, tetrahydrobiopterin and sepiapterin as cofactors. Induction of this enzyme was accompanied by a decrease in the constitutive NO synthase activity, a phenomenon which was prevented or reversed by dexamethasone (1 microM). Thus, human early differentiated megakaryocytic cells can synthesize NO from L-arginine by both the constitutive and the inducible NO synthases. These findings indicate that these enzymes may play an important biological role in megakaryocyte and platelet functions.

Amino Acid Oxidoreductases↗
PubMed1992

Activation of the erythropoietin receptor by the Friend spleen focus-forming virus gp55 glycoprotein induces constitutive protein tyrosine phosphorylation.

The erythropoietin receptor (EPO-R) can be activated to signal cell growth by binding either EPO or gp55, the Friend spleen focus-forming virus (SFFV) glycoprotein. EPO binding induces tyrosine kinase activity and rapid tyrosine phosphorylation of several cellular substrates. To test for gp55-induced tyrosine kinase activity, we performed immunoblots on two murine cell lines that stably express EPO-R and gp55. Stimulation of the parental cell line, Ba/F3, with murine interleukin-3 (IL-3) resulted in rapid, dose-dependent tyrosine phosphorylation of a 97-Kd substrate. Stimulation with IL-3 or EPO of the Ba/F3 cells expressing the recombinant EPO-R (Ba/F3-EPO-R) resulted in tyrosine phosphorylation of the same p97 substrate. These latter cells, when transformed to growth factor-independence by the Friend gp55 glycoprotein, exhibited constitutive tyrosine phosphorylation of the 97-Kd substrate. Other growth factor-independent Ba/F3 subclones, transformed with either the oncoprotein, v-abl, or with a constitutively activated EPO-R, also had constitutive phosphorylation of a 97-Kd substrate. In CTLL-2-EPO-R cells, a T-lymphocyte line stably transfected with the EPO-R, the 97-Kd substrate was tyrosine-phosphorylated in response to IL-2 or EPO. The 97-Kd protein was constitutively phosphorylated in CTLL-2-EPO-R-gp55 cells. In conclusion, a 97-Kd protein found in two murine cell lines is tyrosine-phosphorylated in response to multiple growth factors and viral oncoproteins, and appears to be a central phosphoprotein in signal transduction.

Animals↗
PubMedDate not supplied

Modulation of the constitutive activated STAT3 transcription factor in pancreatic cancer prevention: effects of indole-3-carbinol (I3C) and genistein.

BACKGROUND: The signal transducer and activator of transcription 3 (STAT3) is a latent transcription factor required in proliferation and differentiation. STAT3 is activated constitutively in a number of cancers. MATERIALS AND METHODS: This study was conducted to assess the possible involvement of STAT3 activation in pancreatic cancer and the potential for this pathway as a target in chemopreventive strategy. RESULTS: STAT3 was shown for the first time to be constitutively activated in human pancreatic carcinoma specimens but not in normal pancreatic tissues. Constitutively activated STAT3 was also found in pancreatic tumor cell lines (Panc-1 and MIA PaCa-2) which could be modulated by indole-3-carbinol (13C) and genistein. At concentrations higher than 10 microM, STAT3 constitutive activation is inhibited by both agents. Induction of apoptosis by 13C was also demonstrated. CONCLUSION: Given its critical role in tumorigenesis, our results suggest that STAT3 activation provides an important and appropriate target for chemoprevention in pancreatic cancer treatment.

Adenocarcinoma↗
PubMed2004

Inhibition of constitutively activated nuclear factor-kappaB radiosensitizes human melanoma cells.

