Supplemental patient education for patients taking oral anticoagulants: systematic review and meta-analysis.

OBJECTIVE: Lack of patient knowledge has been associated with poor anticoagulation control, but the effect of patient education on clinical outcomes is unclear. We systematically reviewed the effect of supplemental patient education vs. usual care on hemorrhage, thromboembolic events (TEEs), time in therapeutic range (TTR) and knowledge test scores for all oral anticoagulants. DATA SOURCES: The data sources were electronic databases, including MEDLINE, EMBASE, CENTRAL, CINAHL and IPA, to February 2012 examining any oral anticoagulant. We reviewed references for additional potentially relevant studies. METHODS: Only randomized controlled trials (RCTs) were considered. Data extraction and quality assessment were conducted with GRADE. Pooled relative risks (RRs) were calculated, and heterogeneity was determined by use of χ(2) and I(2) statistics. RESULTS: Seven RCTs (n = 1209) were included in the systematic review, and five RCTs (n = 847) in the meta-analysis. All included studies examined vitamin K antagonists. No significant difference was found for hemorrhage (RR 0.92, 95% confidence interval [CI] 0.04-20.56), TEE (RR 0.66, 95% CI 0.10-4.39), a composite outcome of hemorrhage or TEE (RR 0.48, 95% CI 0.23-1.01), or TTR (mean absolute difference of 2.02%, 95% CI - 2.81 to 6.84). Evidence was conflicting on the impact of supplemental education on test scores. All trials had at least one substantial methodologic limitation. CONCLUSION: Current evidence does not support supplemental patient education as a means to improve patient outcomes, but the quality of this evidence is poor. Larger randomized trials are needed with longer follow-up, recruitment of patients initiating anticoagulation in primary care settings, and clearly defined education interventions.

Hyperdynamic microtubules, cognitive deficits, and pathology are improved in tau transgenic mice with low doses of the microtubule-stabilizing agent BMS-241027.

Tau is a microtubule (MT)-stabilizing protein that is altered in Alzheimer's disease (AD) and other tauopathies. It is hypothesized that the hyperphosphorylated, conformationally altered, and multimeric forms of tau lead to a disruption of MT stability; however, direct evidence is lacking in vivo. In this study, an in vivo stable isotope-mass spectrometric technique was used to measure the turnover, or dynamicity, of MTs in brains of living animals. We demonstrated an age-dependent increase in MT dynamics in two different tau transgenic mouse models, 3xTg and rTg4510. MT hyperdynamicity was dependent on tau expression, since a reduction of transgene expression with doxycycline reversed the MT changes. Treatment of rTg4510 mice with the epothilone, BMS-241027, also restored MT dynamics to baseline levels. In addition, MT stabilization with BMS-241027 had beneficial effects on Morris water maze deficits, tau pathology, and neurodegeneration. Interestingly, pathological and functional benefits of BMS-241027 were observed at doses that only partially reversed MT hyperdynamicity. Together, these data suggest that tau-mediated loss of MT stability may contribute to disease progression and that very low doses of BMS-241027 may be useful in the treatment of AD and other tauopathies.

Assessing psychosocial well-being of adolescents: a systematic review of measuring instruments.

BACKGROUND: The paradigm shift from the clinically deficit-oriented approach to that of educationally strength-based model in assessing adolescents' psychosocial well-being has brought about a recent increase in school-based health promotion and prevention initiatives. This prompted this systematic review of measuring instruments designed to assess psychosocial well-being of children and adolescents. METHODS: Using electronic databases on Academic Search Premier, MEDLINE, PROQUEST, PsycINFO, CINAHL Plus and Psychosocial and Health Instrument, a systematic review of literature of measuring instruments was conducted from their inception to December 2009 using the keywords of child, emotion, assessment, scale and measure. Measuring instruments from selected articles were critically appraised using a predetermined set of quality indicators which guided the rating of the psychometric properties of the instruments into grades of A, B, and C. The constructs of psychosocial well-being from the measuring instruments were categorized into themes. RESULTS: Twenty-nine out of the 908 articles met the inclusion criteria. Seventeen instruments identified from the selected articles were examined using preset quality indicators. In construct building, the themes identified from the strength-based instruments distinguished the construct of psychosocial well-being primarily into the dimensions of personal emotional competency and social functioning. In the ratings of psychometric properties, one instrument was rated 5A, five rated 4A and four rated 3A. For reliability testing, eight measures received grade A when their intraclass correlation is higher than 0.7; whereas only two instruments reported sensitivity and none investigated responsiveness. CONCLUSIONS: Strength-based measures focusing on social emotional behavioural outcomes open up a possibility to link up assessment with promotion of psychosocial well-being, away from clinical settings and into adolescents' homes, schools and community. Future research should focus more on investigating the sensitivity and responsiveness of measuring instruments using longitudinal design in efficacy studies to assess change in adolescents' psychosocial status over extended time.

