Biomedical subjects

P Y Wong

Publications and source records attributed to P Y Wong.

At least 19 recordsLinked to original sources

PubMed2004

p53 dephosphorylation and p21(Cip1/Waf1) translocation correlate with caspase-3 activation in TGF-beta1-induced apoptosis of HuH-7 cells.

The p53 tumor suppressor gene product plays an important role in the regulation of apoptosis. Transforming growth factor beta1 (TGF-beta1)-induced apoptosis in hepatic cells is associated with reduced expression of the retinoblastoma protein (pRb) and subsequent E2F-1-activated expression of apoptosis-related genes. In this study, we explored the potential role of p53 in TGF-beta1-induced apoptosis. HuH-7 human hepatoma cells were either synchronized in G1, S and G2/M phases, or treated with 1 nM TGF-beta1. The results indicated that greater than 90% of the TGF-beta1-treated cells were arrested in G1 phase of the cell cycle. This was associated with enhanced p53 dephosphorylation and p21(Cip1/Waf1) expression, which coincided with decreased Cdk2, Cdk4, and cyclin E expression, compared with synchronized G1 cells. In addition, p53 dephosphorylation coincided with caspase-3 activation, and translocation of p21(Cip1/Waf1) and p27(Kip1) into the cytoplasm, all of which were suppressed by caspase inhibition of TGF-beta1-induced apoptosis. Finally, phosphatase inhibition and pRb overexpression partially inhibited p53-mediated apoptosis. In conclusion, the results demonstrated that TGF-beta1-induced p53 dephosphorylation is associated with caspase-3 activation, and cytosolic translocation of p21(Cip1/Waf1) and p27(Kip1), resulting in decreased expression of Cdks and cyclins. Further, p53 appears to mediate TGF-beta1-induced apoptosis downstream of the pRb/E2F-1 pathway.

Apoptosis↗

PubMed2004

Out-patient chronic pain service in Hong Kong: prospective study.

OBJECTIVE: To examine the profile and referral pattern of patients attending an out-patient pain management service in Hong Kong. DESIGN: Prospective cross-sectional survey. SETTING: Regional public hospitals, Hong Kong. PATIENTS: All patients attending out-patient pain management clinics in the New Territories East public hospitals between 1 September and 31 December 2002. MAIN OUTCOME MEASURES: Demographic profiles, referring specialty, pain diagnosis, pain sites, duration and severity of pain, treatment modality, litigation, compensation, and social welfare status. Data were collected using a standardised questionnaire. RESULTS: Two hundred and forty-eight patients were interviewed. Most patients (70%) were middle-aged, with 21% over 60 years. Seventy-nine percent of patients were referred to the clinics either from orthopaedic surgeons (64.1%), general and other surgeons (14.9%), or general practitioners (3.6%). The median (range) duration of pain was 2.3 (0.08-26.7) years. The most common pain diagnoses were musculoskeletal back pain (46.4%) and neuropathic pain (27.8%). A total of 11.3% of the patients had two pain diagnoses, while 40.7% complained of pain in more than one location. Pain in the limbs was the most frequent complaint followed by the head, neck, and back. Approximately 38% of patients had tried four or more treatment modalities. Oral medication was the most common method (86.7%) of pain-relief treatment. More than half of the patients had also tried physiotherapy and traditional Chinese medicine. Approximately 37% of the patients were unemployed, while 31% were receiving social security subsidy. Eighty-six patients had pain associated with a work-related injury, and of these patients, 80% were involved in compensation claims. CONCLUSIONS: The profile of patients referred to the pain management clinics was complex. Patients were mainly referred from specialists. The economic implication in this group of patients is likely to be significant as many patients utilised multiple treatment modalities, were unemployed and on social welfare benefits, and were involved in compensation and litigation proceedings.

Chronic Disease↗

PubMed2001

Engineered antibodies with increased activity to recruit complement.