Melanoma tumors and cultured cell lines are relatively resistant to the cytotoxic effects of ionizing radiation, thereby limiting the use of radiotherapy for the clinical treatment of melanoma. New strategies for sensitizing melanoma cells therefore deserve examination. In an attempt to identify and target signaling pathways that contribute to radioresistance, we investigated the role of nuclear factor-kappaB (NF-kappaB), a transcription factor known to inhibit apoptosis induced by a variety of stimuli and promote radioresistance. Two human metastatic melanoma cell lines, A375 and MeWo, were used to examine the radiosensitizing effects of inhibitors of the NF-kappaB pathway. Nuclear extracts from these cell lines were tested for active NF-kappaB using the electrophoretic mobility shift assay. Both melanoma cell lines had constitutively activated NF-kappaB as observed by electrophoretic mobility shift assay. In an attempt to reverse NF-kappaB activity, cells were treated either with vehicle alone (DMSO) or with a proteasome inhibitor Z-Leu-Leu-Leu-H (MG132; 10 micromol/L for 2 hours prior to irradiation) that inhibited both constitutive and radiation-induced NF-kappaB activity. The clonogenic cell survival assay showed that pretreatment with MG132 enhanced tumor cell radiosensitivity with the survival factor at 2 Gy being reduced from 48 +/- 0.8% and 48 +/- 1.6% in vehicle-treated cells to 27.7 +/- 0.32% and 34.3 +/- 0.7% in MG132-treated MeWo and A375 cells, respectively. To test the role of NF-kappaB in radioresistance more directly, MeWo cells were stably transfected with a dominant-negative mutant IkappaBalpha construct, which led to the inhibition of both constitutive and radiation-induced NF-kappaB activity. A modest restoration of radiosensitivity was also observed in the stably transfected MeWo cells with survival factor at 2 Gy values being reduced from 47 +/- 0.8% in parental MeWo cells to 32.9 +/- 0.7% in stable transfectants. Because constitutively activated mitogen-activated protein kinase kinase (MEK) pathway has been shown to lead to activated NF-kappaB, we wanted to determine the relative contribution of activated MEK in the human melanoma cells. To test this, MeWo and A375 melanoma cells were exposed to the MEK inhibitor PD184352. Treatment with PD184352 partially reversed NF-kappaB activity but did not impart radiation sensitivity to these cells. Our results indicate that activated NF-kappaB may be one of the pathways responsible for the radioresistance of melanoma cells and that strategies for inhibiting its influence may be useful in restoring the radioresponse of melanomas.

Benzamides↗
PubMed2004

Constitutive expression of human angiostatin in Pichia pastoris using the GAP promoter.

The GAP gene promoter was amplified from P. pastoris GS115 and used to replace the AOX1 promoter (P(AOX1)) on pPIC9K resulting in plasmid pGAP9K. The recombinant expression vector pGAP9K-AS was constructed by inserting the angiostatin gene(AS) into pGAP9K. pGAP9K-AS was then transformed into P. pastoris GS115. The multi-copy integration transformant P. pastoris GS115 (pGAP9K-AS) was used to investigate the constitutive expression of angiostatin in P. pastoris. The expression of angiostatin reached its peak after 4 d of culture in P. pastoris GS115 (pGAP9K-AS) while the angiostatin expressed in P. pastoris GS115 (pPIC9K-AS) after 4 d of induction or 5 d of culture is only 70% of that expressed by P. pastoris GS115 (pGAP9K-AS). The AS expression in inducible system reached the peak after 6 d of induction but the expressed AS was only 86% of that from constitutive system. The results of anti-angiogenic and antitumor activity assay showed that AS expressed from both constitutive and inducible system inhibited the CAM angiogenesis and suppressed B16 melanoma in C57BL/6J mouse and that the tumor inhibition rates reached 90.63% and 90.54%, respectively. The above data indicates that the constitutive promoter P(GAP) can served as an effective alternative to the inductive promoter P(AOX1) to express AS and other proteins in P. pastoris.

Angiogenesis Inhibitors↗
PubMedDate not supplied

[Effect of the constitutive activity of pathogenicity genes in Listeria monocytogenes].