Changes in microtubule turnover accompany synaptic plasticity and memory formation in response to contextual fear conditioning in mice.

Synaptic plasticity plays a crucial role in learning, memory, and cognitive disorders. Cytoskeletal reorganization underlies neuronal synaptic plasticity, but little is known about the regulation of cytoskeletal dynamics in living animals. We used stable isotope labeling to measure the turnover of tubulin in defined microtubule (MT) populations in murine brain. Neuronal MTs generally exhibited low turnover rates in vivo. Basal turnover was highest in tau-associated MTs, intermediate in microtubule-associated protein 2 (MAP2)-associated MTs, and lowest in cold-stable MTs. Labeling of MTs in mature neurons in cell culture yielded similar turnover results. Intracerebroventricular glutamate injection stimulated, via N-methyl-D-aspartic acid receptors, label incorporation (turnover) in cold-stable, tau-associated, and MAP2-associated MTs, the last of which was shown to be dependent on cyclic adenosine-3', 5'-monophosphorothioate-protein kinase A. Contextual fear conditioning, a hippocampus-mediated form of memory formation, was accompanied by increased turnover of hippocampal MAP2-associated and cold-stable MTs. Treatment with the MT-depolymerizing drug nocodazole reversed the conditioning-induced increase in label incorporation in MAP2-associated MTs, reduced dendritic spine density, and impaired memory formation. The effects of nocodazole on MT turnover were prevented by the MT-stabilizing agent Taxol (Sigma-Aldrich, St. Louis, MO, USA) and by brain-derived nerve growth factor, both of which also restored dendritic spine density and memory formation in this model. In conclusion, these results suggest that changes in hippocampal MT turnover are required for, and are a biomarker of, the synaptic plasticity that is involved in memory formation.

Kaempferol stimulates large conductance Ca2+ -activated K+ (BKCa) channels in human umbilical vein endothelial cells via a cAMP/PKA-dependent pathway.

BACKGROUND AND PURPOSE: Kaempferol has been shown to possess a vasodilator effect but its mechanism of action remains unclear. In this study, experiments were carried out to study the effect of kaempferol on K+ channels in endothelial cells. EXPERIMENTAL APPROACH: K+ channel activities in human umbilical vein endothelial cells (HUVECs) were studied by conventional whole cell and cell-attached patch-clamp electrophysiology. KEY RESULTS: Kaempferol stimulated an outward-rectifying current in HUVECs in a dose-dependent manner with an EC50 value of 2.5+/-0.02 microM. This kaempferol-induced current was abolished by large conductance Ca2+ -activated K+ (BKCa) channel blockers, such as iberiotoxin (IbTX) and charybdotoxin (ChTX), whereas the small conductance Ca2+ -activated K+ (SKCa) channel blocker, apamin, and the voltage-dependent K+ (KV) channel blocker, 4-aminopyridine, had no effect. Cell-attached patches demonstrated that kaempferol increased the open probability of BkCa channels in HUVECs. Clamping intracellular Ca2+ did not prevent kaempferol-induced increases in outward current. In addition, the kaempferol-induced current was diminished by the adenylyl cyclase inhibitor SQ22536, the cAMP antagonist Rp-8-Br-cAMP and the PKA inhibitor KT5720, but was not affected by the guanylyl cyclase inhibitor ODQ, the cGMP antagonist Rp-8-Br-cGMP and the PKG inhibitor KT5823. The activation of BKCa channels by kaempferol caused membrane hyperpolarization of HUVECs. CONCLUSION AND IMPLICATIONS: These results demonstrate that kaempferol activates the opening of BKCa channels in HUVECs via a cAMP/PKA-dependent pathway, resulting in membrane hyperpolarization. This mechanism may partly account for the vasodilator effects of kaempferol.

Validation and assessment of a blood-donor arm disinfectant containing chlorhexidine and alcohol.

