Background-Abnormally elevated blood pressure is the most prevalent risk factor for cardiovascular disease. The large-conductance, voltage-and Ca 2ϩ -dependent K ϩ (BK) channel has been proposed as an important effector in the control of vascular tone by linking membrane depolarization and local increases in cytosolic Ca 2ϩ to hyperpolarizing K ϩ outward currents. However, the BK channel may also affect blood pressure by regulating salt and fluid homeostasis, particularly by adjusting the renin-angiotensin-aldosterone system. Methods and Results-Here we report that deletion of the pore-forming BK channel ␣ subunit leads to a significant blood pressure elevation resulting from hyperaldosteronism accompanied by decreased serum K ϩ levels as well as increased vascular tone in small arteries. In smooth muscle from small arteries, deletion of the BK channel leads to a depolarized membrane potential, a complete lack of membrane hyperpolarizing spontaneous K ϩ outward currents, and an attenuated cGMP vasorelaxation associated with a reduced suppression of Ca 2ϩ transients by cGMP. The high level of BK channel expression observed in wild-type adrenal glomerulosa cells, together with unaltered serum renin activities and corticotropin levels in mutant mice, suggests that the hyperaldosteronism results from abnormal adrenal cortical function in BK Ϫ/Ϫ mice. Conclusions-These results identify previously unknown roles of BK channels in blood pressure regulation and raise the possibility that BK channel dysfunction may underlie specific forms of hyperaldosteronism. 6,7 Recent studies raise the possibility that changes in ␤1 subunit expression contribute to the development of hypertension in rat 8 and that gain of function mutation in the same subunit decreases the prevalence of diastolic hypertension in humans. 9 However, even in the absence of functional ␤1 subunits, the ␣ subunit can still form functional channels, which might be activated at physiological potentials if their voltage and Ca 2ϩ sensitivity are increased by other factors such as endothelial factors 10,11 and/or phosphorylation. 12,13 Thus, functional BK channels may be operative in blood vessels even when the ␤1 subunit is lacking. In addition, BK channels in tissues other than vasculature, such as the adrenal gland, 14 may also influence blood pressure regulation. Therefore, we used mice lacking the BK channel ␣ subunit (BK Ϫ/Ϫ 15 to evaluate the global impact of BK channels on blood pressure regulation. MethodsDetails are given in the online-only Data Supplement. Mice BKϪ/Ϫ mice were generated as described. 15 Wild type (WT) and BK Ϫ/Ϫ mice with the hybrid SV129/C57BL6 background (always F2 generation) were used. Either litter-or age-matched animals were randomly assigned to the experimental procedures undertaken in accordance with the German legislation on protection of animals. Immunohistochemistry of Adrenal GlandFor immunofluorescence, on-slide 5-m cryostat slices from nonfixed WT and BK Ϫ/Ϫ adrenal glands were incubated with anti-BK␣ (674 -1115) . BK...

This review covers the topic of cytometric assessment of activation of Ataxia telangiectasia mutated (ATM) protein kinase and histone H2AX phosphorylation on Ser139 in response to DNA damage, particularly the damage that involves formation of DNA double-strand breaks. Briefly described are molecular mechanisms associated with activation of ATM and the downstream events that lead to recruitment of DNA repair machinery, engagement of cell cycle checkpoints, and activation of apoptotic pathway. Examples of multiparameter analysis of ATM activation and H2AX phosphorylation vis-a-vis cell cycle phase position and induction of apoptosis that employ flow-and laser scanning-cytometry are provided. They include cells treated with a variety of exogenous genotoxic agents, such as ionizing and UV radiation, DNA topoisomerase I (topotecan) and II (mitoxantrone, etoposide) inhibitors, nitric oxide-releasing aspirin, DNA replication inhibitors (aphidicolin, hydroxyurea, thymidine), and complex environmental carcinogens such as present in tobacco smoke. Also presented is an approach to identify DNA replicating (BrdU incorporating) cells based on selective photolysis of DNA that triggers H2AX phosphorylation. Listed are strategies to distinguish ATM activation and H2AX phosphorylation induced by primary DNA damage by genotoxic agents from those effects triggered by DNA fragmentation that takes place during apoptosis. While we review most published data, recent new findings also are included. Examples of multivariate analysis of ATM activation and H2AX phosphorylation presented in this review illustrate the advantages of cytometric flow-and image-analysis of these events in terms of offering a sensitive and valuable tool in studies of factors that induce DNA damage and/or affect DNA repair and allow one to explore the linkage between DNA damage, cell cycle checkpoints and initiation of apoptosis. ' International Society for Analytical CytologyKey terms ionizing radiation; DNA topoisomerase inhibitors; DNA double-strand breaks; carcinogens; tobacco smoke; replication stress; genotoxins; DNA photolysis ACTIVATION OF ATM AND PHOSPHORYLATION OF HISTONE H2AX TRIGGERED BY DNA DAMAGE Ataxia telangiectasia mutated (ATM) is a protein kinase that becomes activated in response to DNA damage, particularly when the damage involves formation of DNA double-strand breaks (DSBs) (1-9; Fig. 1). Interestingly, the initial activation of ATM does not takes place at the exact site of the DSB but at some distance from it, and appears to be triggered by a change in the higher order of chromatin structure caused by unwinding and relaxation of the topological stress of the DNA double helix upon induction of the DSB (4). Activation of ATM occurs through its autophosphorylation on Ser1981 and it requires prior ATM acetylation that is mediated by the Tip60 histone acetyltransferase (13). Ser1981 ATM phosphorylation causes dissociation of the inactive ATM dimer or multimer into single monomeric units

