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1.
Bone ; 106: 42-51, 2018 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-28974461

RESUMEN

BACKGROUND: Lost joint range of motion (ROM) is common in chronic osteoarthritis, alters regional weight-bearing across the articular surfaces, and contributes to loss of cartilage and bone alterations. Limited data exist on the regional effects on joints subjected to chronic losses of ROM. OBJECTIVE: To characterize the regional replacement by bone as part of articular cartilage degeneration after prolonged immobilization. METHODS: Eleven rat knees were rigidly-immobilized in flexion for 32weeks with contralateral and sham-operated (n=6) knees as controls. Sagittal medial tibial epiphysis histological sections assessed the anterior (non-weight-bearing), middle and posterior (both weight-bearing) regions. We quantified the distribution of collagen I, collagen II, cartilage thickness, glycosaminoglycan (GAG) staining, Mankin scoring, and subchondral bone plate cross-sectional area. Using immunohistochemistry (IHC), we visualized blood vessels, osteoblasts, and mesenchymal stem cells (MSCs). RESULTS: Immobilized cartilage had increased collagen I content in the anterior tibial region with picrosirius red staining (immobilized=61±20%; contralateral=43±12%, p=0.033; sham=20±10%, p=0.028) and collagen I IHC (immobilized=40±10%; contralateral=11±4%, p=0.003; sham=5±3%, p=0.043). Articular cartilage was thinner anteriorly (18±30µm) in immobilized knees versus contralateral (124±40µm, p<0.001) and sham (125±43µm, p=0.043). GAG staining covered 2±4% of the anterior articular area in immobilized knees versus 28±12% contralaterally (p=0.003) and 26±7% in sham (p=0.043). Mankin scores in immobilized knees were 4.7±1.7 versus 0.2±0.4 and 0±0 for contralateral and sham (p=0.003, p=0.042), respectively. The trabecular bone plate area of anterior and posterior regions showed relative loss of cross-sectional area in immobilized knees compared to controls (immobilized/contralateral area ratios of 0.67 and 0.46 respectively, both p=0.003), while the area in the middle region was preserved. Movat's pentachrome stain and CD31 staining showed chondral vascular ingrowth from subchondral bone. Osteocalcin and CD90 MSC staining were decreased in immobilized knees versus contralateral (p=0.003, p=0.036 respectively). CONCLUSIONS: Bony replacement characterizes articular cartilage degeneration of knees immobilized for 32weeks in the anterior, non-weight bearing region of the tibia. Replacement of cartilage by bone may have been mediated by chondral vascularization, suggesting irreversible changes. These findings stress the importance of weight-bearing and joint motion to maintain cartilage structure.


Asunto(s)
Huesos/patología , Cartílago Articular/metabolismo , Cartílago Articular/fisiología , Articulación de la Rodilla/metabolismo , Articulación de la Rodilla/patología , Animales , Colágeno Tipo I/metabolismo , Articulación de la Rodilla/fisiología , Masculino , Modelos Animales , Osteoartritis de la Rodilla/metabolismo , Osteoartritis de la Rodilla/fisiopatología , Ratas , Ratas Sprague-Dawley , Tibia/metabolismo , Tibia/fisiopatología , Soporte de Peso/fisiología
2.
J Appl Genet ; 53(1): 121-3, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22081432

RESUMEN

Genetic studies in purebred Portuguese water dogs (PWD) have previously identified genetic loci controlling skeleton size. The FH2295 genetic marker was reported to control 43.6% of the size variation in this breed. In the present study, we amplified and sequenced the genomic DNA from female PWD of different sizes in the region of the FH2295 genetic marker. Polymerase chain reaction (PCR) products of 700 and 800 bp were generated and sequencing revealed the presence of a microsatellite marker including either 5 or 24 repeats of the tetranucleotide sequence "CTTT". Dogs were divided into groups based on their genotypes: homozygote for the short allele (II) or homozygote for the long allele (BB) or heterozygote (IB). The smallest dogs were homozygous with 24 repeats and the largest dogs were homozygous with five repeats. Genetic transmission of the microsatellite marker appears to follow Mendelian laws since all puppies born to a homozygous small dog genotyped "BB" included one or two "B" allele. We applied a PCR method to characterize the sequence of the previously identified dog genetic marker FH2295 and propose that the length of the microsatellite identified could be used as a predictor for the body size of female PWD.


Asunto(s)
Tamaño Corporal/genética , Perros/genética , Repeticiones de Microsatélite , Alelos , Animales , Tamaño Corporal/fisiología , ADN/genética , Perros/fisiología , Femenino , Marcadores Genéticos , Genoma , Técnicas de Genotipaje , Homocigoto , Patrón de Herencia , Reacción en Cadena de la Polimerasa
3.
Clin Orthop Relat Res ; 456: 92-7, 2007 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-17194956

RESUMEN

Previous studies have established mechanical stimulation of joints is necessary to maintain the structure and function of the articular cartilage. Immobilization of the rat knee joint induces cartilage degeneration and reduces the joint range of motion, two of the clinical parameters used to define a joint contracture. We hypothesized chondrocytes from articular cartilage increase their expression of the chitinase 3-like protein 1 (CHI3L1) gene in response to joint immobility. We selected the CHI3L1 gene on the basis of its identification as a differentially expressed gene in the articular cartilage obtained from immobilized rat knee joints. Expression of CHI3L1 mRNA was increased after 2 and 4 weeks of immobility. A time-course study revealed that CHI3L1 immuno-reactivity was increased at 2 and 4 weeks and return to basal levels at all later time points. CHI3L1 gene adds to the list of differentially expressed genes defining the response of cartilage to joint immobility. Our data confirm a protective role for CHI3L1 in the initial phase of degeneration induced by immobility.


