Glycerophosphrylocholine (GPC) is a renal medullary compatible organic osmolyte that is derived from choline via phosphatidylcholine, which is catalyzed in part by phospholipase A2 (PLA2) and its degradation by GPC: choline phosphodiesterase (GPC: choline PDE). We found that caffeine elevated intracellular free calcium ([Ca2+]i) and GPC level in cultured MDCK cells, canine kidney epithelial cells, and propose a possible biochemical mechanism. When MDCK cells were incubated for 3 h with 1 to 10 mM caffeine, cellular GPC was elevated in a dose-dependent manner, and this occurred independently of the extracellular osmolality. Caffeine stimulated the rate of [14C]choline incorporation into [14C]GPC and PLA2 activity. Whereas, GPC: choline PDE activity was accompanied by less of increase. These enzyme changes demonstrate the increased net synthesis of MDCK GPC. In order to identify what triggers the PLA2 activation, [Ca2+]i was measured by using a fluorescence dye, Fura-2. Caffeine (10 mM) resulted in a typical transient increase in MDCK [Ca2+]i concentration, and this increase was greatly inhibited by pretreatment of MDCK cells with 10 mM ryanodine for 5 min. Ryanodine (10 mM) also inhibited the caffeine-induced stimulation of PLA2 activity. These findings provide the first evidence that caffeine in MDCK cells causes a ryanodine-inhibitable increase of [Ca2+]i and PLA2 activity, resulting in cellular GPC accumulation.
Animal ; Caffeine/pharmacology* ; Calcium/metabolism* ; Carbon Radioisotopes ; Cell Line ; Choline/metabolism ; Dogs ; Glycerylphosphorylcholine/metabolism* ; Kidney/cytology ; Phospholipases A/metabolism* ; Phospholipases A/drug effects ; Phospholipases A/antagonists & inhibitors ; Phosphoric Diester Hydrolases/metabolism ; Phosphoric Diester Hydrolases/drug effects ; Ryanodine/pharmacology* ; Ryanodine/metabolism

BACKGROUND: Ectonucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1) generates inorganic pyrophosphate, a solute that serves as an essential physiological inhibitor of calcification. Inactivating mutations of ENPP1 are associated with generalized calcification in infancy and an increased risk of developing type 2 diabetes mellitus (T2DM). We hypothesized that the ENPP1 K121Q variant may be associated with increased coronary artery calcification in T2DM patients. METHODS: The study subjects were aged 34 to 85 years and showed no evidence of clinical cardiovascular disease prior to recruitment. A total of 140 patients with T2DM were assessed for their coronary artery calcium (CAC) scores and ENPP1 K121Q polymorphisms were identified. RESULTS: The prevalence of subjects carrying the KQ genotype was 12.9% (n = 18). There were no 121QQ homozygotes. Patients with the KQ genotype did not show a significantly higher CAC score (122 vs. 18; P = 0.858). We matched each patient with the KQ genotype to a respective control with the KK genotype by gender, age, and duration of diabetes. When compared to matched controls, we observed no significant difference in CAC score (P = 0.959). CONCLUSIONS: The ENPP1 K121Q polymorphism does not appear to be associated with coronary artery calcification in patients with T2DM.
Aged ; Calcium ; Cardiovascular Diseases ; Coronary Vessels ; Diabetes Mellitus, Type 2 ; Diphosphates ; Genotype ; Homozygote ; Humans ; Lifting ; Phosphoric Diester Hydrolases ; Prevalence ; Pyrophosphatases

BACKGROUND AND OBJECTIVES: Time-phasic development of nitrate tolerance in cardiovascular diseases is very important because it can contribute to the advent of blunted vasodilation or rebound ischemia even during continuous NTG treatment. In such a condition, we should change the therapeutic regimen of nitrate treatment to prevent the worsening of symptoms. MATERIALS AND METHODS: We created a nitrate-tolerant rat model using an osmotic minipump, and we examined the hemodynamic response to bolus NTG infusion in vivo. We checked the phosphodiesterase (PDE) 1A1 mRNA and protein level by relative quantitative RT-PCR and western blot analysis. We used 8-cpt-cGMP for investigating the development of a time-phasic nitrate tolerance mechanism after nitrate infusion. RESULTS: NTG-treated rats revealed a significant decrease in NTG-induced MAP drop (nitrate tolerance) from 1-day and this continued to the third day. The mRNA and protein levels of PDE1A1 similarly increased during these periods. CONCLUSION: This study revealed the development of time-phasic nitrate tolerance from the the aspects of in vivo hemodynamic responses and PDE 1A1 gene expression, and our work supports the need for further investigation to come up with a different therapeutic strategy and new drugs.
Animals ; Aorta ; Blotting, Western ; Cardiovascular Diseases ; Gene Expression ; Hemodynamics* ; Ischemia ; Models, Animal ; Nitrates ; Phosphoric Diester Hydrolases ; Rats ; RNA, Messenger ; Vasodilation

OBJECTIVETo investigate the changes in the expression of phospholipase C-gamma1tyr783 (PLC-γ1tyr783) in the condylar cartilage of a young rat during functional mandibular protraction. This work also explores the function of PLC-γ1tyr783 in the rat mandibular condylar cartilage bone remodeling, which could provide experimental evidence for clinical bone ortho- pedic work.