The effect of the constitutive expression of pathogenicity factors in L. monocytogenes was studied on the model of the intraperitoneal infection of mice. The constitutive expression was due to a single amino acid substitution in the transcriptional factor PfrA, the master regulator of L. monocytogenes virulence genes. The effective lethal dose (LD50) for the strain with the constitutive expression of pathogenicity factors was 3 times lower than for the isogenic wildtype strain. When introduced in equal doses, this Listeria strain led to the death of the animals 1-2 days earlier than the wild-type stain. The study revealed that the constitutive expression of pathogenicity factors resulted in faster dissemination of bacteria in the body of the animal during the first 24 hours, but led to their earlier elimination from the internal organs at later stages of infection.

Animals↗
PubMed1991

Use of radiation suicide to isolate constitutive and temperature-sensitive conditional Chinese hamster ovary cell mutants with defects in the endocytosis of low density lipoprotein.

A radiation suicide procedure was used to isolate cells with either constitutive or temperature-sensitive (ts) defects in the receptor-mediated endocytosis of low density lipoprotein (LDL). Mutagen-treated Chinese hamster ovary cells maintained at 34 degrees C (permissive temperature) were shifted to 39.5 degrees C (nonpermissive temperature) for 14-26 h and incubated at 39.5 degrees C for an additional 6-8 h with [3H]cholesteryl linoleate LDL. Wild-type cells internalized this lipoprotein via LDL receptors and accumulated [3H]cholesteryl linoleate (1.5-2 dpm/cell). Radiolysis during 80 days of frozen storage killed most of these cells (radiation suicide). Receptor-deficient cells were identified by screening the surviving cells for their inability to internalize and accumulate 125I-LDL using a replica plating assay. From 3.6 x 10(7) tritium-labeled cells, two clones fell into previously defined constitutive and ts complementation groups (ldlA and ldlG, respectively). Another constitutive and two other ts mutants defined two new complementation groups, ldlI (constitutive) and ldlH (ts). This increases to nine the current number of recessive, LDL receptor-deficient, Chinese hamster ovary complementation groups. All of the mutants with ts defects in LDL endocytosis exhibited ts conditional-lethal phenotypes. At the nonpermissive temperature, the rates of loss of LDL receptor activity (t 1/2 = 10-14 h) were significantly faster than the rates of loss of protein synthesis (t 1/2 greater than 24 h), suggesting that the temperature sensitivity of receptor activity was not simply due to the metabolic collapse of dying cells. Detailed analysis of these new classes of mutants should help define gene products and functions required for LDL receptor activity.

Animals↗
PubMed1991

High expression of exogenous cDNAs directed by HIV-1 long terminal repeat in human cells constitutively producing HIV-1 tat and adenovirus E1A/E1B.

A eukaryotic vector-host cell system is described where the additive transactivating effects of HIV-1 tat and adenovirus E1A on HIV-1 long terminal repeat (LTR) are exploited to increase expression of exogenous cDNAs. Human 143B and 293 cells, the latter constitutively producing E1A, were used as host cell lines. The bacterial gene chloramphenicol acetyltransferase (CAT) and the hepatitis B surface antigen (HBs-Ag) gene were employed as reporter genes inserted in pRPneoU3R, an episomal vector containing BK virus replication origin and early region, where cDNAs are expressed under control of HIV-1 LTR. The 293 cells were transformed by tat expression vectors to constitutively express tat. Stable cell clones of 293tat cells, constitutively expressing CAT after transformation with pRPneoU3R-CAT, show a CAT activity 600-fold higher than normal 293 transformed cells. CAT expression obtained in normal 293 cells can be transiently increased 10-fold by transfection by vectors expressing tat. The 293tat cells transformed by pRPneoU3R-HBs, an episomal vector expressing HBs-Ag from HIV LTR, yielded stable cell clones secreting HBs-Ag in the culture medium at a concentration up to 744 ng/ml or 44 ng/10(6) cells/24 h, 48-fold more than normal 293 cells. The use of this system for constitutive or inducible expression of sequences under control of HIV-1 LTR is discussed in view of possible applications for diagnostic, vaccinal and therapeutic purposes.