Asunto(s)
Cartílago Articular/citología , Cartílago Articular/metabolismo , Condrocitos/metabolismo , Glicoproteínas/biosíntesis , Inmovilización , Articulación de la Rodilla/metabolismo , Adipoquinas , Animales , Proteína 1 Similar a Quitinasa-3 , Lectinas , Masculino , Ratas , Ratas Sprague-Dawley
4.
Biochem Biophys Res Commun ; 333(1): 247-52, 2005 Jul 22.
Artículo en Inglés | MEDLINE | ID: mdl-15936722

RESUMEN

The capacity of chondrocytes to synthesize and remodel the extracellular matrix of the articular cartilage is influenced by mechanical forces applied to joints. Either abnormally high or low loads are detrimental to articular cartilage. Experimental work on animals suggests that immobilization can alter proteoglycan synthesis and result in thinning and softening of the articular cartilage. Little is known of the effects of joint immobility on the pattern of genes expressed by chondrocytes. This study focused on the induction of Mcl-1 gene expression in a rat model of knee joint immobilization by the method of differential display PCR. Increase in Mcl-1 gene expression in chondrocytes induced by joint immobilization was confirmed by RT-PCR, Northern blotting, and immunohistochemistry. Our results indicate that chondrocytes respond to the complete absence of joint motion by expressing Mcl-1 gene. This expression may be part of a defense strategy by chondrocytes to overcome the impending chondrocyte death and cartilage degeneration induced by joint immobility.


Asunto(s)
Cartílago Articular/metabolismo , Condrocitos/metabolismo , Regulación de la Expresión Génica/fisiología , Inmovilización/métodos , Articulación de la Rodilla/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Animales , Células Cultivadas , Masculino , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Ratas , Ratas Sprague-Dawley
5.
J Rheumatol ; 28(9): 2066-74, 2001 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-11550976

RESUMEN

OBJECTIVE: To measure the levels of prostaglandin endoperoxide H synthase (PGHS) isozymes (or cyclooxygenase, COX) in vivo during the development of joint contractures secondary to immobilization in rats. METHODS: Rats had one knee joint immobilized for up to 32 weeks. Three groups were compared: 47 rats had knee joints immobilized, 38 animals had sham surgery, and 13 unoperated animals served as controls. Levels of PGHS-1 and PGHS-2 enzymes were characterized in the chondrocytes and synoviocytes of the knee joint by immunohistochemistry. Immunostaining intensity was quantified by microscopy using conventional analysis. RESULTS: PGHS-1 level was lower in synoviocytes of the anterior capsule compared with shams (1.3 vs 2.0; p < 0.05). PGHS-2 level was also lower in synoviocytes of the posterior capsule (1.8 vs 2.3; p < 0.05), but higher in chondrocytes at the anterior aspect of the tibia compared with shams (1.6 vs 0.8; p < 0.05). PGHS-2 staining was increased in chondrocytes at the posterior, opposed, and anterior aspects of the tibia compared with controls (1.1, 0.6, 0.8 vs 0.2, 0.1, 0.2, respectively; all p < 0.05). CONCLUSION: Immobility induced joint contractures are characterized by a contrasting cellular pattern of PGHS enzyme levels: decreased in the synovium and increased in the chondrocytes. These findings suggest that chondrocytic PGHS isoenzymes are important in cartilage degradation of contractured joints.


Asunto(s)
Condrocitos/enzimología , Contractura/enzimología , Contractura/patología , Prostaglandina-Endoperóxido Sintasas/metabolismo , Membrana Sinovial/enzimología , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Immunoblotting , Inmunohistoquímica , Articulación de la Rodilla , Masculino , Probabilidad , Prostaglandina-Endoperóxido Sintasas/análisis , Ratas , Ratas Sprague-Dawley , Valores de Referencia , Sensibilidad y Especificidad , Estadísticas no Paramétricas , Membrana Sinovial/citología
6.
Br J Haematol ; 113(4): 878-85, 2001 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-11442478

