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Changes in intracellular pH (pHi) were measured using the pH indicator, BCECF, in principal cells from split opened cortical collecting tubules (CCTs) derived from rabbits maintained on a normal diet. This monolayer preparation has the advantage of allowing us to visualize the morphological differences in the two major cell types in this nephron segment under transmitted light. The visual identification of the cell types was verified using emission measurements taken from single principal and intercalated cells in the opened tubule which had been exposed to fluorescein isothiocyanate (FITC)-labeled peanut lectin. We confirmed the existence of an amiloride-sensitive Na/H exchange process activated during intracellular acidosis in principal cells. In addition, the exchanger was active under basal conditions and over a wide range of pHi. Because the exchanger was active under basal conditions we tested the hypothesis that changes in intracellular Na (Nai) would alter pHi in a predictable way. Maneuvers designed to alter Nai were without significant effects within a 10-min time frame. Specifically, addition of 100 microM ouabain to increase Nai or exposure of the tubules to 10(-5) M amiloride to decrease luminal Na entry and reduce Nai did not have an effect on pHi. In some experiments we did observe however, after a 30-min exposure to ouabain, a small decrease in pHi. These results suggest that Na/H exchange is a major regulator of pHi in principal cells. However, regulation of Na transport by changes in pHi in principal cells of rabbit CCT via the activity of a Na/H exchanger do not seem to contribute to the feedback control of Na transport. [ABSTRACT FROM AUTHOR]

Chronic lung disease of prematurity (CLD) is a frequent sequela of premature birth and oxygen toxicity is a major associated risk factor. Impaired alveolarization, scarring, and inflammation are hallmarks of CLD. Mast cell hyperplasia is a feature of CLD but the role of mast cells in its pathogenesis is unknown. We hypothesized that mast cell hyperplasia is a consequence of neonatal hyperoxia and contributes to CLD. Additionally, mast cell products may have diagnostic and prognostic value in preterm infants predisposed to CLD. To model CLD, neonatal wild-type and mast cell-deficient mice were placed in an O2 chamber delivering hyperoxic gas mixture [inspired O2 fraction (FiO2 ) of 0.8] (HO) for 2 wk and then returned to room air (RA) for an additional 3 wk. Age-matched controls were kept in RA (FiO2 of 0.21). Lungs from HO mice had increased numbers of mast cells, alveolar simplification and enlargement, and increased lung compliance. Mast cell deficiency proved protective by preserving air space integrity and lung compliance. The mast cell mediators β-hexosaminidase (β-hex), histamine, and elastase increased in the bronchoalveolar lavage fluid of HO wild-type mice. Tracheal aspirate fluids (TAs) from oxygenated and mechanically ventilated preterm infants were analyzed for mast cell products. In TAs from infants with confirmed cases of CLD, β-hex was elevated over time and correlated with FiO2 Mast cell exosomes were also present in the TAs. Collectively, these data show that mast cells play a significant role in hyperoxia-induced lung injury and their products could serve as potential biomarkers in evolving CLD., (Copyright © 2016 the American Physiological Society.)

Background: Injection drug use is a skill learned in social settings. Change the Cycle (CTC), a peer-delivered, one-session intervention, is designed to reduce among people who inject drugs (PIDs) injection initiation-related behaviours (i.e., speaking positively about injecting to non-injectors, injecting in front of non-injectors, explaining or showing a non-injector how to inject) and initiation of non-injectors. We hypothesized that participation in CTC would lead to reductions in initiation-related behaviours six months later., Methods: Using respondent driven sampling (RDS), 98 PIDs were recruited in Toronto, Canada to participate in pilot testing of CTC. The baseline session consisted of a structured interview, the peer-delivered CTC intervention, instructions regarding RDS coupon distribution, and an invitation to return in six months for a follow-up interview. For the 84 PIDs completing the six-month interview, we compared initiation-related behaviours at baseline with six-month follow-up., Results: The proportion of PIDs offering to initiate a non-injector was reduced from 8.4% (95% CI: 2.5, 15.9) at baseline to 1.59% (95% CI: 0.4, 3.7) at 6-month follow-up. The prevalence of speaking positively about injection to non-injectors also decreased significantly. The proportion of PIDs who helped a non-injector with a first injection at baseline was 6.2% (95% CI: 2.1, 11.3) and at follow-up was 3.5% (95% CI: 0.8, 7.1). Paired analyses of initiator baseline versus follow-up data showed a 72.7% reduction in initiation (95%CI: 47.7, 83.1)., Conclusions: While further refinements remain to be tested, pilot study results suggest that CTC holds promise as a prevention intervention., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