BACKGROUND: To minimize the bacterial contamination rate in blood collected from donors, a study was designed to evaluate the suitability of a single-use chlorhexidine-alcohol antiseptic for donor arm preparation at all blood collection venues in Australia. STUDY DESIGN AND METHODS: A prospective study of bacterial load on the skin was performed on 616 blood donors' arms before and after disinfection using a direct swabbing and plating technique. Disinfection was achieved with a swab containing 1 percent chlorhexidine gluconate with 75 percent alcohol, which was applied to the skin in a prescribed method. Feedback from blood donors and staff was obtained using questionnaires. RESULTS: After disinfection, 99 percent of donor arms had bacterial counts of 5 cfu per plate or less, and 99.5 percent had counts of 10 cfu per plate or less, respectively. The mean colony count for all donors after disinfection was 0.39, and the percentage reduction was 99 compared to predisinfection. Sixteen donors (3%) noted transient skin irritation. The majority of staff (64%) preferred not to use the new disinfectant due to the difficulty opening the packaging and an excessive amount of antiseptic solution per pack. CONCLUSION: The bacteriologic study showed that the disinfectant satisfied the requirements of the Australian Red Cross Blood Service for use to prepare blood-donor arms before venesection. An improvement to the packaging was required before it could be acceptable to all staff.

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.

Regulation of epididymal principal cell functions by basal cells: role of transient receptor potential (Trp) proteins and cyclooxygenase-1 (COX-1).

The epithelia lining the epididymides of many species including the human are known to consist of several cell types. Among them, the principal cells are the most abundant and their functions most extensively studied. There are other cell types such as the narrow cells, clear cells, halo cells and basal cells which are scattered along the duct in lesser number. Although these minority cell types have not been studied to the same extent as the principal cells, it is conceivable that their presence are essential to the integrated functions of the epididymis. In the intact epididymis, basal cells can be seen adhering to the basement membrane forming close contact with the principal cells above them. Work in our laboratory has provided evidence that through local formation of prostaglandins, basal cells may regulate electrolyte and water transport by the principal cells. This regulatory process involves two proteins which are exclusively expressed by the basal cells. They are the transient receptor potential (Trp) proteins, which serve as transmembrane pathways for Ca(2+) influx, and cyclooxygenase 1 (COX-1), a key enzyme in the formation of prostaglandins. The role of the two proteins in the integrated functions of the basal cells as humoral regulators of principal cells is discussed.

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.

Secretin controls anion secretion in the rat epididymis in an autocrine/paracrine fashion.

There is growing evidence that secretin, the first hormone discovered in our history, has functions in the brain other than in the gastrointestinal tract. This article reports for the first time that secretin and its receptor mRNAs are produced in distinct cell types within the epididymis. To test if secretin affects electrolyte transport in the epididymis, we measured short-circuit current (Isc) in cultured epididymal epithelia and found secretin dose-dependently stimulated Isc. Ion substitution experiments and use of pharmacological agents inferred that the stimulated Isc is a result of concurrent electrogenic chloride and bicarbonate secretion. It is further shown that secretin and pituitary adenylate cyclase-activating polypeptide (PACAP) function via totally different mechanisms: 1) PACAP works only from the apical side of the epithelium to stimulate chloride and not bicarbonate secretion, while secretin acts on the apical and basolateral sides to stimulate chloride and bicarbonate secretion. 2) the stimulation by PACAP but not secretin requires local prostaglandin synthesis. By immunocytochemical staining, secretin is localized in the principal cells of the initial segment and caput epididymidis, whereas secretin receptor is present in the principal cells of the proximal as well as the distal part of the epididymis. This pattern of distribution appears to be consistent with the idea that secretin is secreted by the proximal epididymis and acts on the proximal and distal epididymis in an autocrine and paracrine fashion. Its function is to control secretion of electrolytes and water.

Regulation of cell growth by selective COX-2 inhibitors in oral carcinoma cell lines.