RESUMEN

Thromboxane A2 (TXA2), synthesized in platelets, is a powerful aggregating agent and vasoconstrictor. To induce platelet aggregation, the platelets' enzyme, prostaglandin endoperoxide H synthase-1 (PGHS-1), first converts arachidonic acid (AA) into prostaglandin H2 (PGH2). PGH2 is then converted by the enzyme thromboxane synthase into TXA2. Finally, TXA2 is secreted and can activate the TXA2 receptor on the platelet surface. The importance of TXA2 in haemostasis has been demonstrated by the presence of a bleeding tendency in patients showing an inherited defect in the TXA2 production pathway. We studied an 18-year-old woman with a lifelong bleeding disorder, moderate thrombocytopenia (55-71 x 109/l) and a prolonged bleeding time (12.5 min). Her platelets aggregated in the presence of both PGH2 and a stable TXA2 analogue, but did not aggregate in the presence of AA. The activity of PGHS-1 in platelets, measured using thin-layer chromatography and radioactive counting of TXA2 formation from [14C]-AA, was reduced to 13% of the activity measured in control subjects. PGHS-1 protein levels, measured using Western blot analysis, were also markedly reduced to 10% of control values. Such levels of PGHS-1 enzyme were too low to sustain platelet aggregation in the patient, even if the enzyme was active. The PGHS-1 protein level was also reduced in the patient's immortalized B lymphocytes, suggesting a systemic expression defect. Northern blot analysis was then carried out with poly (A)+ RNA extracted from the patient's immortalized B lymphocytes. PGHS-1 mRNA was detected as a 2.8-kb band in both the patient and control. The intensity of the band representing the patient's PGHS-1 mRNA was similar to that of the control subject. The Northern blot result suggests a normal transcriptional rate of the PGHS-1 gene for the patient. Therefore, the defect responsible for the reduced levels of PGHS-1 protein is probably post-transcriptional.


Asunto(s)
Trastornos de la Coagulación Sanguínea/enzimología , Prostaglandina-Endoperóxido Sintasas/deficiencia , Adulto , Ácido Araquidónico/metabolismo , Autorradiografía , Linfocitos B/enzimología , Plaquetas/enzimología , Northern Blotting , Western Blotting , Estudios de Casos y Controles , Línea Celular , Femenino , Humanos , Masculino , Persona de Mediana Edad , Agregación Plaquetaria , Prostaglandina-Endoperóxido Sintasas/genética , Prostaglandina-Endoperóxido Sintasas/metabolismo , ARN Mensajero/análisis , Tromboxano A2/biosíntesis , Tromboxano B2/metabolismo
7.
Am J Physiol Renal Physiol ; 281(1): F123-32, 2001 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-11399653

RESUMEN

Our present study has investigated the effect of cyclooxygenase-2 (COX-2) inhibition on prostaglandin E2 (PGE2) receptor expression in M-1 cortical collecting duct cells and measured their response to PGE2. Using a semiquantitative titration analysis method, we show that following the addition of the COX-2-specific inhibitor NS-398, E-prostanoid receptor subtype (EP3 and EP4) mRNA expression was found to increase threefold each vs. the vehicle-treated control. We also observed that EP1 but not EP2 is expressed in M-1 cells and EP2 levels are not induced by NS-398. To determine the status of the PGE2 response on exposure to NS-398, we measured cAMP levels in cells after stimulation with varying concentrations of PGE2, then pretreated the cells with 10 microM NS-398 before PGE2 exposure and found a significant rise in the stimulatory effect of PGE2 on cAMP production. Finally, Western blot analysis of the levels of the EP4 receptor protein in control vs. NS-398-treated cells revealed an induction in protein levels in these cells, correlating with the induction in EP4 mRNA. We conclude that NS-398 upregulates the expression of EP3 and EP4 mRNA in M-1 cells. Also, EP4 protein levels are increased, resulting in an increased stimulation of cAMP production by PGE2.


Asunto(s)
Isoenzimas/antagonistas & inhibidores , Túbulos Renales Colectores/efectos de los fármacos , Nitrobencenos/farmacología , Receptores de Prostaglandina E/metabolismo , Sulfonamidas/farmacología , Animales , Western Blotting , Línea Celular , AMP Cíclico/análisis , Ciclooxigenasa 2 , Dinoprostona/farmacología , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Túbulos Renales Colectores/metabolismo , Ratones , Prostaglandina-Endoperóxido Sintasas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/análisis , Receptores de Prostaglandina E/biosíntesis , Receptores de Prostaglandina E/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
8.
Exp Hematol ; 28(4): 411-21, 2000 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-10781899