This manuscript describes two sites in a human IgG1 that, when mutated individually or in combination, result in a dramatic increase in C1q binding and complement-dependent cytotoxicity activity. These two residues, K326 and E333, are located at the extreme ends of the C1q binding epicenter in the C(H)2 domain of a human IgG. A mutation to tryptophan at K326 debilitates Ab-dependent cell-mediated cytotoxicity activity. In addition, substitutions of the residues E333 with serine and of K326 with tryptophan in a human IgG2 confer biological activity in the complement-dependent cytotoxicity assay in which the wild-type IgG2 is inactive. This study reveals that the residues K326 and E333 play a significant role in the control of the biological activity of an IgG molecule and can rescue the activity of an inactive IgG isotype.

Amino Acid Substitution↗

PubMed2001

Pattern of cytokine and adhesion molecule mRNA in hapten-induced relapsing colon inflammation in the rat.

We examined the mRNA expression of cytokines, chemokines, integrins, and selectins in colon lesions of rat colitis with a semi-quantitative RT-PCR assay. Rat colitis was induced by trinitrobenzene sulfonic acid (TNBS) in 50% ethanol. Within 24 h, an acute inflammation occurred with hyperemia, edema, necrosis and an intense infiltration of granulocytes in the mucosa. The lesion proceeded into a T-lymphocyte/monocyte-driven chronic inflammation for two weeks and healed in 6 weeks. An acute inflammation recurred at the same site when the recovered animals were systemically injected with TNBS. We isolated RNA from colon tissue at 24 h, 1, 2, 4, 6 weeks after TNBS treatment and from the relapsed animals. The mRNA for cytokines IL-1beta, IL-6, IL-10 and the chemokines CINC, MIP-1alpha, MCP-1 were significantly (P < 0.05) elevated and persisted for 2 weeks, decreased in 6 weeks and increased again during relapse. IFN-gamma mRNA stayed at control levels initially, but increased dramatically in the second weeks of chronic inflammation as well as in relapse. The mRNA levels of adhesion molecules, ICAM-1, VCAM-1, the mucosal homing integrin beta7 as well as P- and E-selectin were greatly enhanced between 1 and 3 weeks. The data showed that the chronically inflamed tissue expresses a time-dependent changing pattern of TH1 cytokines and adhesion molecules that maintain the infiltration and activation of inflammatory cells and tissue injury.

Animals↗

PubMed2001

Coordination exercise and postural stability in elderly people: Effect of Tai Chi Chuan.

OBJECTIVE: To evaluate the effects of coordination exercise on postural stability in older individuals by Chinese shadow boxing, Tai Chi Chuan (TCC). DESIGN: Cross-sectional study. SETTING: Research project in a hospital-based biomechanical laboratory. PARTICIPANTS: The TCC group (n = 25) had been practicing TCC regularly for 2 to 35 years. The control group (n = 14) included healthy and active older subjects. INTERVENTION: Static postural stability test: progressively harder sequential tests with 6 combinations of vision (eyes open, eyes closed, sway-referenced) and support (fixed, sway-referenced); and dynamic balance test: 3 tests of weight shifting (left to right, forward-backward, multidirectional) at 3 speeds. MAIN OUTCOME MEASURES: Static and dynamic balance of Sensory Organization Testing (SOT) of the Smart Balance Master System. RESULTS: In static postural control, the results showed no differences between the TCC or control group in the more simple conditions, but in the more complicated SOT (eyes closed with sway surface, sway vision with sway surface), the TCC group had significantly better results than the control group. The TCC group also had significantly better results in the rhythmic forward-backward weight-shifting test. Duration of practice did not seem to affect the stability of elder people. CONCLUSION: The elderly people who regularly practiced TCC showed better postural stability in the more challenged conditions than those who do not (eg, the condition with simultaneous disturbance of vision and proprioception). TCC as a coordination exercise may reduce the risk of a fall through maintaining the ability of posture control.

Adolescent↗

PubMed2001

Expression of cystic fibrosis transmembrane conductance regulator in rat efferent duct epithelium.