METHODSA total of 60 four-week-old male Sprague-Dawley (SD) rats were used in this study. The rats were divided equally and randomly into experimental group and control group. The functional appliances that were fitted to the upper incisors of the animals in the experimental group were worn 24 h a day after the rats were fed for 7 d with homemade pellet feed. The animals in the experimental group, along with their matched controls, were sacrificed after 1, 3, 7, 14, 21, and 28 d. The bilateral condylar was fixed, decalcified, dehyded, and then conventional paraffin embedded. Immunohisto- chemistry of PLC-γ1tyr783 was applied to observe its express distribution and variation.

RESULTSThe expression of PLC-γ1tyr783 decreased gradually in the control group, which showed age-related changes (P > 0.05). On the 14th day, PLC-γ1tyr783 expres- sion in the experimental group was significantly higher than that in the control group. PLC-γ1tyr783 expression began to appear statistically and significantly different between the two groups (P < 0.01).

CONCLUSIONPLC-γ1tyr783 is involved in the bone remodeling process of the rat condylar cartilage after functional mandibular-protraction.

Androgen has been claimed for so long as a pivotal hormone in regulating male sexual function, acting both at the central and peripheral level. We believe that androgen is indeed the main synchronizer of sexual activity regulating libido and enzymes as nitric oxide synthase (NOS) and phosphodiesterase type 5 ( PDE5) , which are crucial for the erectile process. The main action of androgen is to timely adjust the erectile process as a function manifestation of sexual desire, therefore finalizing erection to sex.
Androgens ; pharmacology ; physiology ; Animals ; Male ; Nitric Oxide Synthase ; metabolism ; Penile Erection ; drug effects ; physiology ; Phosphoric Diester Hydrolases ; metabolism ; Rats

PURPOSE: Nitric oxide (NO) and phosphodiesterases (PDEs) play key roles in mediating relaxation of corpus cavernosal smooth muscle by increasing intracellular cGMP level. Here, we investigated effects of NO-donor (sodium nitroprusside, SNP) and penile specific type-V PDE inhibitor (zaprinast) in human and rabbit corpus cavernosal cells and tissues in vitro. MATERIALS AND METHODS: The cultured smooth muscle cells and tissues of human and rabbit corpus cavernosum were treated with increasing concentrations of SNP or zaprinast for 5 and 20 minutes, respectively, and intracellular cGMP levels were measured by radioimmunoassay. Organ bath study was performed to measure the relaxation effects of drugs on precontracted corpus cavernosal muscle strips. RESULTS: Although both NO-donor and type-V PDE inhibitor effectively stimulated the accumulation of cGMP in a dose-dependent manner, magnitude of cGMP increase and specificity of drug were found to be species-dependent. In human corpus cavernosal tissues, cGMP was increased upto 10- and 5-folds by SNP and zaprinast, respectively. However, magnitude of increase was much less in cultured smooth muscle cells. In rabbit, SNP effect was most prominent in cultured cells and effects of SNP and zaprinast were modest in tissues. Both agents also resulted in effective relaxation of human and rabbit cavernosal tissue strips. Similar patterns of dose-response curves were shown between results from the organ bath studies and cGMP radioimmunoassay with cavernosal smooth muscle cells. CONCLUSIONS: Present results show that effects of SNP and zaprinast are not coincident in different species, suggesting possible species-specificities of these two agents. Measurement of cGMP changes in cultured cavernosal smooth muscles cells could be reflected to the relaxation effects of drugs on corpus cavernosal muscle strips.
Baths ; Cells, Cultured ; Cyclic GMP ; Erectile Dysfunction ; Humans* ; Male ; Muscle, Smooth* ; Myocytes, Smooth Muscle ; Negotiating ; Nitric Oxide ; Nitroprusside ; Phosphoric Diester Hydrolases ; Radioimmunoassay ; Relaxation* ; Sensitivity and Specificity ; Signal Transduction*