RESUMEN

OBJECTIVE: Because the prostaglandin endoperoxide H synthase-1 (PGHS-1)-dependent formation of thromboxane A(2) is an important modulator of platelet function, this pathway represents a pharmacologic target for the inhibition of platelet function by aspirin. The objective of our research was to study how PGHS-1 expression is regulated in platelets. MATERIALS AND METHODS: Because platelets are anucleated, their protein content is a consequence of gene expression in precursor cells known as megakaryocytes. We used the immortalized human megakaryoblastic cell line MEG-01 as a model to study the expression of PGHS-1, because MEG-01 cells can be induced to differentiate into platelet-like structures by adding nanomolar concentrations of 12-0-tetradecanoylphorbol-13-acetate (TPA). We determined the expression profiles of PGHS-1 protein and mRNA in the cells comprising the three different populations of MEG-01 cultures: nucleated floating, nucleated attached, and platelet-like structures. RESULTS: We determined that PGHS-1 protein levels were higher in the nucleated adherent population than in the nucleated floating population. PGHS-1 protein levels were greatest in the anucleated platelet-like population. In contrast, we found that PGHS-1 mRNA levels were highest in the cells that comprised the nucleated adherent population. Addition of TPA induced the expression of PGHS-1 protein and mRNA in all three populations but did not change the relationship of the amount of PGHS-1 protein or mRNA expressed in a given population relative to the other two fractions. We measured the expression of PGHS-1 protein on a cell-by-cell basis in the nucleated MEG-01 populations. We found that the percentage of MEG-01 cells expressing PGHS-1 protein in the adherent population was greater than in the floating population. We measured a time-dependent increase in the percentage of cells that expressed PGHS-1 over a period of 8 days after singular addition of TPA (1.6x10(-8)M). Importantly, we observed that TPA treatment stimulated floating MEG-01 to adhere to the surface of the tissue culture vessel and that, after such treatment, only floating MEG-01 cells suffered a compromised viability. We found that a high percentage of control cells expressed glycoprotein IIb/IIIa and that TPA treatment did not significantly alter this percentage. We did not detect glycoprotein Ib in control cells but did measure a slight increase in the percentage of MEG-01 cells that expressed this antigen in the TPA-treated population. CONCLUSION: We established a correlation between the level of PGHS-1 expression and the overall level of differentiation of MEG-01 cells. PGHS-1 protein expression, which increases consistently over the full course of differentiation, now may be used as an additional and perhaps better index by which to survey megakaryocytes.


Asunto(s)
Diferenciación Celular/fisiología , Isoenzimas/biosíntesis , Megacariocitos/enzimología , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Adhesión Celular , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Ciclooxigenasa 1 , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Isoenzimas/genética , Megacariocitos/citología , Megacariocitos/efectos de los fármacos , Proteínas de la Membrana , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/biosíntesis , Complejo GPIb-IX de Glicoproteína Plaquetaria/biosíntesis , Prostaglandina-Endoperóxido Sintasas/genética , ARN Mensajero/biosíntesis , Acetato de Tetradecanoilforbol/farmacología
9.
Biochem J ; 344 Pt 3: 677-85, 1999 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-10585854

RESUMEN

The enzyme prostaglandin endoperoxide H synthase (PGHS) has a pivotal role in the prostanoid biosynthetic pathway because it catalyses the formation of prostaglandin H(2) (PGH(2)), the common precursor of prostanoids. Two PGHS isoforms have been reported, PGHS-1 and PGHS-2, which have 61% identity (at the amino acid level) and 73% similarity (at the nucleotide level) between the two human enzymes. Transcription of the PGHS-1 gene leads to the formation of two transcripts (2.8 and 5.1 kb); two transcripts of 2.8 and 4.5 kb are produced from the PGHS-2 gene. By Northern blot analysis with the entire coding region of human PGHS-1, 2.8 and 5.1 kb transcripts as well as a novel 4.5 kb transcript were detected in the human megakaryoblastic cell line MEG-01. We designed a strategy to characterize the 4.5 kb PGHS transcript. Probes specific for each PGHS-1 and PGHS-2 were designed on the basis of the 3' untranslated region (3' UTR), where no similarity is present. The 4.5 kb transcript was detected only with the PGHS-1-specific 3' UTR probes and not with the PGHS-2-specific 3' UTR probe. To investigate the origin of the 4.5 kb PGHS-1 transcript, the remaining 947 bp of the 5.1 kb PGHS-1 transcript was generated by 3' rapid amplification of cDNA ends (3' RACE) and sequenced. A non-canonical polyadenylation signal (AAGAAA) located upstream of a potential cleavage site (CA) was found and could generate the 4.5 kb PGHS-1 transcript. Analysis of the sequence also produced several possible G/U-rich elements downstream of the potential cleavage site. An RNA dot-blot with 50 different human tissues was probed with the 4.5 and 5.1 kb PGHS-1-specific probes. A signal for the 4.5 kb PGHS-1 transcript was detected in the bladder and appendix. Signals of lower intensity were detected in the colon, bone marrow, small intestine, uterus, prostate, peripheral leucocyte, lymph node and stomach. In conclusion, our results suggest that the cell line MEG-01, the bladder and the appendix contain a new PGHS-1 transcript of 4.5 kb that can be produced from the PGHS-1 gene and we provide a better strategy for distinguishing PGHS-1 transcripts from PGHS-2.


Asunto(s)
Prostaglandina-Endoperóxido Sintasas/genética , Apéndice/metabolismo , Secuencia de Bases , Plaquetas/enzimología , Línea Celular , Clonación Molecular , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Lipopolisacáridos/farmacología , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Factores de Tiempo , Vejiga Urinaria/metabolismo
10.
J Am Soc Nephrol ; 10(11): 2261-71, 1999 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-10541284