The expression of cystic fibrosis transmembrane conductance regulator (CFTR) was studied in rat efferent ducts. Under whole-cell patch-clamp condition, efferent duct cells responded to intracellular cAMP with a rise in inward current. The cAMP-activated current exhibited a linear I-V relationship and time- and voltage-independent characteristics. The current was inhibited by the Cl(-) channel blocker diphenylamine 2,2'-dicarboxylic acid (DPC) in a voltage-dependent manner and reversed at 24 +/- 0.5 mV, close to the equilibrium potential for Cl(-) (30 mV), suggesting that the current was Cl(-) selective. The cAMP-activated current displayed a permeability sequence of Br(-) > Cl(-) > I(-). Short-circuit current measurement in cultured rat efferent duct epithelia also revealed a cAMP-activated inward current inhibitable by DPC. These electrophysiological properties of the cAMP-activated Cl(-) conductance in the efferent duct were consistent with those reported for CFTR. In support of the functional studies, reverse transcription polymerase chain reaction revealed the presence of CFTR message in cultured efferent duct epithelium. Immunohistochemical studies in intact rats also demonstrated CFTR protein at the apical membrane of the principal cells of efferent duct. CFTR may play a role in modulating fluid transport in the efferent duct.

Animals↗

PubMed2001

Expression of the cystic fibrosis transmembrane conductance regulator in rat spermatids: implication for the site of action of antispermatogenic agents.

To establish whether cystic fibrosis transmembrane conductance regulator (CFTR) is functionally expressed in the testis, we subjected spermatogenic cells from rat testes to analysis of CFTR mRNA, protein and channel activity. CFTR mRNA was detected in the testes of mature but not immature rats using reverse transcription-polymerase chain reaction analysis. Western blot analysis performed with a CFTR specific antibody revealed immunoreactivity in the membrane extract of spermatogenic cells. Immunohistochemical studies localized CFTR in round and elongated spermatids, but not in the fully developed spermatozoa. Using a whole-cell patch clamp technique, we recorded an inward current activated by intracellular cAMP (100 micromol/l) in round spermatids. The current displayed a linear I / V relationship and was inhibited by diphenylamine-2-carboxylate (DPC), a chloride channel blocker. Transfection of the rat germ cell CFTR cDNA into human embryonic kidney (HEK) 293 cells caused the expression of a cAMP-activated chloride current with CFTR characteristics. The current was completely blocked by the antispermatogenic agents 1-(2,4-dichlorobenzyl)-indazole-3-carboxylic acid, lonidamine (500 micromol/l) and 1-(2,4-dichlorobenzyl)-indazole-3-acrylic acid, AF2785 (250 micromol/l). These results taken together provide evidence that CFTR is differentially expressed in spermatids during spermiogenesis. We speculate that CFTR may interact with aquaporin to bring about cytoplasmic volume contraction which is an essential feature of spermiogenesis.

Animals↗

PubMed2001

Expression of multiple Na+/H+ exchanger isoforms in cultured epithelial cells from rat efferent duct and cauda epididymidis.

Although earlier work has pointed to the presence of Na+/H+ exchangers (NHEs) in the rat epididymis, little is known about the regional distribution of various NHE isoforms and their functions. In the present work, expression of different isoforms of NHE in cultured epithelia of the efferent duct and cauda epdidymidis were studied. Reverse transcription-polymerase chain reaction revealed the presence of NHE1, NHE2, and NHE3, but not NHE4, message in both cultured epithelia. Western blot analysis detected the presence of NHE1 and NHE2 proteins in both cultured epithelia, but NHE3 protein was only detected in the cultured epithelial cells from the efferent duct. Immunohistochemical studies demonstrated that NHE2 was localized in the cytoplasm of the ciliated cells, whereas NHE3 was localized at the apical membrane of the principal cells of the efferent duct. The NHE activities in both cultured epithelia were inhibited by 10 microM HOE-694 (3-methylsulphonyl-4-piperidinobenzoyl guanidine methanesulphonate), a NHE1 inhibitor, by approximately 76%. The HOE-694-resistant NHE activities in the cultured epithelia of efferent duct and cauda epididymidis were completely inhibited by 20 microM S3226 (3-[2-(3-guanidino-2-methyl-3-oxo-propenyl)-5-methyl-phenyl]-N:-isopropylidene-2-methyl-acrylamide dihydrochloride), a NHE3 inhibitor, and 300 microM HOE-694 (a dose that can completely block NHE2), respectively. These results indicated that NHE1, NHE2, and NHE3 were expressed in the cultured epithelial cells of the efferent duct, whereas only NHE1 and NHE2 were expressed in the cultured epithelial cells of the cauda epididymidis. It is suggested that NHE1 may provide "housekeeping" functions in both epithelia, whereas NHE2 in the cauda epididymidis and NHE3 in the efferent duct may be involved in Na+ reabsorption and regulation of pH of the luminal fluid.