Adenovirus Early Proteins↗
PubMed1990

The normal erbB-2 product is an atypical receptor-like tyrosine kinase with constitutive activity in the absence of ligand.

Overexpression of the erbB-2/neu gene is frequently detected in human cancers. When overexpressed in NIH 3T3 cells, the normal erbB-2 product, gp185erbB-2, displays potent transforming ability as well as constitutively elevated levels of tyrosine kinase activity in the absence of exogenously added ligand. To investigate the basis for its chronic activation we sought evidence of a ligand for gp185erbB-2 either in serum or produced by NIH 3T3 cells in an autocrine manner. We demonstrate that a putative ligand for gp185erbB-2 is not contained in serum. Chimeric molecules composed of the extracellular domain of gp185erbB-2 and the intracellular portion of the epidermal growth factor receptor (EGFR) did not show any transforming ability or constitutive autophosphorylation when they were expressed in NIH 3T3 cells. However, they were able to transduce a mitogenic signal when triggered by a monoclonal antibody directed against the extracellular domain of erbB-2. These results provide evidence against the idea that an erbB-2 ligand is produced by NIH 3T3 cells. Furthermore, we obtained direct evidence of the constitutive enzymative activity of gp185erbB-2 by demonstrating that the erbB-2 kinase remained active in a chimeric configuration with the extracellular domain of the EGFR, in the absence of any detectable ligand for the EGFR. Thus, under conditions of overexpression, the normal gp185erbB-2 is a constitutively active kinase able to transform NIH 3T3 cells in the absence of ligand.

Animals↗
PubMed1978

The regulation of urea amidolyase of Saccharomyces cerevisiae: mating type influence on a constitutivity mutation acting in cis.

Constitutivity for the synthesis of the urea amidolyase bienzymatic complex is obtained by durOh mutations located in the regulatory genetic region adjacent to the dur1, dur2 gene cluster. The durOh mutations act only in cis and are a new case of cis effect strongly cancelled in alpha/a diploid, similar to cargA+Oh mutation shown previously to lead to arginase constitutivity. Illegitimate diploids do not show such a cancellation of constitutivity. The constitutivity produced by durOh mutation comprises the process of induction and the release of the glutamine effect. It does not impair the NH+4 effect.

Allophanate Hydrolase↗
PubMed1981

Ursodeoxycholic acid oral tolerance test in patients with constitutional hyperbilirubinemias and effect of phenobarbital.

An oral tolerance test using a 25-mg dose of ursodeoxycholic acid was carried out in 46 patients with constitutional hyperbilirubinemias: 26 with Gilbert's syndrome, 9 with Dubin-Johnson syndrome, 7 with Rotor Syndrome, 3 with constitutional indocyanine green intolerance, and 1 with Crigler-Najjar syndrome type II; they were compared with 14 healthy control subjects. The integrated area under the curve of increments of serum UDCA above the base line between 0 and 2 h after oral administration was significantly increased in patients with Gilbert's syndrome and Dubin-Johnson syndrome, whereas it was normal in patients with Rotor syndrome and constitutional indocyanine green intolerance. As the area under the curve reflects the hepatic clearance of intestinally absorbed ursodeoxycholic acid and therefore gives a quantitative estimate of hepatic ursodeoxycholic acid clearance, the hepatic clearance of ursodeoxycholic acid seems to be impaired in patients with Gilbert's syndrome and Dubin-Johnson syndrome. Administration of phenobarbital, 100 mg/day for 2 wk, improved the ursodeoxycholic acid tolerance test in concordance with the reduction in serum bilirubin in 9 patients with Gilbert's syndrome studied. If the turnover of ursodeoxycholic acid is representative of that of other bile acids, these findings suggest that the hepatic transport of bile acid is impaired like other anions in patients with certain constitutional hyperbilirubinemias.