We propose a new approach to address the question of how a single quantum of neurotransmitter is secreted from a presynaptic terminal whose clustered secretory vesicles are locally bathed in high levels of calcium ions [Proceedings of the Symposium on Bioelectrogenesis (1961) 297-309; The Physiology of Synapses (1964) Chapters 1, 4, 5, 6; How the Self Controls its Brain (1994) Chapters 1, 4, 5, 6; Science 256 (1992) 677-679]. This hypothesis, which we term 'porocytosis', posits that the post-synaptic quantal response results from transmitter secreted through an array of docked vesicle/secretory pore complexes. The transient increase in calcium ions, which results from the voltage activated calcium channels, stimulates the array of secretory pores to simultaneously flicker open to pulse transmitter. Porocytosis is consistent with the quantal nature of presynaptic secretion and transmission, and with available biochemical, morphological and physiological evidence. It explains the frequency dependency of quantal size as a function of the secretion process. It permits a signature amount of transmitter release for different frequencies allowing a given synapse to be employed in different behavioral responses. The porocytosis hypothesis permits fidelity of secretion and the seemingly apposed characteristic of synaptic plasticity. The dynamics inherent in an array insure a constant quantal size as a function of the number of units within the array. In this hypothesis, plasticity is a consequence of concurrent pre- and post-synaptic changes due to a change in array size. Changes in the number of docked vesicle-secretory pore complexes composing the array can explain facilitation, depletion, graded excitation-secretion and long term plasticity.

We coexpressed Kaposi's sarcoma-associated herpesvirus G protein-coupled receptors (KSHV-GPCRs) with thyrotropin-releasing hormone (TRH) receptors or m1-muscarinic-cholinergic receptors in Xenopus oocytes and in mammalian cells. In oocytes, KSHV-GPCR expression resulted in pronounced (81%) inhibition (heterologous desensitization) of Ca(2+)-activated chloride current responses to TRH and acetylcholine. Similar inhibitions of cytoplasmic free Ca(2+) responses to TRH were observed in human embryonic kidney HEK 293 EM cells and in mouse pituitary AtT20 cells. Further study of oocytes showed that this inhibition was partially reversed by interferon-gamma-inducible protein 10 (IP-10), an inverse agonist of KSHV-GPCR. The basal rate of (45)Ca(2+) efflux in oocytes expressing KSHV-GPCRs was 4.4 times greater than in control oocytes, and IP-10 rapidly inhibited increased (45)Ca(2+) efflux. In the absence of IP-10, growth-related oncogene alpha caused a further 2-fold increase in (45)Ca(2+) efflux. In KSHV-GPCR-expressing oocytes, responses to microinjected inositol 1,4,5-trisphosphate were inhibited by 74%, and this effect was partially reversed by interferon-gamma-inducible protein 10. Treatment with thapsigargin suggested that the pool of calcium available for mobilization by TRH was decreased in oocytes coexpressing KSHV-GPCRs. These results suggest that constitutive signaling by KSHV-GPCR causes heterologous desensitization of responses mediated by other receptors, which signal via the phosphoinositide/calcium pathway, which is caused by depletion of intracellular calcium pools.

Intercalated cells (ICs) from kidney collecting ducts contain proton-transporting ATPases (H(+)-ATPases) whose plasma membrane expression is regulated under a variety of conditions. It has been shown that net proton secretion occurs in the distal nephron from chronically K(+)-depleted rats and that upregulation of tubular H(+)- ATPase is involved in this process. However, regulation of this protein at the level of individual cells has not so far been examined. In the present study, H(+)-ATPase activity was determined in individually identified ICs from control and chronically K(+)-depleted rats (9-14 days on a low-K(+) diet) by monitoring K(+)- and Na(+)-independent H(+) extrusion rates after an acute acid load. Split-open rat cortical collecting tubules were loaded with the intracellular pH (pH(i)) indicator 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, and pH(i) was determined by using ratiometric fluorescence imaging. The rate of pH(i) recovery in ICs in response to an acute acid load, a measure of plasma membrane H(+)-ATPase activity, was increased after K(+) depletion to almost three times that of controls. Furthermore, the lag time before the start of pH(i) recovery after the cells were maximally acidified fell from 93.5 +/- 13.7 s in controls to 24.5 +/- 2.1 s in K(+)-depleted rats. In all ICs tested, Na(+)- and K(+)-independent pH(i) recovery was abolished in the presence of bafilomycin (100 nM), an inhibitor of the H(+)-ATPase. Analysis of the cell-to-cell variability in the rate of pH(i) recovery reveals a change in the distribution of membrane-bound proton pumps in the IC population of cortical collecting duct from K(+)-depleted rats. Immunocytochemical analysis of collecting ducts from control and K(+)-depleted rats showed that K(+)-depletion increased the number of ICs with tight apical H(+)ATPase staining and decreased the number of cells with diffuse or basolateral H(+)-ATPase staining. Taken together, these data indicate that chronic K(+) depletion induces a marked increase in plasma membrane H(+)ATPase activity in individual ICs.