Amiloride↗

PubMed2001

Na+ reabsorption in cultured rat epididymal epithelium via the Na+/nucleoside cotransporter.

The effect of nucleoside on Na+ reabsorption via Na+/nucleoside cotransporter in cultured rat epididymal epithelia was studied by short-circuit current (Isc) technique. Guanosine added apically stimulated Isc in a dose-dependent manner, with a median effective concentration (EC50) of 7 +/- 2 microM (mean +/- SEM). Removal of Na+ from the apical bathing solution or pretreatment with a nonspecific Na+/nucleoside cotransporter inhibitor, phloridzin, completely blocked the Isc response to guanosine. Moreover, the guanosine response was abolished by pretreatment of the tissue with ouabain, a Na+/K+-ATPase inhibitor, suggesting the involvement of Na+/nucleoside cotransporter on the apical side and Na+/K+-ATPase on the basolateral side in Na+ reabsorption. In contrast, the Isc response to guanosine was not affected after desensitization of purinoceptors by ATP. Addition of the Na+/K+/2Cl- symport inhibitor bumetanide to the basolateral side or the nonspecific Cl- channel blocker diphenylamine-2-carboxylate to the apical side showed no effect on the Isc response to guanosine, excluding stimulation of Cl- secretion by guanosine as the cause of the guanosine-induced Isc. The Isc response to purine nucleoside (guanosine and inosine) was much higher than that to pyrimidine nucleoside (thymidine and cytidine). Consistent with substrate specificity, results of reverse transcription-polymerase chain reaction revealed mRNA for concentrative nucleoside transporter (CNT2), which is a purine nucleoside-selective Na+/nucleoside cotransporter in the epididymis, but not for CNT1. It is suggested that the Na+/nucleoside cotransporter (i.e., CNT2) may be one of the elements involved in Na+ and fluid reabsorption in the epididymis, thereby providing an optimal microenvironment for the maturation and storage of spermatozoa.

Absorption↗

PubMed2001

Characterization of nucleoside transport systems in cultured rat epididymal epithelium.

The nucleoside transport systems in cultured epididymal epithelium were characterized and found to be similar between the proximal (caput and corpus) and distal (cauda) regions of the epididymis. Functional studies revealed that 70% of the total nucleoside uptake was Na(+) dependent, while 30% was Na(+) independent. The Na(+)-independent nucleoside transport was mediated by both the equilibrative nitrobenzylthioinosine (NBMPR)-sensitive system (40%) and the NBMPR-insensitive system (60%), which was supported by a biphasic dose response to NBMPR inhibition. The Na(+)-dependent [(3)H]uridine uptake was selectively inhibited 80% by purine nucleosides, indicating that the purine nucleoside-selective N1 system is predominant. Since Na(+)-dependent [(3)H]guanosine uptake was inhibited by thymidine by 20% and Na(+)-dependent [(3)H]thymidine uptake was broadly inhibited by purine and pyrimidine nucleosides, this suggested the presence of the broadly selective N3 system accounting for 20% of Na(+)-dependent nucleoside uptake. Results of RT-PCR confirmed the presence of mRNA for equilibrative nucleoside transporter (ENT) 1, ENT2, and concentrative nucleoside transporter (CNT) 2 and the absence of CNT1. It is suggested that the nucleoside transporters in epididymis may be important for sperm maturation by regulating the extracellular concentration of adenosine in epididymal plasma.