Phosphodiesterases (PDEs) are functionally diverse enzymes with wide organ and tissue distributions. Of these enzymes, PDE5 has received particular attention largely because of the introduction and widespread use of the selective PDE5 inhibitor sildenafil citrate (Viagra(R)) as an oral therapy for erectile dysfunction(ED). Within the corpus cavernosum of the penis, PDE5 influences regulation of vascular and trabecular smooth muscle contractile tone by enzymatically degrading the cyclic 3',5'guanosine monophosphate(cGMP), the key second messenger. By reversibly inhibiting this enzymatic activity, the competitive inhibitors of PDE5, including sildenafil and newly introducing tadalafil(Cialis(TM)) and vardenafil(Lebitralm(TM)) act as potent 'agonists' of the erectile response. Data from separate 12-week multicenter, randomized, doubleblind, placebocontrolled trials involving sildenafil, as well as tadalafil and vardenafil, have demonstrated that approximately 80% (or more) of men reported improved erections after treatment with each of these PDE5 inhibitors at the upper end of the dosing range. PDE5 inhibitors have been well tolerated. In clinical studies, vasodilator effects(e.g. headache and flushing) have generally been mild, transient, and infrequently associated with premature study discontinuation. The present article reviews the characteristics of new PDE5 inhibitors in experimental and clinical studies.
Citric Acid ; Erectile Dysfunction* ; Headache ; Humans ; Male ; Muscle, Smooth ; Penis ; Phosphodiesterase 5 Inhibitors ; Phosphoric Diester Hydrolases ; Second Messenger Systems ; Sildenafil Citrate ; Tadalafil ; Tissue Distribution ; Vardenafil Dihydrochloride

Uncovering enzyme (UCE), encoded by the human NAGPA, is a trans-Golgi enzyme that adds the mannose-6-phosphate recognition tag on lysosomal enzymes destined for the lysosome. Mutations in NAGPA are known to cause stuttering, a common speech disorder with unknown etiology. The human NAGPA gene is transcribed into two different forms, probably due to alternative splicing. One of them, known as a brain isoform, is lacking exon 8 (102-bp). We performed quantitative real-time PCR for the NAGPA brain and non-brain isoforms in a cDNA panel originating from 16 human tissues and 24 sub-brain regions. According to our findings, the relative quantity of the NAGPA brain isoform in the brain was 4.7 times more than that in the control cDNA, a pooled mixture of equal amounts of cDNAs from the 16 different tissues. Further analysis using the cDNA panel originating from 24 different sub-brain regions revealed that the cerebral cortex contained the largest amount of NAGPA brain isoform. Relative quantity in the cerebral cortex was 8.6 times more than that in the control cDNA (P=0.00004). The lowest quantity of this isoform was detected in cDNA from the pituitary gland. In conclusion, findings of the current study suggest that the cerebral cortex, expressing the highest quantity of the NAGPA brain isoform, might be the region associated with speech function.
Alternative Splicing ; Brain ; Cerebral Cortex ; DNA, Complementary* ; Exons ; Humans* ; Lysosomes ; Mannosephosphates ; Phosphoric Diester Hydrolases ; Pituitary Gland ; Protein Isoforms* ; Real-Time Polymerase Chain Reaction ; Stuttering

Animals ; Anti-Asthmatic Agents ; therapeutic use ; Asthma ; drug therapy ; etiology ; Humans ; Phosphodiesterase Inhibitors ; pharmacology ; Phospholipase D ; physiology ; Phosphoric Diester Hydrolases ; physiology

Increased FcepsilonR1alpha expression with upregulated CD203c expression on peripheral basophils is seen in patients with chronic urticaria (CU). However, there has been no published report on the association between CD203c expression level and clinical disease activity in CU patients. To investigate whether the increase of basophil activation is associated with the disease activity of CU, we measured basophil CD203c expression using a tricolor flow cytometric method in 82 CU patients and 21 normal controls. The relationship between the percentage of CD203c-expressing basophils and clinical parameters was analyzed. The mean basophil CD203c expression was significantly higher in CU patients than in healthy controls (57.5% vs 11.6%, P < 0.001). The basophil CD203c expression in severe CU patients was significantly higher than in non-severe CU (66.5% +/- 23.3% vs 54.0% +/- 23.3%, P = 0.033). Multiple logistic regression analysis indicated that both > or = 72% basophil CD203c expression and urticaria activity score (UAS)> or = 13 were significant predictors of severe CU (P = 0.005 and P = 0.032, respectively). These findings suggest that the quantification of basophil activation with CD203c at baseline may be used as a potential predictor of severe CU requiring another treatment option beyond antihistamines.
Adult ; Autoantibodies/blood ; Basophils/*immunology ; Female ; Flow Cytometry ; Humans ; Immunoglobulin E/blood/immunology ; Male ; Phosphoric Diester Hydrolases/biosynthesis/*immunology ; Pyrophosphatases/biosynthesis/*immunology ; Receptors, IgE/biosynthesis ; Urticaria/*immunology