Activation of endonucleases that cleave chromosomal DNA preferentially at internucleosomal sections is a hallmark of apoptosis. DNA fragmentation revealed by the presence of a multitude of DNA strand breaks, therefore, is considered to be the gold standard for identification apoptotic cells. Several variants of the methodology that is based on fluorochrome-labeling of 3′-OH termini of DNA strand breaks in situ with the use of exogenous terminal deoxynucleotidyl transferase (TdT), commonly defined as the TUNEL assay, have been developed by us. This Chapter describes the variant based on strand breaks labeling with Br-dUTP that is subsequently detected immunocytochemically with Br-dU Ab. Compared with other TUNEL variants the Br-dU-labeling assay offers the greatest sensitivity in detecting DNA breaks. Described also are modifications of the protocol that allow one to use other than Br-dUTP fluorochrome-tagged deoxynucleotides to label DNA breaks. Concurrent staining of DNA with propidium or 4′,6-diamidino-2-phenylindole (DAPI) and multiparameter analysis of cells by flow-or laser scanning-cytometry enables one to correlate induction of apoptosis with the cell cycle phase.

A B S T R A C T The pancreatic duct secretes alkaline fluid that is rich in HCO 3-and poor in C1-. The molecular mechanisms that mediate ductal secretion and are responsible for the axial gradients of C1-and HCO3-along the ductal tree are not well understood because H + and HCO3-transport by duct cells have not been characterized or localized. To address these questions, we microdissected the intralobular, main, and common segments of the rat pancreatic duct. H + and HCO3-transporters were characterized and localized by following intracellular pH while perfusing the bath and the lumen of the ducts. In intralobular ducts, Na+-dependent and amiloride-sensitive recovery from acid load in the absence of HCO3-was used to localize a Na+/H + exchanger to the basolateral membrane (BLM). Modification of C1-gradients across the luminal (LM) and BLM in the presence of HCO~-showed the presence of C1-/HCO3-exchangers on both membranes of intralobular duct cells. Measurement of the effect of C1-on one side of the membrane on the rate and extent of pHi changes caused by removal and addition of C1-to the opposite side suggested that both exchangers are present in the same cell. In the presence of HCO3-, intralobular duct cells used three separate mechanisms to extrude H+: (a) BLM-Iocated Na+/H + exchange, (b) Na+-independent vacuolar-type H + pump, and (c) BLM-located, Na+-dependent, amilorideinsensitive, and 4',4'-diisothiocyanatostilbene-2,2'-disulfonic acid sensitive mechanism, possibly a Na+-dependent HCO:c transporter.The main and common segments of the duct displayed similar mechanisms and localization of H + and HCO3-transporters to the extent studied in the present work. In addition to the transporters found in intralobular ducts, the main and common ducts showed Na+/H + exchange activity in the LM. Three tests were used to exclude a significant luminal to basolateral Na + leak as the cause for an apparent luminal Na+/H + exchange in an HCO3-secreting cells: (a) addition of amiloride and removal of Na + from the LM had a profound effect on Na+/H + exchange activity on the BLM and vice versa; (b) inhibition of all transporters in the BLM by bathing the duct in the inert hydrocarbon Fluorinert FC-75 did not prevent cytosolic acidification caused by removal of luminal Na+; and (c) luminal Na + did not activate the basolateral Na+-dependent HCO3-transporter.An Na+-independent, bafilomycin-sensitive H + pumping activity was marginal in the absence of HCO3-. Exposing all segments of the duct to COz/HCO3-significantly increased the appearance of H + pumping in the plasma membrane. This J. GEN. PHYSIOL. 9 The Rockefeller University Press