RESUMEN

The cortical collecting duct (CCD) is a major site of intrarenal prostaglandin E2 (PGE2) synthesis. This study examines the expression and regulation of the prostaglandin synthesizing enzymes cyclooxygenase-1 (COX-1) and -2 in the CCD. By indirect immunofluorescence using isoform-specific antibodies, COX-1 and -2 immunoreactivity was localized to all cell types of the murine M-1 CCD cell line. By immunohistochemistry, both COX-1 and COX-2 were localized to intercalated cells of the CCD on paraffin-embedded mouse kidney sections. When COX enzyme activity was measured in the M-1 cells, both indomethacin (COX-1 and -2 inhibitor) and the specific COX-2 inhibitor NS-398 effectively blocked PGE2 synthesis. These results demonstrate that COX-2 is the major contributor to the pool of PGE2 synthesized by the CCD. By Western blot analysis, COX-2 expression was significantly upregulated by incubation with either indomethacin or NS-398. These drugs did not affect COX-1 protein expression. Evaluation of COX-2 mRNA expression by Northern blot analysis after NS-398 treatment demonstrated that the COX-2 protein upregulation occurred independently of any change in COX-2 mRNA expression. These studies have for the first time localized COX-2 to the CCD and provided evidence that the intercalated cells of the CCD express both COX-1 and COX-2. The results also demonstrate that constitutively expressed COX-2 is the major COX isoform contributing to PGE2 synthesis by the M-1 CCD cell line. Inhibition of COX-2 activity in the M-1 cell line results in an upregulation of COX-2 protein expression.


Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Inhibidores de la Ciclooxigenasa/farmacología , Isoenzimas/biosíntesis , Corteza Renal/efectos de los fármacos , Túbulos Renales Colectores/efectos de los fármacos , Nitrobencenos/farmacología , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Sulfonamidas/farmacología , Animales , Células Cultivadas , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Dinoprostona/biosíntesis , Isoenzimas/genética , Corteza Renal/enzimología , Túbulos Renales Colectores/enzimología , Proteínas de la Membrana , Ratones , Prostaglandina-Endoperóxido Sintasas/genética , ARN Mensajero/análisis , Regulación hacia Arriba
11.
Am J Physiol ; 275(6): F904-14, 1998 12.
Artículo en Inglés | MEDLINE | ID: mdl-9843907

RESUMEN

It is widely held that only one prostacyclin (IP) receptor exists that can couple to guanine stimulatory nucleotide binding proteins (Gs) leading to activation of adenyl cyclase. Although IP receptor mRNA is expressed in vascular arterial smooth muscle cells and platelets, with lower level expression in mature thymocytes, splenic lymphocytes, and megakaryocytes, there is no molecular evidence for IP receptor expression in renal epithelial cells. The purpose of the present study was to obtain molecular evidence for the expression and localization of the IP receptor and to study the signaling pathways of IP receptor in rat medullary thick ascending limb (MTAL). Biochemical studies showed that IP prostanoids do not increase cAMP in rat MTAL. However, in the presence of vasopressin, inhibition of cAMP formation by prostacyclin (PGI2) analogs is pertussis toxin sensitive and does not activate protein kinase C. In situ hybridization studies localized IP receptor mRNA expression to MTAL in the rat kidney outer medulla. The results of RT-PCR of freshly isolated RNA from MTAL, with primers specific for the mouse IP receptor cDNA, produced an amplification product of the correct predicted size that contained an expected Nco I endonuclease restriction site. We conclude that rat renal epithelial cells express the IP receptor, coupled to inhibition of cAMP production.


Asunto(s)
Asa de la Nefrona/metabolismo , Prostaglandinas/fisiología , Receptores de Prostaglandina/metabolismo , Transducción de Señal/fisiología , Toxina de Adenilato Ciclasa , Animales , Arginina Vasopresina/farmacología , AMP Cíclico/antagonistas & inhibidores , AMP Cíclico/metabolismo , Activación Enzimática/efectos de los fármacos , Epoprostenol/análogos & derivados , Epoprostenol/farmacología , Asa de la Nefrona/citología , Masculino , Ratones , Toxina del Pertussis , Prostaglandinas/metabolismo , Prostaglandinas/farmacología , Proteína Quinasa C/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Epoprostenol , Receptores de Prostaglandina/genética , Distribución Tisular/fisiología , Factores de Virulencia de Bordetella/farmacología
13.
J Biol Chem ; 271(47): 29569-75, 1996 Nov 22.
Artículo en Inglés | MEDLINE | ID: mdl-8939885

RESUMEN

Prostaglandin endoperoxide H synthase-1 (PGHS-1) is expressed constitutively in murine NIH 3T3 cells and RAW 264.7 cells. PGHS-2 is inducibly expressed in these cells following stimulation with serum or bacterial lipopolysaccharide (LPS), respectively. Reverse transcription-polymerase chain reaction (RT-PCR) analysis established that a variety of G protein-linked and peroxisomal proliferator-activated prostanoid receptors are expressed in both of these cell types. The levels of the EP2 and EP4 prostaglandin E2 (PGE2) receptors and the prostaglandin I2 receptor were changed in these cells by serum or LPS stimulation. Quantitative RT-PCR indicated that the mRNA for the murine EP4 receptor, the butaprost-insensitive PGE2 receptor that couples to Gs, increases 1.5-3-fold in response to serum (NIH 3T3) or LPS (RAW 264.7) with a time course approximating the induction of PGHS-2 expression. To study expression of the EP4 receptor we isolated the mouse EP4 receptor gene; the gene is 10 kilobase pairs (kb) in length and, like other known prostanoid receptor genes, contains three exons and two introns. The first intron is 0.5 kb and is located 16 base pairs (bp) downstream of the translational start site. This is a different location than that of the first introns of other prostanoid receptor genes. The second intron is located immediately following the sixth transmembrane domain at the same position as the second intron of the thromboxane A2 receptor, prostaglandin D2 receptor, prostaglandin I2 receptor, and one of the PGE2 (EP1) receptor genes. A major transcriptional start was detected at -142 bp upstream of the translational start. There are a variety of putative cis-acting elements within 1.5 kb upstream of the translational start site and within the first intron. Promoter analyses of the EP4 receptor gene promoter in RAW 264.7 cells indicated that there is a constitutive negative regulatory region between -992 and -928 bp, a constitutive positive region between -928 and -554 bp, and an LPS/serum-responsive region between -554 and -116 bp.


