Effects of extracellular metabolic acidosis and out-of-equilibrium CO/HCO solutions on intracellular pH in cultured rat hippocampal neurons.

Metabolic acidosis (MAc)-an extracellular pH (pH) decrease caused by a [HCO ] decrease at constant [CO]-usually causes intracellular pH (pH) to fall. Here we determine the extent to which the pH decrease depends on the pH decrease vs the concomitant [HCO ] decrease. We use rapid-mixing to generate out-of-equilibrium CO/HCO solutions in which we stabilize [CO] and [HCO ] while decreasing pH (pure acidosis, pAc), or stabilize [CO] and pH while decreasing [HCO ] (pure metabolic/down, pMet↓).

Capsaicin induces NKCC1 internalization and inhibits chloride secretion in colonic epithelial cells independently of TRPV1.

Colonic chloride secretion is regulated via the neurohormonal and immune systems. Exogenous chemicals (e.g.

Secreted protein acidic and rich in cysteine is a matrix scavenger chaperone.

Secreted Protein Acidic and Rich in Cysteine (SPARC) is one of the major non-structural proteins of the extracellular matrix (ECM) in remodeling tissues. The functional significance of SPARC is emphasized by its origin in the first multicellular organisms and its high degree of evolutionary conservation. Although SPARC has been shown to act as a critical modulator of ECM remodeling with profound effects on tissue physiology and architecture, no plausible molecular mechanism of its action has been proposed.

Activated PKC{delta} and PKC{epsilon} inhibit epithelial chloride secretion response to cAMP via inducing internalization of the Na+-K+-2Cl- cotransporter NKCC1.

The basolateral Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) is a key determinant of transepithelial chloride secretion and dysregulation of chloride secretion is a common feature of many diseases including secretory diarrhea. We have previously shown that activation of protein kinase C (PKC) markedly reduces transepithelial chloride secretion in human colonic T84 cells, which correlates with both functional inhibition and loss of the NKCC1 surface expression. In the present study, we defined the specific roles of PKC isoforms in regulating epithelial NKCC1 and chloride secretion utilizing adenoviral vectors that express shRNAs targeting human PKC isoforms (α, δ, ε) (shPKCs) or LacZ (shLacZ, non-targeting control).

Phorbol 12-myristate 13-acetate-induced endocytosis of the Na-K-2Cl cotransporter in MDCK cells is associated with a clathrin-dependent pathway.

In secretory epithelial cells, the basolateral Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) plays a major role in salt and fluid secretion. Our laboratory has identified NKCC1 surface expression as an important regulatory mechanism for Cl(-) secretion in the colonic crypt cell line T84, a process also present in native human colonic crypts. We previously showed that activation of protein kinase C (PKC) by carbachol and phorbol 12-myristate 13-acetate (PMA) decreases NKCC1 surface expression in T84 cells.

Cloning and characterization of novel human SLC4A8 gene products encoding Na+-driven Cl-/HCO3(-) exchanger variants NDCBE-A, -C, and -D.

The reported sequences of the human and mouse Na+-driven Cl-/HCO3(-) exchangers (NDCBEs) differ greatly in their extreme cytosolic COOH termini (Ct). In human NDCBE (NDCBE-B), a 17-amino acid (aa) sequence replaces 66 aa at the equivalent position in mouse NDCBE (NDCBE-A). We performed 5'- and 3'-rapid amplification of cDNA ends (RACE) on human brain cDNA, followed by PCR of full-length cDNAs to determine whether the human SLC4A8 gene was capable of producing the mouselike Ct sequence.

Role of the AT1A receptor in the CO2-induced stimulation of HCO3- reabsorption by renal proximal tubules.

The proximal tubule (PT) is major site for the reabsorption of filtered HCO(3)(-). Previous work on the rabbit PT showed that 1) increases in basolateral (BL) CO(2) concentration ([CO(2)](BL)) raise the HCO(3)(-) reabsorption rate (J(HCO(3))), and 2) the increase that luminal angiotensin II (ANG II) produces in J(HCO(3)) is greatest at 0% [CO(2)](BL) and falls to nearly zero at 20%. Here, we investigate the role of angiotensin receptors in the [CO(2)](BL) dependence of J(HCO(3)) in isolated perfused PTs.

