Sodium Coupled Bicarbonate Influx Regulates Intracellular and Apical pH in Cultured Rat Caput Epididymal Epithelium

The epithelium lining the epididymis provides an optimal acidic fluid microenvironment in the epididymal tract that enable spermatozoa to complete the maturation process. The present study aims to investigate the functional role of Na(+)/HCO(3)(-) cotransporter in the pH regulation in rat epididymis.Immunofluorescence staining of pan cytokeratin in the primary culture of rat caput epididymal epithelium showed that the system was a suitable model for investigating the function of epididymal epithelium. Intracellular and apical pH were measured using the fluorescent pH sensitive probe carboxy-seminaphthorhodafluor-4F acetoxymethyl ester (SNARF-4F) and sparklet pH electrode respectively to explore the functional role of rat epididymal epithelium. In the HEPES buffered Krebs-Henseleit (KH) solution, the intracellular pH (pHi) recovery from NH(4)Cl induced acidification in the cultured caput epididymal epithelium was completely inhibited by amiloride, the inhibitor of Na(+)/H(+) exchanger (NHE). Immediately changing of the KH solution from HEPES buffered to HCO(3)(-) buffered would cause another pHi recovery. The pHi recovery in HCO(3)(-) buffered KH solution was inhibited by 4, 4diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), the inhibitor of HCO(3)(-) transporter or by removal of extracellular Na(+). The extracellular pH measurement showed that the apical pH would increase when adding DIDS to the apical side of epididymal epithelial monolayer, however adding DIDS to the basolateral side had no effect on apical pH.The present study shows that sodium coupled bicarbonate influx regulates intracellular and apical pH in cultured caput epididymal epithelium

Merkel Cells as Putative Regulatory Cells in Skin Disorders: An In Vitro Study

Merkel cells (MCs) are involved in mechanoreception, but several lines of evidence suggest that they may also participate in skin disorders through the release of neuropeptides and hormones. In addition, MC hyperplasias have been reported in inflammatory skin diseases. However, neither proliferation nor reactions to the epidermal environment have been demonstrated. We established a culture model enriched in swine MCs to analyze their proliferative capability and to discover MC survival factors and modulators of MC neuroendocrine properties. In culture, MCs reacted to bFGF by extending outgrowths. Conversely, neurotrophins failed to induce cell spreading, suggesting that they do not act as a growth factor for MCs. For the first time, we provide evidence of proliferation in culture through Ki-67 immunoreactivity. We also found that MCs reacted to histamine or activation of the proton gated/osmoreceptor TRPV4 by releasing vasoactive intestinal peptide (VIP). Since VIP is involved in many pathophysiological processes, its release suggests a putative regulatory role for MCs in skin disorders. Moreover, in contrast to mechanotransduction, neuropeptide exocytosis was Ca2+-independent, as inhibition of Ca2+ channels or culture in the absence of Ca2+ failed to decrease the amount of VIP released. We conclude that neuropeptide release and neurotransmitter exocytosis may be two distinct pathways that are differentially regulated

Global patient outcomes after elective surgery: prospective cohort study in 27 low-, middle- and high-income countries.

BACKGROUND: As global initiatives increase patient access to surgical treatments, there remains a need to understand the adverse effects of surgery and define appropriate levels of perioperative care. METHODS: We designed a prospective international 7-day cohort study of outcomes following elective adult inpatient surgery in 27 countries. The primary outcome was in-hospital complications. Secondary outcomes were death following a complication (failure to rescue) and death in hospital. Process measures were admission to critical care immediately after surgery or to treat a complication and duration of hospital stay. A single definition of critical care was used for all countries. RESULTS: A total of 474 hospitals in 19 high-, 7 middle- and 1 low-income country were included in the primary analysis. Data included 44 814 patients with a median hospital stay of 4 (range 2-7) days. A total of 7508 patients (16.8%) developed one or more postoperative complication and 207 died (0.5%). The overall mortality among patients who developed complications was 2.8%. Mortality following complications ranged from 2.4% for pulmonary embolism to 43.9% for cardiac arrest. A total of 4360 (9.7%) patients were admitted to a critical care unit as routine immediately after surgery, of whom 2198 (50.4%) developed a complication, with 105 (2.4%) deaths. A total of 1233 patients (16.4%) were admitted to a critical care unit to treat complications, with 119 (9.7%) deaths. Despite lower baseline risk, outcomes were similar in low- and middle-income compared with high-income countries. CONCLUSIONS: Poor patient outcomes are common after inpatient surgery. Global initiatives to increase access to surgical treatments should also address the need for safe perioperative care. STUDY REGISTRATION: ISRCTN5181700