Animals↗

PubMed2001

Nongenomic effect of testosterone on chloride secretion in cultured rat efferent duct epithelia.

Short-circuit current (I(sc)) technique was used to investigate the role of testosterone in the regulation of chloride secretion in cultured rat efferent duct epithelia. Among the steroids tested, only testosterone, and to a lesser extent, 5alpha-dihydrotestosterone (5alpha-DHT), reduced the basal and forskolin-induced I(sc) in cultured rat efferent duct epithelia when added to the apical bathing solution. Indomethacin, a 3alpha-hydroxysteroid dehydrogenase, did not affect the inhibitory effect of 5alpha-DHT. The effect of testosterone occurred within 10-20 s upon application and was dose dependent with apparent IC(50) value of 1 microM. The effect was abolished by removal of Cl(-) but not HCO from the normal Krebs-Henseleit solution, suggesting that testosterone mainly inhibited Cl(-) secretion. The efferent duct was found to be most sensitive to testosterone, while the caput and the cauda epididymidis were only mildly sensitive. Cyproterone acetate, a steroidal antiandrogen, or flutamide, a nonsteroidal antiandrogen, did not block the effect of testosterone on the forskolin-induced I(sc), nor did protein synthesis inhibitors, cycloheximide, or actinomycin D. However, pertussis toxin, a G(i) protein inhibitor, attenuated the inhibition of forskolin-induced I(sc) by testosterone. Testosterone caused a dose-dependent inhibition of forskolin-induced rise in cAMP in efferent duct cells. It is suggested that the rapid effect of testosterone was mediated through a membrane receptor that is negatively coupled to adenylate cyclase via G(i) protein. The role of nongenomic action of testosterone in the regulation of electrolyte and fluid transport in the efferent duct is discussed.

1-Methyl-3-isobutylxanthine↗

PubMed2001

Colon epithelial cell death in 2,4,6-trinitrobenzenesulfonic acid-induced colitis is associated with increased inducible nitric-oxide synthase expression and peroxynitrite production.

Peroxynitrite, derived from the reaction of nitric oxide (NO(.)) with superoxide (O(2)), is a potent nitrating and oxidizing agent that can induce apoptosis in a variety of different cell types. In the present study, we investigated the possible role of peroxynitrite as a mediator of colon epithelial cell death in rat colitis. Rat colon inflammation was induced by intracolonic administration of 2,4,6-trinitrobenzenesulfonic acid (TNBS) and rats were sacrificed 24 h after TNBS administration. Expression of inducible nitric-oxide synthase (iNOS) was detected by reverse transcription-polymerase chain reaction and immunohistochemistry. The enzymatic activities of Ca(2+)-independent iNOS and Ca(2+)-dependent constitutive nitric-oxide synthase were determined biochemically. Evidence of peroxynitrite-mediated cell injury was detected by immunostaining of nitrotyrosine. Apoptosis was examined by in situ terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay and DNA gel electrophoresis. To evaluate the specific contribution of peroxynitrite to the observed cell injury, a selective iNOS inhibitor, L-N(G)-[1-iminoethyl]lysine (L-NIL), was administered after TNBS induction. Morphological examination and analysis of TUNEL/cytokeratin double immunofluorescence revealed significant apoptosis in mucosal epithelial cells. Nitrotyrosine was colocalized with TUNEL, strongly demonstrating the association of peroxynitrite with the apoptotic death of colon epithelial cells. The administration of L-NIL reduced iNOS activity in 24-h lesions by 92% and also significantly attenuated both nitrotyrosine staining and apoptotic cell counts in the colon epithelium. These results strongly suggest that local elevated level of peroxynitrite produced from increased iNOS expression and activity is a major contributor to colon epithelial apoptosis during colon inflammation.

Animals↗

PubMed2001

Body weight, serum uric acid and lipid profile one year after spinal cord injury.