Administration, Oral↗
PubMed1981

Constitutive and carcinogen-derived DNA binding as a basis for the assessment of potency of chemical carcinogens.

1. The hypothesis is presented that a ground-level DNA damage is unavoidable and must be regarding constitutive to a cell. The genotoxic agents responsible for this type of initiation comprise a number of physiological chemists which are known or suspected to be degraded via chemically reactive species, nitrosamines present in the diet or formed from amines in the stomach, ubiquitous carcinogenic metals, UV- and alpha-irradiation, viruses, and other sources in diet and environment. 2. It is concluded that what is normally called spontaneous tumor incidence is partly due to this constitutive DNA damage. 3. Under this assumption, carcinogens can be divided into two classes, the initiating (genotoxic, DNA-damaging) carcinogens and the non-genotoxic carcinogens which act by modulating any of a number of reactions and side reactions that lead to an increase of the constitutive DNA damage or enhance the chance for the constitutive DNA damage to proceed to a tumor. 4. The carcinogenic potency of a carcinogen is described as a product of persistent DNA damage x mutagenicity x non-genotoxic modulation of the DNA damage, and short-term tests are proposed to determine the contributions of the first two parameters to the potency of an initiating carcinogen. 5. As an approximation to this theoretical approach, a correlation is shown of DNA binding in vivo in the form of a "Covalent Binding Index" to the carcinogenic potency as derived from long-term bioassays. An astonishingly good linear correlation is found with an approximate uncertainty of the estimate of a factor of 10 with a total span of values of 10(6). 6. It is concluded that DNA binding in vivo provides a useful first look at the potency of initiating carcinogens but that additional knowledge is required to assess the organ specificity of initiating carcinogens. 7. The non-genotoxic carcinogens seem to be much less potent than the initiating carcinogens if administered alone. They cannot, however, be spotted on the basis of chemical structure, and there is no short-term test which would allow a good quantitative approach to their carcinogenic potency as was demonstrated for the initiating carcinogens. One main reason for this lack is the wide variety of different mechanisms of action of non-genotoxic carcinogens.

Animals↗
PubMed1995

Identification of constitutive and inducible forms of nitric oxide synthase in human platelets.

A number of cell types possess an L-arginine-nitric oxide (NO) pathway. We studied the presence of constitutive and inducible forms of NO synthase in human platelets. N omega-nitro-L-arginine, an inhibitor of NO synthase, potentiated thrombin-induced aggregation of washed human platelets, whereas L-arginine inhibited it. The direct evidence for the presence of constitutive form of NO synthase came from the observation of conversion of tritium-labeled L-arginine to tritium-labeled L-citrulline by washed platelets suspended in Ca(++)-rich but not in Ca(++)-free buffer. Incubation of washed platelets in Ca(++)-free buffer with cytokines (tumor necrosis factor-alpha and interferon-gamma) or cytokines plus lipopolysaccharide caused a marked increase in the conversion of [3H]L-arginine to [3H]L-citrulline, suggesting the presence of inducible form of NO synthase. Gel electrophoresis identified an approximately 130 kd protein band with NO synthase in the platelet cytosol, which on isolation converted [3H]L-arginine to [3H]L-citrulline. This 130 kd protein required the presence of Ca++, reduced nicotinamide adenine dinucleotide phosphate tetrahydro-L-biopterin, and flavin adenine dinucleotide for expression of NO synthase activity. Platelet sonicates demonstrated presence of nitrite, and its concentrations were lowered by preincubation of platelets with NG-nitro-L-arginine methyl ester and enhanced in cytokine-treated platelets. Reverse-transcription polymerase chain reaction demonstrated messenger RNA expression of the constitutive endothelial (but not brain) and inducible isoforms of NO synthase in platelets. These observations indicate that human platelet cytosol possesses both constitutive and inducible forms of NO synthase.

Amino Acid Oxidoreductases↗
PubMed1994

[Acquired and constitutional neutropenia in children].