Evidence indicates that NSAIDs that inhibit prostaglandin (PG) synthesis can reduce the incidence of colorectal cancers and that inhibition of cyclooxygenase-2 (COX-2) may be the underlying mechanism. The objective of this study was to investigate this putative mechanism by examining the effect of selective COX-2 inhibitors (Celebrex, DFU, NS-398) and COX-1 inhibitors (Aspirin) on the growth of two human oral carcinoma cell lines (OEC-M1 and KB) and one normal fibroblast cell line (NF). We found that the growth of OEC-M1 cells could be significantly inhibited by DFU concentrations above 30 microM (31%) after 4 days, and above 50 microM (35%) after 2 days in culture; by Celebrex at concentrations above 20 microM (52%) after 6 days, above 30 microM (36%) after 5 days, and above 40 microM (33%) after 4 days in culture; and by NS-398 above 1 microM (30%) after 6 days, and above 10 microM (35%) after 5 days in culture. The growth of KB cells could be significantly inhibited by DFU concentrations above 10 microM (33%) after 6 days, above 20 microM (35%) after 4 days in culture; and by Celebrex at concentrations above 10 microM (33%) after 5 days, and above 50 microM (30%) after 4 days in culture; and by NS-398 above 1 microM (45%) after 5 days, above 20 microM (36%) after 4 days in culture. The growth of NF cells could be significantly inhibited by DFU above 30 microM (45%) after 6 days, and above 40 microM (32%) after 3 days in culture, and by Celebrex at concentrations above 10 microM (42%) after 6 days, above 30 microM (31%) after 4 days, above 50 microM (32%) after 3 days in culture, and by NS-398 above 0.1 microM (35%) after 4 days, and above 1 microM (32%) after 3 days in culture. The growth-inhibitory concentration (IC50) values for DFU on OEC-M1, KB, and NF cells were about 39.1, 14.8, and 42.9 microM at 144 h, respectively, and on KB was about 45.2 microM at 120 h. The IC50 values for Celebrex on OEC-M1, KB, and NF cells were about 19.1, 8.6, and 15.8 microM at 144 h, respectively, and on KB and NF were about 27.7 and 35.3 microM, respectively, at 120 h. The IC50 values for NS-398 on OEC-M1, KB, and NF were about 18.9, 0.7 and 1 microM, respectively, at 144 h; on KB and NF values were about 10.8 and 1.4 microM, respectively, at 120 h and on KB and NF were about 26.6 and 4.1 microM, respectively, at 96 h. The results show that the growth of these cell lines is inhibited by three COX-2 selective inhibitors but not by any COX-1 selective inhibitors. These findings suggest that COX-2 may play an important role in the generation of biochemical mediators that stimulate the growth of human oral cancer and normal fibroblast cell lines.

Chemistry of buttermilk solid antioxidant activity.

Antioxidant activity of buttermilk solids was assessed by analyzing for relative reducing activity, sulfhydryl content, and ferrous and ferric iron binding affinity. These experiments were followed by monitoring the affinity of buttermilk solids to scavenge both hydroxyl and peroxyl radicals in vitro. Notable relative reducing activity of buttermilk solids to L-ascorbic acid (43.80 to 85.85% over a range of 5.0 to 10.0 mg) was attributed in part to the sulfhydryl content (28.8 microM). Buttermilk solids sequestering activity was greater for ferrous than ferric ion. These chemical properties of buttermilk solids corresponded to a significant affinity to scavenge Fenton-induced hydroxyl radical over a range of 5 to 10 mg. A significant affinity of buttermilk solids to protect against lipid peroxidation, tested using an in vitro model lipid system, was also observed at both 0.1 and 0.2% (wt/vol). These findings demonstrated that buttermilk solids possess significant antioxidant activity, thereby suggesting potential use as a value-added ingredient for stabilizing food matrixes against lipid peroxidation reactions.

A comparison of the buttermilk solids functional properties to nonfat dried milk, soy protein isolate, dried egg white, and egg yolk powders.

Physicochemical (i.e., sulfhydryl group, protein, and total solubility) as well as functional properties (i.e., water-holding and fat-absorption capacity, foaming and emulsification capacity, and stability) of commercial buttermilk solids (BMS) were compared to nonfat dried milk, soy protein isolate, and dried egg yolk and egg white powders on an equivalent protein basis. BMS showed limited functional properties in water-holding capacity (0.75 g water/g protein) and fat-absorption capacity (1.2 g of oil/g of protein), and foaming capacity (0.5 ml of foam/ml of solution) and stability. However, emulsifying capacity and stability of BMS was not significantly different from other dried protein powders. Results indicated that 0.9 g of protein (approximately 0.45%, wt/vol, concentration) from BMS was needed to emulsify a maximum oil concentration of 50% in water at temperatures up to 50 degrees C. Denaturation of protein, quantified by free sulfhydryl groups, was a critical factor affecting the functionality of BMS and all other protein powders tested. The milk fat globule membrane present in BMS did not enhance either emulsifying capacity or stability.