BACKGROUND: Survey of the serum lipoprotein profile, uric acid levels and body weight in Chinese patients with spinal cord injury (SCI) one year after injury, and to report the data of patients with SCI at different levels. METHODS: Seventy-seven Chinese patients with SCI categorized into cervical SCI, thoracic SCI, and lumbar SCI groups were enrolled in the study. The serum uric acid and lipoprotein profiles of these patients were evaluated one year after injury. All of the data in those different groups of SCI patients were compared with normal levels. Body weight before and after injury were also evaluated. RESULTS: Serum uric acid was within normal limits in all three SCI groups. A similar lower in serum high density lipoprotein cholesterol (HDL-C) level was observed in all three groups of SCI patients. Higher serum levels were observed in total cholesterol (TC) in the lumbar SCI group, triglyceride and very low density lipoprotein cholesterol in the thoracic and lumbar SCI group, and TC/HDL-C ratio in all three SCI groups. Decrease of body weight were noted in cervical and thoracic SCI patients. CONCLUSION: Further long-term follow up was necessary to determine the serum lipid and body weight change in patients with SCI at different levels.

Adult↗

PubMed2000

Characterization of Lonidamine and AF2785 blockade of the cyclic AMP-activated chloride current in rat epididymal cells.

It has been shown previously that the antifertility agents Lonidamine and its analogue AF2785, [1-(2,4-dichlorobenzyl)-indazole-3-acrylic acid] are potent inhibitors of the cAMP-activated chloride channel (CFTR) in rat epididymal cells. In this study, we further characterized the blocking actions of these two compounds and compared them with the known chloride channel blocker diphenylamine-2-carboxylate (DPC). Results show that the order of potency in blocking the cAMP-activated current is AF2785 > Lonidamine > DPC. All three compounds shared similar blocking characteristics. Firstly, their blockade of the current exhibited voltage dependence; all three agents blocked the current more markedly at negative than at positive membrane potentials. Secondly, they blocked the channels from the outside of the cell. Thirdly, their blocking efficacies were maximal at low extracellular pH. Lastly, the time course of the block by AF2785 and DPC appeared to be more rapid than that of Lonidamine. It is hoped that further studies with other indazole compounds will add knowledge to the physiology and pharmacology of CFTR in the epididymis. Such information will be of great importance to our quest for novel male contraceptives.

Animals↗

PubMed2000

Mapping of the C1q binding site on rituxan, a chimeric antibody with a human IgG1 Fc.

Rituxan (Rituximab) is a chimeric mAb with human IgG1 constant domains used in the therapy of non-Hodgkin's B cell lymphomas. This Ab targets B cells by binding to the cell-surface receptor, CD20. In our investigation of the mechanism of B cell depletion mediated by Rituximab, we first constructed mutants of Rituximab defective in complement activation but with all other effector functions intact. Our results demonstrate that the previously described C1q binding motif in murine IgG2b constituting residues E318, K320, and K322 is not applicable to a human IgG1 when challenged with either human, rabbit, or guinea pig complement. Alanine substitution at positions E318 and K320 in Rituximab had little or no effect on C1q binding and complement activation, whereas alanine substitution at positions D270, K322, P329, and P331 significantly reduced the ability of Rituximab to bind C1q and activate complement. We have also observed that concentrations of complement approaching physiological levels are able to rescue >60% of the activity of these mutant Abs with low affinity for C1q. These data localize the C1q binding epicenter on human IgG1 and suggest that there are species-specific differences in the C1q binding site of Igs.

Alanine↗

PubMed2000

Immunopathogenesis of hepatitis B virus recurrence after liver transplantation.

