Expression of human phosphodiesterase 3A gene using baculovirus expression system in insect cell / 药学学报

<p><b>AIM</b>To investigate the expression of recombinant human phosphodiesterase 3A (HPDE3A) using baculovirus expression system in Tn cell line.</p><p><b>METHODS</b>The HPDE3A cDNA was recombined with baculovirus, and then the recombinant was transfected into Tn cell line. The expression of HPDE3A in Tn cell line was detected and identified by the RT-PCR, SDS-PAGE and Western blotting.</p><p><b>RESULTS</b>The recombinant HPDE3A protein was stably expressed in Tn cell line and detected by the distinct morphological changes of Tn cell, RT-PCR, SDS-PAGE and Western blotting using polyclonal antibody. The M(w) of the recombinant protein was about 120 kD.</p><p><b>CONCLUSION</b>Recombinant HPDE3A can be expressed in Tn cell line using the baculovirus expression system, and thus provided the basic material for studying its bioactivity and application in screening for HPDE3A inhibitor.</p>

Effects of phosphodiesterase 4 inhibitor on cough response in guinea pigs sensitized and challenged with ovalbumin / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>There is currently considerable interest in the potential value of selective inhibitors of cyclic nucleotide phosphodiesterase 4 in the treatment of asthma. However, whether they influence eosinophilic airway inflammation-associated cough remains unclear. The objective of this study was to investigate the effects of selective phosphodiesterase 4 inhibitor SB207499 on cough response and airway inflammation in guinea pigs sensitized and challenged with ovalbumin.</p><p><b>METHODS</b>Forty sensitized guinea pigs were randomly divided into four groups: control (n = 10), challenge (n = 10), SB207499 (n = 10) and aminophylline (n = 10), then challenged with aerosol of 1% ovalbumin or saline. Two hours later, animals were intraperitoneally injected with either saline, 25 mg/kg of SB207499 or aminophylline. At the 24th hour, the injection was repeated with 2.5 mg/kg and 25 mg/kg SB207499 or aminophylline, then cough response to inhaled capsaicin and airway responsiveness to methacholine inducing a 150% of the peak airway pressure to the baseline (PC150) was measured. Finally, total cell number and differentials in bronchoalveolar lavage fluid were analysed.</p><p><b>RESULTS</b>The cough frequency per 3 minutes and PC150 in the challenge group were (22 +/- 4) times/3 minutes and (198 +/- 54) microg/ml, which were significantly different from (6 +/- 2) times/3 minutes and (691 +/- 81) microg/ml in the control group (P < 0.05, respectively). The injection of 25 mg/kg SB207499 significantly inhibited the increased cough response and airway hyperresponsiveness, the cough frequency and PC150 in guinea pigs were (13 +/- 2) times/3 minutes and (680 +/- 81) microg/ml (P < 0.05), which differed significantly from (18 +/- 2) times/3 minutes and (400 +/- 86) microg/ml after the administration of the same dose of aminophylline (P < 0.05). The inhibition of SB207499 on cough response was dose-dependent. Similarly, SB207499 decreased the total cell number and percentage of eosinophils in bronchoalveolar lavage fluid to (2.1 +/- 0.5) x 10(6)/ml and (20 +/- 5)% respectively, which were significantly different from (3.2 +/- 0.5) x 10(6)/ml and (29 +/- 5)% in the aminophylline group (P < 0.05, respectively) or (4.2 +/- 0.7) x 10(6)/ml and (35 +/- 4)% in the challenge group (P < 0.05, respectively).</p><p><b>CONCLUSION</b>Phosphodiesterase 4 inhibitor may be more useful than aminophylline for cough associated with eosinophilic airway inflammation via inhibiting airway inflammation and airway hyperresponsiveness.</p>

Adaptation of cAMP signaling system in SH-SY5Y neuroblastoma cells following expression of a constitutively active stimulatory G protein alpha, Q227L Gsalpha

