Functional and molecular characterization of the fluid secretion mechanism in human parotid acinar cells

2007 ◽  
Vol 292 (6) ◽  
pp. R2380-R2390 ◽  
Author(s):  
Tetsuji Nakamoto ◽  
Alaka Srivastava ◽  
Victor G. Romanenko ◽  
Catherine E. Ovitt ◽  
Patricia Perez-Cornejo ◽  
...  
Keyword(s):  
Salivary Gland ◽  
Ion Transport ◽  
Acinar Cells ◽  
Fluid Secretion ◽  
K Channel ◽  
Resting Membrane Potential ◽  
Specific Cell ◽  
Parotid Glands ◽  
Voltage Dependent ◽  
Intracellular Ca

The strategies available for treating salivary gland hypofunction are limited because relatively little is known about the secretion process in humans. An initial microarray screen detected ion transport proteins generally accepted to be critically involved in salivation. We tested for the activity of some of these proteins, as well as for specific cell properties required to support fluid secretion. The resting membrane potential of human acinar cells was near −51 mV, while the intracellular [Cl−] was ∼62 mM, about fourfold higher than expected if Cl ions were passively distributed. Active Cl− uptake mechanisms included a bumetanide-sensitive Na+-K+-2Cl− cotransporter and paired DIDS-sensitive Cl−/HCO3− and EIPA-sensitive Na+/H+ exchangers that correlated with expression of NKCC1, AE2, and NHE1 transcripts, respectively. Intracellular Ca2+ stimulated a niflumic acid-sensitive Cl− current with properties similar to the Ca2+-gated Cl channel BEST2. In addition, intracellular Ca2+ stimulated a paxilline-sensitive and voltage-dependent, large-conductance K channel and a clotrimazole-sensitive, intermediate-conductance K channel, consistent with the detection of transcripts for KCNMA1 and KCNN4, respectively. Our results demonstrate that the ion transport mechanisms in human parotid glands are equivalent to those in the mouse, confirming that animal models provide valuable systems for testing therapies to prevent salivary gland dysfunction.

Cl-/HCO3- exchange is acetazolamide sensitive and activated by a muscarinic receptor-induced [Ca2+]i increase in salivary acinar cells

2004 ◽  
Vol 286 (2) ◽  
pp. G312-G320 ◽  
Author(s):  
Ha-Van Nguyen ◽  
Alan Stuart-Tilley ◽  
Seth L. Alper ◽  
James E. Melvin
Keyword(s):  
Salivary Gland ◽  
Carbonic Anhydrase ◽  
Muscarinic Receptor ◽  
Plasma Membranes ◽  
Acinar Cells ◽  
Parotid Glands ◽  
Atpase Inhibitor ◽  
Uptake Mechanism ◽  
Receptor Stimulation ◽  
Intracellular Ca

Large volumes of saliva are generated by transepithelial Cl- movement during parasympathetic muscarinic receptor stimulation. To gain further insight into a major Cl- uptake mechanism involved in this process, we have characterized the anion exchanger (AE) activity in mouse serous parotid and mucous sublingual salivary gland acinar cells. The AE activity in acinar cells was Na+ independent, electroneutral, and sensitive to the anion exchange inhibitor DIDS, properties consistent with the AE members of the SLC4A gene family. Localization studies using a specific antibody to the ubiquitously expressed AE2 isoform labeled acini in both parotid and sublingual glands. Western blot analysis detected an ∼170-kDa protein that was more highly expressed in the plasma membranes of sublingual than in parotid glands. Correspondingly, the DIDS-sensitive [Formula: see text] exchanger activity was significantly greater in sublingual acinar cells. The carbonic anhydrase antagonist acetazolamide markedly inhibited, whereas muscarinic receptor stimulation enhanced, the [Formula: see text] exchanger activity in acinar cells from both glands. Intracellular Ca2+ chelation prevented muscarinic receptor-induced upregulation of the AE, whereas raising the intracellular Ca2+ concentration with the Ca2+-ATPase inhibitor thapsigargin mimicked the effects of muscarinic receptor stimulation. In summary, carbonic anhydrase activity was essential for regulating [Formula: see text] exchange in salivary gland acinar cells. Moreover, muscarinic receptor stimulation enhanced AE activity through a Ca2+-dependent mechanism. Such forms of regulation may play important roles in modulating fluid and electrolyte secretion by salivary gland acinar cells.