The evaluation of a neutropenia first must document its etiology. Besides the particular etiological aspects in the newborn, neutropenia in a child may be 1) acquired, 2) constitutional, part of a complex genetic disease, 3) constitutional, isolated. Primary acquired neutropenia, also called benign chronic neutropenia, is the most frequent cause of chronic neutropenia in children; it is usually well tolerated and has a frequent favorable outcome in 12-14 months. Many complex genetic diseases include a neutropenia, among which several immunologic disorders that must be ruled out before considering the diagnosis of isolated constitutional neutropenia. Infantile agranulocytosis is the main primary constitutional neutropenia. It may be sporadic or hereditary (autosomal recessive or dominant inheritance) and is present at birth. It is profound, usually < 0.5 G/l (< 500/mm3) and exposes to severe pyogenic and fungal infections. In the neonatal period neutropenia must primarily suggest a bacterial infection, although other etiologies have to be known, particularly neonatal neutropenia caused by passive transfer of maternal antibodies and neutropenia related to gravidic maternal hypertension. The treatment of severe chronic neutropenia is directed towards the prevention of infections. It includes prophylactic antibiotherapy, the most commonly used one being the trimetroprim-sulfamethoxazole association, and granulocyte colony stimulating factor (G-CSF). G-CSF has considerably improved the condition of patients; it is usually well tolerated, but secondary effects have been reported (hypersplenism, glomerulonephritis, osteoporosis, vasculitis), and a potential leukemogenic risk has been evoked.

Child↗
PubMed1994

Structural and functional domains critical for constitutive activation of the HGF-receptor (Met).

The MET gene, encoding the tyrosine kinase receptor for Hepatocyte Growth Factor, is a potentially harmful oncogene overexpressed in a significant fraction of human cancers. To study the molecular mechanisms responsible for oncogenic activation, the biochemical and biological properties of a number of MET constructs were analysed. The native heterodimeric receptor (alpha beta), the beta chain alone, as well as a kinase defective mutant did not transform rodent fibroblasts upon transfection. The cytoplasmic domain, truncated immediately below the transmembrane region, acquired constitutive tyrosine kinase activity in vivo, produced foci of transformation, and was tumorigenic in nude mice. Removal of the first 39 amino acids of the juxtamembrane domain resulted in loss of constitutive activation in vivo and transforming potential, without impairment of the in vitro kinase activity. Replacement of the juxtamembrane domain with 5' TPR sequences restored constitutive kinase activation and transforming properties. Site-directed mutagenesis of either of the two tyrosine residues involved in the positive regulation of the catalytic activity upon phosphorylation (Y1234 or Y1235 in the kinase domain of the HGF receptor), strongly impaired TRP-MET transforming potential. These data show that: (1) the truncated cytoplasmic HGF receptor has constitutive kinase activity and is oncogenic; (2) the first 39 amino acids of the juxtamembrane domain and (3) the regulatory tyrosines in the catalytic domain are required to unleash its transforming potential.

3T3 Cells↗
PubMedDate not supplied

Specific pattern of unconjugated bilirubin during fasting can identify constitutional hyperbilirubinemia.

The pattern of individual bilirubin pigments during a 24 hour caloric restriction (400 Kcal) was investigated in three groups of patients: one group with constitutional hyperbilirubinaemia (n = 29), another with microcytaemia and signs of haemolysis (n = 15) and the third consisting of patients without signs of hepatic disease (n = 11). The different bilirubin fractions (unconjugated, mono- and di-conjugated) were separated as methylated tetrapyrroles by t.l.c. following alkaline methanolysis. In all patients fasting induced an enhancement of the unconjugated bilirubin while the concentration of mono- and di-methyl esters of conjugated bilirubins remained within the normal range. When a pre-fasting concentration of total bilirubin equal to 1.2 mg/dl was used as a discriminating point, two different patterns in the fasting-induced increase in unconjugated bilirubin were identified. An increase of more than 1 mg/dl was found in 30 patients and 28 of these had constitutional hyperbilirubinaemia as diagnosed by exclusion of other disorders. The group with an increase of less than 1 mg/dl was composed of 25 patients, only one of whom had constitutional hyperbilirubinaemia: 14 were affected by microcytaemia and 10 were outpatients without signs of disease. The "fasting-induced increase in unconjugated bilirubin" has a specificity of 78%, a sensitivity of 84% and a positive and negative predictive value of 85 and 76%, respectively, for the diagnosis of constitutional hyperbilirubinaemia.