Colony-stimulating factor-1 increases osteoclast intracellular pH and promotes survival via the electroneutral Na/HCO3 cotransporter NBCn1.

Colony-stimulating factor-1 (CSF-1) promotes the survival of osteoclasts, short-lived cells that resorb bone. Although a rise in intracellular pH (pH(i)) has been linked to inhibition of apoptosis, the effect of CSF-1 on pH(i) in osteoclasts has not been reported. The present study shows that, in the absence of CO(2)/HCO(3)(-), CSF-1 causes little change in osteoclast pH(i).

Role of a tyrosine kinase in the CO2-induced stimulation of HCO3- reabsorption by rabbit S2 proximal tubules.

A previous study demonstrated that proximal tubule cells regulate HCO(3)(-) reabsorption by sensing acute changes in basolateral CO(2) concentration, suggesting that there is some sort of CO(2) sensor at or near the basolateral membrane (Zhou Y, Zhao J, Bouyer P, and Boron WF Proc Natl Acad Sci USA 102: 3875-3880, 2005). Here, we hypothesized that an early element in the CO(2) signal-transduction cascade might be either a receptor tyrosine kinase (RTK) or a receptor-associated (or soluble) tyrosine kinase (sTK). In our experiments, we found, first, that basolateral 17.

Effects of angiotensin II on the CO2 dependence of HCO3- reabsorption by the rabbit S2 renal proximal tubule.

Previous authors showed that, at low doses, both basolateral and luminal ANG II increase the proximal tubule's HCO(3)(-) reabsorption rate (J(HCO(3))). Using out-of-equilibrium CO(2)/HCO(3)(-) solutions, we demonstrated that basolateral CO(2) increases J(HCO(3)). Here, we examine interactions between ANG II and CO(2) in isolated, perfused rabbit S2 segments.

Evidence from renal proximal tubules that HCO3- and solute reabsorption are acutely regulated not by pH but by basolateral HCO3- and CO2.

Respiratory acidosis, a decrease in blood pH caused by a rise in [CO(2)], rapidly triggers a compensatory response in which the kidney markedly increases its secretion of H(+) from blood to urine. However, in this and other acid-base disturbances, the equilibrium CO(2) + H(2)O HCO(3)(-) + H(+) makes it impossible to determine whether the critical parameter is [CO(2)], [HCO(3)(-)], and/or pH. Here, we used out-of-equilibrium CO(2)/HCO(3)(-) solutions to alter basolateral (BL) [HCO(3)(-)], [CO(2)], or pH, systematically and one at a time, on isolated perfused S2 rabbit proximal tubules.

Effect of extracellular acid-base disturbances on the intracellular pH of neurones cultured from rat medullary raphe or hippocampus.

Previous reports suggest that an important characteristic of chemosensitive neurones is an unusually large change of steady-state intracellular pH in response to a change in extracellular pH (DeltapH(i)/DeltapH(o)). To determine whether such a correlation exists between neurones from the medullary raphe (a chemosensitive brain region) and hippocampus (a non-chemosensitive region), we used BCECF to monitor pH(i) in cultured neurones subjected to extracellular acid-base disturbances. In medullary raphe neurones, respiratory acidosis (5%--> 9% CO(2)) caused a rapid fall in pH(i) (DeltapH(i) approximately 0.

An increase in intracellular calcium concentration that is induced by basolateral CO2 in rabbit renal proximal tubule.

Working with isolated perfused S2 proximal tubules, we asked whether the basolateral CO2 sensor acts, in part, by raising intracellular Ca2+ concentration ([Ca2+]i), monitored with the dye fura 2 (or fura-PE3). In paired experiments, adding 5% CO2/22 mM HCO3- (constant pH 7.40) to the bath (basolateral) solution caused [Ca2+]i to increase from 57 +/- 3 to 97 +/- 9 nM(n = 8, P < 0.

Transport of volatile solutes through AQP1.

For almost a century it was generally assumed that the lipid phases of all biological membranes are freely permeable to gases. However, recent observations challenge this dogma. The apical membranes of epithelial cells exposed to hostile environments, such as gastric glands, have no demonstrable permeability to the gases CO2 and NH3.