The ROMK family of proteins has biophysical properties and distribution within the kidney similar to those of secretory potassium channels of the distal nephron. To study the regulation of ROMK during variations in dietary potassium, we measured the abundance of ROMK protein in rat kidney by immunoblotting. Neither 2 nor 5 days of a high-potassium diet had an effect on protein abundance in the cortex or medulla. Potassium deprivation (2 or 5 days) decreased ROMK protein content in both cortical and medullary fractions, to 51 and 40% of controls, respectively. To see whether the Na-K-2Cl cotransporter is similarly affected by potassium restriction, we analyzed immunoblots by using an antibody for the rat type 1 bumetanide-sensitive cotransporter (BSC-1). Like ROMK, BSC-1 protein content was found to decrease significantly in the renal medulla of potassium-deprived rats. In the thick ascending limb of Henle's loop, a decrease in ROMK and BSC-1 could result in decreased reabsorption of NaCl, a finding associated with hypokalemia.

Molecular cloning experiments have identified the existence of two H+-K+-ATPases (HKAs), colonic and gastric. Recent functional and molecular studies indicate the presence of both transporters in the kidney, which are presumed to mediate the exchange of intracellular H+ for extracellular K+. On the basis of these studies, a picture is evolving that indicates differential regulation of HKAs at the molecular level in acid-base and electrolyte disorders. Of the two transporters, gastric HKA is expressed constitutively along the length of the collecting duct and is responsible for H+ secretion and K+ reabsorption under normal conditions and may be stimulated with acid-base perturbations and/or K+ depletion. This regulation may be species specific. To date there are no data to indicate that the colonic HKA (HKAc) plays a role in H+ secretion or K+ reabsorption under normal conditions. However, HKAc shows adaptive regulation in pathophysiological conditions such as K+ depletion, NaCl deficiency, and proximal renal tubular acidosis, suggesting an important role for this exchanger in potassium, HCO-3, and sodium (or chloride) reabsorption in disease states. The purpose of this review is to summarize recent functional and molecular studies on the regulation of HKAs in physiological and pathophysiological states. Possible signals responsible for regulation of HKAs in these conditions will be discussed. Furthermore, the role of these transporters in acid-base and electrolyte homeostasis will be evaluated in the context of genetically altered animals deficient in HKAc.

We imaged the horizontal semicircular canal (HSCC) crista and cupula of toadfish, Opsanus tau, by using a) confocal light microscopy of isolated vital HSCC; b) serial sections of fixed, trichrome-stained HSCC; and c) scanning electron microscopy of fixed HSCCs. HSCC were dissections which included an ampulla and an attached canal tube (long and slender canal portion), and, in some cases, a small portion of the utricular wall. Cupulae were seen as multipartite mucus connective tissue shells rising from the crista and extending toward the ampullary roof. They were composed of several refractile bands traversing the cupulae perpendicular to longitudinal fibers extending from the cupular base to its apex. Alcian green-stained cupulae showed an asymmetric alcianphilic, dark, X-shaped structure, indicating that the pillar is rich in mucin and carbohydrate, an interpretation supported by images of trichrome-stained sections. The cupular antrum is devoid of prominent refractile fibers. No tubes or channels were observed in the cupula or antrum of vital preparations. Cupular shell fibers cover the surface of the crista, are roughly parallel, and are associated with a translucent material having a refractive index greater than the surrounding endolymph. Stereocilia were thin, 100-microm-long structures, with little longitudinal curvature, which end with no end bulb. No strands extend from stereocilia to the roof or other portions of the cupular antrum. Gross movements of stereocilia were not seen in mechanically quiescent preparations. Within the cupular antrum, stereocilia were parallel to connective tissue fibers, all embedded in an isotropic gel. This fiber-reinforced gel and cupular matrix are sensitive to N-acetlyneuraminidase and beta-N-acetyl glucosaminidase, and minimally sensitive to beta-N-acetyl hexosaminidase. Connective tissue fibers may serve to stiffen the gel, whose matrix would restrict lateral motion of embedded fibers and stereocilia thereby providing mechanical support for stereocilia.