Cyclooxygenase-2 regulates apoptosis in rat epididymis through prostaglandin D 2 1

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) E 2; 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 PGD 2 and partially by PGE 2. In addition, the protective effect of PGD 2 against COX-2 inhibition was significantly blocked by a PGDP-receptor antagonist, BWA868C. These results indicate that the COX-2 products PGD 2 and, to a lesser extent, PGE 2 control apoptosis in cultured rat epididymal cells in vitro.link_to_subscribed_fulltex

Association of EGFR gene fragments with nuclear matrices in glioblastoma cell lines

The association of epidermal growth factor receptor DNA fragments with nuclear matrix in glioblastoma cell lines was investigated. Two different DNA fragments (primer I-III, 940 bp and primer IV-V, 110 bp) were amplified in both genomic DNA and nuclear matrix-associated DNA. It was found that the 110 bp DNA fragment (primer IV-V) from a non-encoding region was more closely associated with the nuclear matrices of cell line U343, U373, A172, and T98 than with U87. The other DNA fragment (primer I-III) was found in both the genomic DNA and NM DNA from cell line of U343 and U87. Another long DNA fragment (primer I-II, 1015 bp) was not detected in the DNA of all cell lines. Our findings suggest that the attachment of the 110 bp DNA fragments to nuclear matrix may contribute to the growth and malignancy of gliblastoma.link_to_subscribed_fulltex

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 μM 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 μM S3226 (3-[2-(3-guanidino-2-methyl-3-oxo-propenyl)-5-methyl-phenyl]-N-isopropylidene -2-methyl-acrylamide dihydrochloride), a NHE3 inhibitor, and 300 μM 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.link_to_OA_fulltex

Polarized distribution of NHE1 and NHE2 in the rat epididymis

Previous studies from our laboratory have provided evidence that the rat epididymis utilizes the Na+/H+ exchanger to transport acid and base. The present study was undertaken to use immunohistochemistry for investigating the localization (apical versus basolateral) and distribution of NHE1 and NHE2 proteins along intact rat epididymis. Both proteins were found to be exclusively localized within the epithelium. Immunoreactivity for NHE1 was detected on the basolateral surface, whereas NHE2 immunoreactivity was detected on the apical side of the epithelium. Interestingly, NHE1 was found along the entire length of the epididymal tubule whereas NHE2 was absent in the initial segment but present in the caput, corpus, and cauda regions. These results, when interpreted along with those of previous functional studies, may suggest that the apical NHE2 is involved in Na+ reabsorption and the basolateral NHE1 in HCO3 - secretion in the rat epididymis.link_to_subscribed_fulltex

Nuclear matrix of human nasopharyngeal carcinoma cells in culture: a preliminary report

Nuclear matrix is a scaffold-like structure of the nucleus after sequential treatments with detergents, DNase, and high salt buffers. Its involvement in various cellular activities has been emphasised in various studies. In this study, nuclear matrix of two human nasopharyngeal carcinoma cell lines were compared and analysed in terms of their morphological appearance and electrophoretic patterns of proteins. The stability of nuclear matrices against enzymatic digestion was found to be similar in these two cell lines. However, cell line specific nuclear matrix proteins were detected in each cell line. The significance of these proteins in cellular function and oncogenesis will be investigated in due course.link_to_subscribed_fulltex