Asunto(s)
Receptores de Prostaglandina E/genética , Células 3T3 , Animales , Secuencia de Bases , Intrones , Ratones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , ARN Mensajero/genética , Subtipo EP4 de Receptores de Prostaglandina E , Transcripción Genética
14.
J Biol Chem ; 270(33): 19330-6, 1995 Aug 18.
Artículo en Inglés | MEDLINE | ID: mdl-7642610

RESUMEN

Human prostaglandin-endoperoxide H synthase-1 and -2 (hPGHS-1 and hPGHS-2) were expressed by transient transfection of COS-1 cells. Microsomes prepared from the transfected cells were used to measure the rates of oxygenation of several 18- and 20-carbon polyunsaturated fatty acid substrates including eicosapentaenoic, arachidonic, dihomo-gamma-linolenic > alpha-linolenic (delta 9, 12, 15), gamma-linolenic, and linoleic acids. Comparisons of kcat/Km values indicate that the order of efficiency of oxygenation is arachidonate > dihomo-gamma-linolenate > linoleate > alpha-linolenate for both isozymes; while the order of efficiency was the same for hPGHS-1 and hPGHS-2, alpha-linolenate was a particularly poor substrate for hPGHS-1. Gamma-Linolenate and eicosapentaenoate were poor substrates for both isozymes, but in each case, these two fatty acids were better substrates for hPGHS-2 than hPGHS-1. These studies of substrate specificities are consistent with previous studies of the interactions of PGHS isozymes with nonsteroidal anti-inflammatory drugs that have indicated that the cyclooxygenase active site of PGHS-2 is somewhat larger and more accommodating than that of PGHS-1. The major products formed from linoleate and alpha-linolenate were characterized. 13-Hydroxy-(9Z,11E)-octadecadienoic acid was found to be the main product formed from alpha-linoleate by both isozymes. The major products of oxygenation of alpha-linolenate were determined by mass spectrometry to be 12-hydroxy-(9Z,13E/Z,15Z)-octadecatrienoic acids. This result suggests that alpha-linolenate is positioned in the cyclooxygenase active site with a kink in the carbon chain such that hydrogen abstraction occurs from the omega 5-position in contrast to abstraction of the omega 8-hydrogen from other substrates.


Asunto(s)
Ácidos Grasos Insaturados/metabolismo , Isoenzimas/metabolismo , Ácidos Linoleicos/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Animales , Línea Celular , Humanos , Cinética , Ácido Linoleico , Espectrometría de Masas , Oxígeno/metabolismo , Ovinos , Especificidad por Sustrato
15.
J Pharmacol Exp Ther ; 271(2): 927-34, 1994 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-7965814

RESUMEN

We developed an in vitro expression system for accurate kinetic analyses of the inhibition of the human prostaglandin H synthase isozymes (hPGHS-1 and -2) by nonsteroidal anti-inflammatory drugs (NSAIDs). Assays of instantaneous inhibition in which enzyme, 10 microM arachidonate, and an NSAID were mixed simultaneously were used to determine apparent affinities of 14 common NSAIDs for hPGHS-1 and hPGHS-2. All NSAIDs except salicylate had appreciable apparent affinities (IC50 < or = 100 microM) for hPGHS-1. Most NSAIDs also exhibited appreciable affinities toward hPGHS-2, but three prominent exceptions were indomethacin, piroxicam and phenylbutazone. We subsequently performed measurements of time-dependent inhibition in which either (a) enzyme and an NSAID were preincubated before substrate was added to initiate the reactions or (b) recovery of activity after time-dependent inhibition was measured using intact cells preincubated with various NSAIDs. Indomethacin, flurbiprofen, meclofenamate and diclofenac, but not ibuprofen, piroxicam or phenylbutazone, caused time-dependent inhibition of both hPGHS-1 and -2 in vitro. For cells pretreated with flurbiprofen or meclofenamate, hPGHS-2 activities, but not hPGHS-1 activities, were recovered relatively rapidly; with indomethacin, recoveries of hPGHS-1 and hPGHS-2 activities were both slow. hPGHS-2 is thought to be the target of NSAIDs acting as anti-inflammatory agents. However, our results indicate that neither measurements of affinities of NSAIDs for hPGHS-2 conducted in vitro with 10 microM arachidonate nor measurements of time-dependent inhibition of hPGHS-2 always predict whether a compound (e.g., piroxicam or phenylbutazone) has anti-inflammatory activity in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)


Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Inhibidores de la Ciclooxigenasa/farmacología , Isoenzimas/antagonistas & inhibidores , Línea Celular , Humanos , Isoenzimas/análisis , Prostaglandina-Endoperóxido Sintasas/análisis , Factores de Tiempo
16.
J Biol Chem ; 269(18): 13207-15, 1994 May 06.
Artículo en Inglés | MEDLINE | ID: mdl-8175750

RESUMEN

Aspirin (acetylsalicylate) treatment of human (h) prostaglandin endoperoxide H synthase (PGHS)-1 expressed in cos-1 cells caused a time-dependent inactivation of oxygenase activity. Aspirin treatment of hPGHS-2 produced an enzyme which retained oxygenase activity but formed exclusively 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) instead of PGH2. The 15-HETE was exclusively of the 15R configuration. The Km values for arachidonate of native and aspirin-treated hPGHS-2 were about the same suggesting that arachidonate binds to both aspirin-treated and native hPGHS-2 in a similar manner. If, as expected, the formation of 15R-HETE proceeds through abstraction of the 13proS hydrogen from arachidonate, O2 insertion must occur from the same side as the hydrogen abstraction; with all other lipoxygenases and cyclooxygenases, O2 addition is antarafacial. When microsomal hPGHS-2 was incubated with [acetyl-14C]aspirin, the enzyme was acetylated. An S516A mutant of hPGHS-2, which retains enzyme activity, was not acetylated. This indicates that Ser-516 is the site of aspirin acetylation of hPGHS-2; this residue is homologous to the "active site" serine of PGHS-1. An S516N mutant of hPGHS-2 was catalytically active; in contrast, an S516Q mutant lacked cyclooxygenase but retained peroxidase activity. Because in the case of PGHS-1 a smaller asparagine substitution is sufficient to eliminate cyclooxygenase activity, we conclude that the active site of PGHS-2 is slightly larger than that of PGHS-1. An S516M mutant of hPGHS-2 was obtained which resembled aspirin-acetylated hPGHS-2 in that this mutant made 15R-HETE as its major product; however, unlike the aspirin-acetylated hPGHS-2, the Km value of the S516M mutant for arachidonate was 100 times that of native hPGHS-2.


Asunto(s)
Aspirina/metabolismo , Inhibidores de la Ciclooxigenasa/metabolismo , Isoenzimas/metabolismo , Acetilación , Secuencia de Bases , Línea Celular , Humanos , Ácidos Hidroxieicosatetraenoicos/biosíntesis , Isoenzimas/genética , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos
17.
Biochem J ; 295 ( Pt 2): 393-7, 1993 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-8240236

RESUMEN

We have previously shown that the hepoxilins are capable of increasing the intracellular free concentration of calcium ([Ca2+]i) in human neutrophils through a pertussis toxin-sensitive, extracellular calcium-independent pathway involving the mobilization of calcium from internal stores. A subsequent hepoxilin-induced and extracellular calcium-dependent influx of calcium is observed. In an effort to investigate further the role of these compounds in the human neutrophil, we investigated their potential effects on the action of known agonists such as formyl-methionine-leucine-phenylalanine (fMLP), platelet-activating factor (PAF) and leukotriene B4 (LTB4) on the mobilization of calcium. Hepoxilis dose-dependently inhibited the increases in [Ca2+]i induced by fMLP, PAF and LTB4. The hepoxilin concentration required for inhibition was around 100 ng/ml (3 x 10(-7) M). This concentration of hepoxilin did not cause any measurable change in [Ca2+]i. The extent of inhibition of the agonist-evoked rise in [Ca2+]i by hepoxilins was proportional to the increase in the calcium response evoked by hepoxilin beyond its threshold concentration. Additional experiments were carried out to investigate the mechanism for the hepoxilin effect. Using calcium-free medium and in the presence of sufficient amounts of thapsigargin (200 ng/ml) to maximally block the calcium pump (thereby achieving a constant rate of calcium leakage from stores), hepoxilin A3 increased further this rate of calcium leakage, indicating that hepoxilin acts by rapidly draining calcium from stores. Its potential (additional) thapsigargin-like action in blocking the pump, however, cannot be ruled out by these experiments. These observations suggest that the hepoxilins may serve an important negative regulatory function in the agonist-induced mobilization of calcium in these cells by depleting calcium stores.


Asunto(s)
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Calcio/metabolismo , Leucotrieno B4/antagonistas & inhibidores , N-Formilmetionina Leucil-Fenilalanina/antagonistas & inhibidores , Factor de Activación Plaquetaria/antagonistas & inhibidores , Ácido 8,11,14-Eicosatrienoico/farmacología , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Humanos , Técnicas In Vitro , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Estereoisomerismo , Terpenos/farmacología , Tapsigargina
18.
Br J Pharmacol ; 107(3): 808-12, 1992 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-1335339