Synergistic effects of cystic fibrosis transmembrane conductance regulator and aquaporin-9 in the rat epididymis.

The cystic fibrosis transmembrane conductance regulator (CFTR) and aquaporin-9 (AQP-9) are present in the luminal membrane of the epididymis, where they play an important role in formation of the epididymal fluid. Evidence is accumulating that CFTR regulates other membrane transport proteins besides functioning as a cAMP-activated chloride channel. We have explored the possible interaction between epididymal CFTR and AQP-9 by cloning them from the rat epididymis and expressing them in Xenopus oocytes. The effects of the expressed proteins on oocyte water permeability were studied by immersing oocytes in a hypo-osmotic solution, and the ensuing water flow was measured using a gravimetric method. The results show that AQP-9 alone caused an increase in oocyte water permeability, which could be further potentiated by CFTR. This potentiation was markedly reduced by phloretin and lonidamine (inhibitors of AQP-9 and CFTR, respectively). The regulation of water permeability by CFTR was also demonstrated in intact rat epididymis luminally perfused with a hypo-osmotic solution. Osmotic water reabsorption across the epididymal tubule was reduced by phloretin and lonidamine. Elevation of intracellular cAMP with 3-isobutyl-1-methylxanthine increased osmotic water permeability, whereas inhibiting protein kinase A with H-89 (N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinoline sulfonamide hydrochloride) reduced it. These results are consistent with a role for CFTR in controlling water permeability in the epididymis in vivo. We conclude that this additional role of CFTR in controlling water permeability may have an impact on the genetic disease cystic fibrosis, in which men with a mutated CFTR gene have abnormal epididymis and infertility.

Indazole inhibition of cystic fibrosis transmembrane conductance regulator Cl(-) channels in rat epididymal epithelial cells.

Previous studies have shown that two indazole compounds, lonidamine [1-(2,4-dichlorobenzyl)-indazole-3-carboxylic acid] and its analogue AF2785 [(1-(2,4-dichlorobenzyl)-indazol-3-acrylic acid], suppress fertility in male rats. We also found that these compounds inhibit the cystic fibrosis transmembrane conductance regulator chloride (CFTR-Cl(-)) current in epididymal epithelial cells. To further investigate how lonidamine and AF2785 inhibit the current, we used a spectral analysis protocol to study whole-cell CFTR current variance. Application of lonidamine or AF2785 to the extracellular membrane of rat epididymal epithelial cells introduced a new component to the whole-cell current variance. Spectral analysis of this variance suggested a block at a rate of 3.68 micro mol(-1)/sec(-1) and an off rate of 69.01 sec(-1) for lonidamine, and an on rate of 3.27 micro mol(-1)/sec(-1) and an off rate of 108 sec(-1) for AF2785. Single CFTR-Cl(-) channel activity using excised inside-out membrane patches from rat epididymal epithelial cells revealed that addition of lonidamine to the intracellular solution caused a flickery block (a reduction in channel-open time) at lower concentration (10 micro M) without any effect on open channel probability or single-channel current amplitude. At higher concentrations (50 and 100 micro M), lonidamine showed a flickery block and a decrease in open-channel probability. The flickery block by lonidamine was both voltage-dependent and concentration-dependent. These results suggest that lonidamine and AF2785, which are open-channel blockers of CFTR at low concentrations, also affect CFTR gating at high concentrations. We conclude that these indazole compounds provide new pharmacological tools for the investigation of CFTR. By virtue of their interference with reproductive processes, these drugs have the potential for being developed into novel male contraceptives.

COX-dependent and -independent pathways in bradykinin-induced anion secretion in rat epididymis.