Adolescent↗
PubMed1993

Constitutively active mutants of the alpha 2-adrenergic receptor.

We have mutated a single residue, Thr373 [corrected], in the C-terminal portion of the third intracellular loop of the alpha 2C10-adrenergic receptor into five different amino acids. In analogy with the effect of similar mutations in the alpha 1B- and beta 2-adrenergic receptors, these substitutions resulted in two major biochemical modifications: 1) increased constitutive activity of the alpha 2-adrenergic receptor leading to agonist-independent inhibition of adenylyl cyclase and 2) increased affinity of the receptor for binding agonist but not antagonists. The increased constitutive activity of the mutated alpha 2-adrenergic receptors could be inhibited by pertussis toxin, clearly indicating that it results from spontaneous ligand-independent receptor coupling to Gi. In contrast, the increased affinity of the mutant receptors for binding agonists was unaffected by pertussis toxin treatment, indicating that this is an inherent property of the receptors not dependent on interaction with Gi. Coexpression of the receptor mutants with the receptor-specific kinase, beta ARK1, indicated that the constitutively active alpha 2-adrenergic receptors are substrates for beta-adrenergic receptor kinase (beta ARK)-mediated phosphorylation even in the absence of agonist. These findings strengthen the idea that constitutively active adrenergic receptors mimic the "active" state of a G protein-coupled receptor adopting conformations similar to those induced by agonist when it binds to wild type receptors. In addition, these results extend the notion that in the adrenergic receptor family the C-terminal portion of the third intracellular loop plays a general role in the processes involved in receptor activation.

Adenylate Cyclase Toxin↗
PubMed1995

Vectorial secretion by constitutive and regulated secretory pathways in mammary epithelial cells.

Lactating mammary epithelial cells synthesize large quantities of milk proteins, which they secrete vectorially at the apical membrane into the alveolar lumen of the gland. Recent work suggested that mammary protein secretion is not wholly constitutive, but may also occur in part through a regulated secretory pathway. This study used mouse mammary epithelial cells cultured on Engelbreth-Holm-Swarm (EHS) matrix to compare the proportions of basally and apically-directed proteins secreted constitutively or in a regulated manner. On EHS matrix, mammary cells formed mammospheres, multicellular structures enshrouded in matrix material, within which they became polarised, formed tight intercellular junctions, and secreted milk proteins vectorially. Protein secreted basolaterally was collected in culture medium, whereas apically-secreted milk proteins accumulated in a closed lumen within the mammosphere, and were recovered by EGTA treatment of the cells in situ. Protein secretion was measured by following the release of radiolabelled protein after pulse-labelling with [35S]-methionine. Basolateral and apical secretion of [35S]-protein appeared complete within 1 h of pulse-labelling, consistent with immediate secretion through constitutive secretory pathways. However, addition of the calcium ionophore ionomycin induced a second wave of secretion in both directions. Ca(2+)-stimulated secretion occurred within 15 min of ionomycin addition, doubled the extent of basolateral and apical secretion, but did not change the populations of proteins secreted. Ionomycin treatment did not affect mammosphere morphology or mammary cell ultrastructure. The results suggest that lactating mammary epithelial cells secrete proteins apically and basolaterally by two pathways, one a Ca(2+)-independent constitutive pathway, the other a regulated pathway stimulated by elevation of intracellular Ca2+.

Animals↗