Heterotrimeric GTP-binding proteins (G protein) are known to participate in the transduction of signals from ligand activated receptors to effector molecules to elicit cellular responses. Sustained activation of cAMP-G protein signaling system by agonist results in desensitization of the pathway at receptor levels, however it is not clear whether such receptor responses induce other changes in post-receptor signaling path that are associated with maintenance of AMP levels, i.e. cAMP-forming adenylate cyclase (AC), cAMP-degrading cyclic nucleotide phosphodiesterase (PDE) and cAMP-dependent protein kinase (PKA). Experiments were performed to determine the expression of AC, PDE, and PKA isoforms in SH-SY5Y neuroblastoma cells, in which cAMP system was activated by expressing a constitutively activated mutant of stimulatory G protein (Q227L Gsalpha). Expression of ACI mRNA was increased, but levels of ACVIII and ACIX mRNA were decreased. All of the 4 expressed isoforms of PDE (PDE1C, PDE2, PDE 4A, and PDE4B) were increased in mRNA expression; the levels of PKA RIalpha, RIbeta, and RIIbeta were increased moderately, however, those of RIIalpha and Calpha were increased remarkably. The activities of AC, PDE and PKA were also increased in the SH-SY5Y cells expressing Q227L Gsalpha. The similar changes in expression and activity of AC, PDE and PKA were observed in the SH-SY5Y cells treated with dbcAMP for 6 days. Consequently, it is concluded that the cAMP system adapts at the post-receptor level to a sustained activation of the system by differential expression of the isoforms of AC, PDE, and PKA in SH-SY5Y neuroblastoma. We also showed that an increase in cellular cAMP concentration might mediate the observed changes in the cAMP system.

Effect of acute administration of isoproterenol on submandibular salivary gland of female rat.

The functions of salivary glands are under the regulation of both sympathetic as well as parasympathetic nerve fibers. Further, it has also been demonstrated that chronic administration of a beta-adrenergic agonist isoproterenol (IPR) results in hypertrophy and hyperplasia of submandibular gland [Schneyer C A, Am J Physiol, 203 (1962) 232]. Specific purpose of the present attempt was to look for metabolic responses of submandibular gland of oestrous female rats at very short intervals after 10 min of administration of 5, 10 and 15 micrograms of IPR to females in oestrous condition; pharmacological action and clearance time being only 8 min. The results indicated significant reduction in case of enzymic activities of phosphorylase, total ATPase and Na(+)-K+ ATPase. Cyclic AMP-specific phosphodiesterase and succinate dehydrogenase activities were suppressed only with 5 micrograms dose, but with rising dose levels the effect was not so apparent. Protein content of the gland was reduced slightly by administration of IPR. Hence, it became clear that submandibular gland responds rapidly to IPR administration. Implications of these observations are discussed.

Differential changes in the expression of cyclic nucleotide phosphodiesterase isoforms in rat brains by chronic treatment with electroconvulsive shock

Electroconvulsive shock (ECS) has been suggested to affect cAMP signaling pathways to exert therapeutic effects. ECS was recently reported to increase the expression of PDE4 isoforms in rat brain, however, these studies were limited to PDE4 family in the cerebral cortex and hippocampus. Thus, for comprehensive understanding of how ECS regulates PDE activity, the present study was performed to determine whether chronic ECS treatment induces differential changes in the expression of all the PDE isoforms in rat brains. We analyzed the mRNA expression of PDE isoforms in the rat hippocampus and striatum using reverse transcription polymerase chain reaction. We found chronic ECS treatment induced differential changes in the expression of PDE isoform 1, 2, 3, 4, 5 and 7 at the rat hippocampus and striatum. In the hippocampus, the expression of PDE1A/B (694%), PDE4A (158%), PDE4B (323 %), and PDE4D (181%) isoforms was increased from the controls, but the expression of PDE2 (62.8%) and PDE7 (37.8%) decreased by chronic ECS treatment. In the striatum, the expression of PDE1A/B (179%), PDE4A (223%), PDE4B (171%), and PDE4D (327%) was increased by chronic ECS treatment with the concomitant decrease in the expression of PDE2 (78.4%) and PDE3A (67.1%). In conclusion, chronic ECS treatment induces differential changes in the expression of most PDE isoforms including PDE1, PDE2, PDE3, PDE4, PDE5, and PDE7 in the rat hippocampus and striatum in an isoform- and brain region-specific manner. Such differential change is suggested to play an important role in regulation of the activity of PDE and cAMP system by ECS.

The Effect of Pentoxifylline on IL-1beta and TNF-alpha mRNA Gene Expression in Hypoxic-Ischemic Brain Injury of Immature Rat / 대한소아신경학회지