RESUMEN

1. Hepoxilin A3 (8R and 8S isomers) (HxA3), hepoxilin A3-C (8R and 8S isomers) (HxA3-C) and trioxilin A3 (8S isomer) (TrXA3, the stable derivative of HxA3) were tested for their effects on helicoidal strips of guinea-pig isolated tracheae. 2. None of the compounds (10(-9)-10(-6) M) tested had a direct effect on resting tension of trachea. 3. HxA3 (8S) and HxA3-C (8R) (10(-8) M) produced a significant leftward shift of the log concentration-response curves to neurokinin A (NKA) (EC50 (nM), control = 29.0 +/- 2.8, HxA3 (8S) = 21.7 +/- 3.7, HxA3-C (8R) = 13.8 +/- 3.8, n = 6 for each). Also the maximal response to NKA was significantly increased when the tissues were exposed to these hepoxilins (% of the maximal response to NKA, control = 100, HxA3 (8S) = 114.5 +/- 5.3, HxA3-C (8R) = 139.0 +/- 6.2, n = 6 for each). The threshold concentrations for both hepoxilins was 10(-8) M and their effects were dose-related. 4. Stereochemical specificity was observed. The 8S-isomer of HxA3 was active in potentiating the NKA-induced contraction of the trachea while the 8R isomer was inactive. In contrast, the 8R isomer of HxA3-C was active while the 8S isomer was inactive. The trihydroxy metabolite of the active isomer of HxA3 (8S), i.e. TrXA3 (8S) (10(-6) M), was inactive in potentiating the NKA-induced contraction of the trachea. 5. It is concluded that hepoxilins sensitize the guinea-pig isolated trachea to the potent bronchoconstrictor, NKA.


Asunto(s)
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Músculo Liso/efectos de los fármacos , Neuroquinina A/farmacología , Ácido 8,11,14-Eicosatrienoico/farmacología , Animales , Broncoconstricción/efectos de los fármacos , Sinergismo Farmacológico , Cobayas , Técnicas In Vitro , Masculino , Contracción Muscular/efectos de los fármacos , Estereoisomerismo , Tráquea/efectos de los fármacos
19.
Br J Pharmacol ; 105(2): 297-304, 1992 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-1559126

RESUMEN

1. The vascular activity of two stereoisomers of hepoxilin A3 (HxA3) (8R and 8S) and of its glutathione conjugate, hepoxilin A3-C (HxA3-C) (8R and 8S), was investigated on rat helicoidal strips of thoracic aorta and longitudinal strips of portal vein. 2. Neither of the hepoxilins tested had a direct effect on the tone of the aortic strip or on the spontaneous contractions of the portal vein. However, the noradrenaline (NA)-induced response of these vessels, as expressed by the dose required for half maximal contraction, (EC50) was greater in HxA3 (8S)- and HxA3-C (8R)-treated aorta. Increased frequency and strength of spontaneous contractions of the portal vein were detected at lower concentrations of NA in the presence of hepoxilins. 3. The threshold dose for both hepoxilins was 10(-8) M and their effect was not dose-related beyond 10(-8) M. The effect of hepoxilin appeared after a 45 min incubation period and could be observed even if the compounds were washed out after 15 min. 4. Stereochemical specificity was observed. The 8S isomer of HxA3 was active in potentiating the NA-induced contraction of these vessels while the 8R isomer was inactive. In contrast, the 8R isomer of HxA3-C was active while the 8S isomer was inactive. In both tissues, HxA3 (8S) was more potent than its glutathione conjugate, HxA3-C (8R). 5. In calcium-free buffer or in the presence of a calcium channel blocker (nifedipine 1 microM), no potentiation of NA-induced contraction by hepoxilins could be observed, suggesting the involvement of extracellular calcium in the actions of hepoxilins.6. These experiments suggest that hepoxilins may be involved in the modulation of vascular tone and contractility.


Asunto(s)
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Músculo Liso Vascular/efectos de los fármacos , Norepinefrina/farmacología , Ácido 8,11,14-Eicosatrienoico/farmacología , Animales , Aorta Torácica/efectos de los fármacos , Calcio/fisiología , Sinergismo Farmacológico , Glutatión/farmacología , Técnicas In Vitro , Masculino , Contracción Muscular/efectos de los fármacos , Nifedipino/farmacología , Vena Porta/efectos de los fármacos , Ratas , Ratas Endogámicas , Estereoisomerismo
20.
Biochem Biophys Res Commun ; 179(1): 52-6, 1991 Aug 30.
Artículo en Inglés | MEDLINE | ID: mdl-1883378

RESUMEN

In this paper we show that hepoxilin A3 induces the expression of heat shock protein expression in human neutrophils at a concentration of 100 nM using Western blotting techniques employing the use of a commercial monoclonal antibody to HSP72. No regiospecificity was observed as the 8S enantiomer of HxA3 was as active as the 8R enantiomer of HxA3. Comparison of the effects of HxA3 with 12S-HETE and PGA1 indicated that HxA3 was as effective as 12S-HETE although PGA1 was essentially inactive at the same concentration used for these 12-lipoxygenase products.


Asunto(s)
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Proteínas de Choque Térmico/sangre , Neutrófilos/fisiología , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico , Ácido 8,11,14-Eicosatrienoico/farmacología , Anticuerpos Monoclonales , Western Blotting , Citosol/metabolismo , Proteínas de Choque Térmico/biosíntesis , Humanos , Ácidos Hidroxieicosatetraenoicos/farmacología , Técnicas In Vitro , Cinética , Neutrófilos/efectos de los fármacos , Prostaglandinas A/farmacología
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