Lysylbradykinin (LBK) added to the apical or basolateral side of cultured rat epididymal monolayers stimulated a rise in short-circuit current (Isc) due to anion secretion. The concentration-response relationships for the apical and basolateral applications have EC50 value of 0.001 microM. The responses to apical or basolateral application of LBK were blocked by WIN64338, a specific B2 receptor antagonist, but not by Des-Arg9,[Leu8]-BK, a specific B1 receptor antagonist, indicating that the LBK effects were mediated through B2 bradykinin receptors. Experiments to desensitize the B2 receptors by repeated stimulation have demonstrated that the responses to apical or basolateral LBK were due to discrete receptors on the apical or basolateral surface. In epithelia clamped in the Ussing chambers, addition of LBK to the apical or basolateral surface evoked release of PGE2 into the apical and basolateral bathing solutions over the first 10 min following hormone addition. LBK added to the basolateral side elicited a greater release than it was added to the apical side. Pretreatment of the epithelia with piroxicam (5 microM) abolished PGE2 release elicited by apical or basolateral LBK and abrogated the Isc induced by basolateral LBK. However, the rise in Isc induced by apical LBK was reduced by 31.3% only. The anion secretion response to apical LBK was not affected by MDL-12330A, an adenylate cyclase inhibitor, but greatly attenuated by thapsigargin, an inhibitor of intracellular Ca2+ release. However, the reverse effects were seen for basolateral LBK. It is concluded that distinct pathways are involved in the stimulation of anion secretion by apical or basolateral LBK. The response to basolateral LBK was COX-dependent, mediated by PGE2 and involves cAMP as second messenger. In contrast, the response to apical LBK is largely COX-independent, not mediated by PCE2 and involves Ca2+ as intracellular messenger.

A BK(Ca) to K(v) switch during spermatogenesis in the rat seminiferous tubules.

Spermatogenesis is a complex cellular event during which the diploid germ cells differentiate and divide by mitosis and meiosis at specific time points along the spermatogenic cycle to generate the haploid spermatozoa. For this complex event to go in an orderly manner, cell differentiation and division must be precisely controlled by signals arising from within and outside the seminiferous tubules. Changes in the membrane potential of the germ cells are likely to be an important part of the signaling mechanism. We have applied the whole-cell patch clamp technique to identify and characterize ion channels in different spermatogenic cells from immature and mature rat testes fractionated by discontinuous Percoll gradient. A voltage- and Ca(2+)- dependent, outwardly rectifying current with gating and pharmacologic properties resembling the large conductance K(+) channels (BK(Ca)) was recorded from the spermatogonia and primary spermatocytes. Another voltage-dependent, outwardly rectifying current that was sensitive to 4-aminopyridine, a K(v) channel blocker, was detected in spermatocytes and early spermatids. This current is likely caused by the smaller conductance, voltage-sensitive K(+) channels (K(v)). In some spermatogonia, both the BK(Ca) channels and the K(v) channels could be simultaneously detected in the same cell. It appears that during the course of spermatogenesis, there is up-regulation of K(v) but down-regulation of BK(Ca). Reverse transcription-polymerase chain reaction, Western blot analysis, and immunohistochemistry further confirmed the differential expression of the ion channels in different spermatogenic cells. We conclude that these ion channels may play an important role in the control of spermatogenesis.

Cyclooxygenase-2 regulates apoptosis in rat epididymis through prostaglandin D2.

In previous studies, cyclooxygenase (COX)-1 and COX-2 isozymes have been detected in the rat epididymis. COX-1 mediates electrolyte and fluid secretion induced by a number of peptide hormones, including bradykinin, angiotensin, and endothelin, via local formation of prostaglandin (PG) E2; however, the physiological role of COX-2 remains largely unknown. Marked apoptotic cell death in the rat epididymis following androgen depletion has been reported. Because expression of both COX isozymes is dependent on androgen, we investigated whether these isozymes control apoptosis in the epididymis. Apoptosis was detected in rat epididymal epithelial cells by in situ staining using the TUNEL method and by the presence of internucleosomal DNA fragmentation using capillary electrophoresis with laser-induced fluorescence detection. Specific COX inhibitors were used to delineate the roles of the 2 isozymes. There was no significant apoptotic cell death in normal and specific COX-1 inhibitor (SC-560)-treated epididymal cells. However, application of a specific COX-2 inhibitor (NS-398) induced apoptosis in a dose- and time-dependent manner. A similar apoptotic effect of COX-2 inhibitor was seen in the in vivo study. The drastic DNA fragmentation induced by COX-2 inhibitor could be reversed completely by PGD2 and partially by PGE2. In addition, the protective effect of PGD2 against COX-2 inhibition was significantly blocked by a PGDP-receptor antagonist, BWA868C. These results indicate that the COX-2 products PGD2 and, to a lesser extent, PGE2 control apoptosis in cultured rat epididymal cells in vitro.