PURPOSE: Interleukin-1beta(IL-1beta) and Tumor necrosis factor-alpha(TNF-alpha) are multifunctional cytokines that may play important roles both in the normal development of central nervous system and in the response of brain to diverse forms of injury. IL-1beta and TNF-alpha have potent proinflammatory action and the potential to modulate cell growth. Cerebral hypoxia-ischemia selectively stimulates IL-1beta and TNF-alpha gene expression in brain regions susceptible to irreversible injury in perinatal rats. Pentoxifylline, a cAMP phosphodiesterase inhibitor, attenuates hypoxic-ischemic brain injury in immature rats and inhibits TNF-alpha expression at the transcription level. We hypothesize that pentoxifylline would attenuate the expression of IL-1beta and TNF-alpha mRNA gene expression on hypoxic-ischemic brain injury in immature rats. METHODS: To elicit focal hypoxic-ischemic brain injury, 7-d-old(P7) rats underwent right carotid artery ligation, followed by 3 hr of hypoxia(fractional concentration of inspired O2=0.08). In 3 rats, pentoxifylline(40mg/kg) was injected into the intraperitoneal cavity immediately before and after hypoxia. The other 4 rats were given PBS solutions. IL-1beta and TNF-alpha mRNA content were measured by reverse transcription followed by polymerase chain reaction amplification(RT-PCR) in the samples prepared from the lesioned and contralateral hemispheres killed 4 hr post-hypoxia. cDNA were amplified with primers specific for IL-1beta and TNF-alpha. and also amplified with GAPDH primers which served as an internal control. RESULTS: In control group, hypoxia-ischemia induced IL-1beta and TNF-alpha mRNA expression from the lesioned hemisphere in immature rat brain. In pentoxifylline treated group, IL-1beta and TNF-alpha mRNA expression were attenuated at 4 hr post hypoxia- ischemia. CONCLUSION: Preteatment with pentoxifylline decreased incidence and severity of hypoxic-ischemic injury in immature rat brain. Pentoxifylline attenuated the expression of IL-1beta and TNF-alpha gene on hypoxic-ischemic injury in immature rat brain. IL-1beta and TNF-alpha may play important roles in the response of the developing brain to acute hypoxic-ischemic injury.

Effect of milrinone on ethanol and halothane induced cardiodepression

Se estudió el efecto inotrópico de milrinona en preparaciones de corazón aislado de rata y de gato. En aurícula de rata con latido espontáneo se observó un efecto inotrópico positivo inicial dosis-dependiente, con aumento máximo en la tensión desarrolada (TD) de +26.5 ñ 2.8% con la dosis de 25 microng/ml y un aumento máximo de la frecuencia espontánea de 60 ñ 6% con la dosis de 80 microng/ml. Sin embargo, en aurícula de rata elétricamente dirigida, el aumento máximo de TD fue de +39.2 ñ 3.4%. La diferencia entre ambas preparaciones puede ser consecuencia del fenómeno de la escalera negativa que limita el aumento en TD en aurícula de rata con latido espontáneo. En aurícula aislada de gato eléctricamente dirigida, el efecto inotrópico positivo de milrinona se inició con la dosis de 0.5 microng/ml y el efecto máximo (36% de aumento en TD) se obtuvo con la dosis de 8 microng/ml. En músculo papilar de gato la relación dosis-respuesta se desplazó hacia arriba y hacia la ezquierda si se compara con preparaciones de aurícula de rata, y el aumento máximo de la TD fue de alrededor de 90%. En preparaciones de aurículas aisladas de rata el etanol (22 y 44 mM) produjo una significativa disminución de la TD dosis-relacionada, que fue revertida por milrinona y su relación dosis-efecto fue similar tanto en presencia como en ausencia de etanol. En músculo papilar aislado de gato, el etanol (rr mM) produjo una disminución de la TD de 15.1 ñ 1.8%. Milrinona en concentraciones bajas de hasta 8 microng/ml prod

Cyclic amp, adenylate cyclase and cyclic amp-phosphodiesterase activities in diabetic rat adipocytes

En el presente trabajo se estudió el efecto de la diabetes inducida por estreptozotocina sobre el contenido de AMP cíclico, y las actividades de adenilato ciclasa y fosfodiesterasa de AMP cíclico en adipocitos de rata. Los resultados muestran un aumento del contenido intracelular de AMP cíclico en células grasas de animales diabéticos. La actividad de adenilato ciclasa basal y en respuesta a la noradrenalina fue mayor en membranas de adipocitos provenientes de ratas diabéticas. La actividad de adenilato ciclasa estimulada por fluoruro fue la misma en ambas preparaciones de membrana (normales y diabéticas). Por otra parte, los adipocitos de ratas diabéticas tuvieron una mayor actividad de fosfodiesterasa de AMP cíclico (bajo y alto Km) con respecto a los controles. Los resultados sugieren que la deficiencia de insulina presente en el estado diabético induce un aumento en la actividad de adenilato ciclasa, la cual estimula la producción de AMP cíclico. Esta mayor producción de AMP cíclico promueve una mayor actividad de fosfodiesterasa, la cual no alcanza a hidrolizar todo el AMP cíclico formado como consecuencia de una mayor actividad de adenilato ciclasa