BACKGROUND AND AIMS: Hepatitis B virus (HBV) recurrence after orthotopic liver transplantation is associated with inflammatory graft changes, despite immunosuppression and donor/recipient HLA mismatch. We investigated whether immune mechanisms are involved in the pathogenesis of hepatitis B after liver transplantation. METHODS: The virus-specific T helper (Th) cell response, activation of Th1/Th2 subpopulations, donor/recipient HLA, and expression of tumor necrosis factor (TNF)-alpha/TNF receptors were determined in 28 patients who underwent transplantation for HBV-related cirrhosis (17 with HBV recurrence and 11 without recurrence) in comparison to 30 nontransplant patients with chronic hepatitis B. RESULTS: Orthotopic liver transplantation recipients with HBV recurrence showed significant hepatitis B core antigen-specific T-cell proliferation, comparable to nontransplant patients, which was not present in transplant recipients without recurrence. In addition, hepatic and serum interleukin (IL)-2, interferon-gamma, and TNF-alpha were enhanced, without changes in IL-4 and IL-10. Phenotypically, hepatic infiltrates in allografts with HBV recurrence were comprised of CD4+ lymphocytes and macrophages with a correlation between interferon-gamma- and TNF-alpha-producing cells and the degree of necroinflammatory activity. There was a marked up-regulation of both TNF-alpha receptors, significantly greater than in nontransplant patients. CONCLUSIONS: These findings suggest that despite immunosuppression, HLA class I-independent immune mechanisms have a significant pathogenic role in liver damage associated with HBV recurrence after liver transplantation.

Adult↗

PubMed2000

Testicular hormonal regulation of the renin-angiotensin system in the rat epididymis.

Evidence for the existence of an intrinsic angiotensin system based on locally formed angiotensinogen as a precursor for angiotensin production has been demonstrated in the rat epididymis. The data strongly support the presence of an epididymal renin-angiotensin system (RAS) which may be important for epididymal and sperm functions. In the present study, the effects of castration and testicular hormonal replacement on the expression of RAS components from the rat epididymis are investigated at the gene and protein levels. Results from northern blot and western blot analyses consistently showed that the expression of angiotensinogen mRNA and protein was apparently abolished by castration whereas their expression was completely restored to control levels when the castrated rats were hormonally replaced with either testosterone alone or with combined testosterone and estradiol. Northern blot did not detect any signal for angiotensinogen mRNA while western blot could detect a weak signal for angiotensinogen protein when the castrated rats were replaced with estradiol alone. Renin could be detected neither in control, castrated nor hormonally replaced rats. Moreover, the expression of angiotensin II receptor, type I (AT1) was almost abolished by castration as demonstrated by northern blot and reverse transcription-polymerase chain reaction. These data indicate that the expression of RAS by the rat epididymis at the levels of its precursor angiotensinogen and its receptor AT1, is subject to the regulation of testicular hormones and its expression appears to be predominantly testosterone-dependent.

Angiotensinogen↗

PubMed2000

Mechanism and signal transduction of 14 (R), 15 (S)-epoxyeicosatrienoic acid (14,15-EET) binding in guinea pig monocytes.

14(R), 15(S)-epoxyeicosatrienoic acid (14,15-EET) is a cytochrome P-450 monooxygenase (epoxygenase) metabolite of arachidonic acid (AA). In this study, we have identified a population of specific high affinity binding sites for 14,15-EET in the guinea pig mononuclear (GPM) cells. The results of competition studies showed that 14(R), 15(S)-EET was an effective competing ligand with a Ki of 226.3 nM followed by 11(R), 12(S)-EET, 14(S), 15(R)-EET, 14,15 thia(S)-ET, and 14,15-aza(N)-ET. The binding was sensitive to various protease treatments suggesting that the binding site is protein in nature. Cholera toxin (CT) and dibutyryl cAMP attenuated 14,15-EET binding in GPM cells. Mean binding site density (Bmax), decreased 32.0% and 19.1% by the pretreatment with cholera toxin (200 micrograms/ml) and dibutyryl cAMP (100 nM), respectively, without changing the dissociation constant. A specific protein kinase A (PKA) inhibitor, H-89, but not the PKC inhibitor K252a reversed the down regulation of 14,15-EET receptor binding caused by dibutyryl cAMP in GPM cells. Thus, the results sug-gest that the specific binding site of 14,15-EET in GPM cells be associated with a receptor that could be down regulated through an increase in intracellular cAMP and activation of a PKA signal trans-duction. We propose that the signal transduction mechanism begins with the binding of 14,15-EET to its receptor that leads to increase intracellular cAMP levels and the activation of PKA, and finally, with the down regulation of 14,15-EET receptor binding.

8,11,14-Eicosatrienoic Acid↗