Chloride Channels and Salivary Gland Function

1999 ◽  
Vol 10 (2) ◽  
pp. 199-209 ◽  
Author(s):  
J.E. Melvin
Keyword(s):  
Salivary Gland ◽  
Chloride Channels ◽  
Acinar Cells ◽  
Fluid Secretion ◽  
Ductal Cells ◽  
Salivary Gland Cells ◽  
Intracellular Ca ◽  
Cl Channels ◽  
Gland Function ◽  
Induced Changes

Fluid and electrolyte transport is driven by transepithelial Cl- movement. The opening of Cl- channels in the apical membrane of salivary gland acinar cells initiates the fluid secretion process, whereas the activation of Cl- channels in both the apical and the basolateral membranes of ductal cells is thought to be necessary for NaCl re-absorption. Saliva formation can be evoked by sympathetic and parasympathetic stimulation. The composition and flow rate vary greatly, depending on the type of stimulation. As many as five classes of Cl- channels with distinct gating mechanisms have been identified in salivary cells. One of these Cl- channels is activated by intracellular Ca2+, while another is gated by cAMP. An increase in the intracellular free Ca2+ concentration is the dominant mechanism triggering fluid secretion from acinar cells, while cAMP may be required for efficient NaCl re-absorption in many ductal cells. In addition to cAMP- and Ca 2+-gated Cl- channels, agonist-induced changes in membrane potential and cell volume activate different Cl- channels that likely play a role in modulating fluid and electrolyte movement. In this review, the properties of the different types of Cl- currents expressed in salivary gland cells are described, and functions are proposed based on the unique properties of these channels.

scholarly journals Electrogenic NBCe1 (SLC4A4), but not electroneutral NBCn1 (SLC4A7), cotransporter undergoes cholinergic-stimulated endocytosis in salivary ParC5 cells

2008 ◽  
Vol 295 (5) ◽  
pp. C1385-C1398 ◽  
Author(s):  
Clint Perry ◽  
David O. Quissell ◽  
Mary E. Reyland ◽  
Irina I. Grichtchenko
Keyword(s):  
Membrane Trafficking ◽  
Basolateral Membrane ◽  
Fluorescent Microscopy ◽  
Acinar Cells ◽  
Fluid Secretion ◽  
Parotid Glands ◽  
Calmodulin Antagonist ◽  
Parotid Acinar Cells ◽  
Early Endosomes ◽  
Gf 109203X

Cholinergic agonists are major stimuli for fluid secretion in parotid acinar cells. Saliva bicarbonate is essential for maintaining oral health. Electrogenic and electroneutral Na+-HCO3− cotransporters (NBCe1 and NBCn1) are abundant in parotid glands. We previously reported that angiotensin regulates NBCe1 by endocytosis in Xenopus oocytes. Here, we studied cholinergic regulation of NBCe1 and NBCn1 membrane trafficking by confocal fluorescent microscopy and surface biotinylation in parotid epithelial cells. NBCe1 and NBCn1 colocalized with E-cadherin monoclonal antibody at the basolateral membrane (BLM) in polarized ParC5 cells. Inhibition of constitutive recycling with the carboxylic ionophore monensin or the calmodulin antagonist W-13 caused NBCe1 to accumulate in early endosomes with a parallel loss from the BLM, suggesting that NBCe1 is constitutively endocytosed. Carbachol and PMA likewise caused redistribution of NBCe1 from BLM to early endosomes. The PKC inhibitor, GF-109203X, blocked this redistribution, indicating a role for PKC. In contrast, BLM NBCn1 was not downregulated in parotid acinar cells treated with constitutive recycling inhibitors, cholinergic stimulators, or PMA. We likewise demonstrate striking differences in regulation of membrane trafficking of NBCe1 vs. NBCn1 in resting and stimulated cells. We speculate that endocytosis of NBCe1, which coincides with the transition to a steady-state phase of stimulated fluid secretion, could be a part of acinar cell adjustment to a continuous secretory response. Stable association of NBCn1 at the membrane may facilitate constitutive uptake of HCO3− across the BLM, thus supporting HCO3− luminal secretion and/or maintaining acid-base homeostasis in stimulated cells.

Characterization of a novel 132-bp exon of the human maxi-K channel

2001 ◽  
Vol 281 (1) ◽  
pp. C361-C367 ◽  
Author(s):  
Victoria P. Korovkina ◽  
Daniel J. Fergus ◽  
Amanda J. Holdiman ◽  
Sarah K. England
Keyword(s):  
Alternative Splicing ◽  
Single Channel ◽  
K Channel ◽  
Distribution Analysis ◽  
Protein Distribution ◽  
Intracellular Loop ◽  
Cell Excitability ◽  
Muscle Tissues ◽  
Voltage Dependent ◽  
Intracellular Ca

The large-conductance Ca2+-activated voltage-dependent K+ channel (maxi-K channel) induces a significant repolarizing current that buffers cell excitability. This channel can derive its diversity by alternative splicing of its transcript-producing isoforms that differ in their sensitivity to voltage and intracellular Ca2+. We have identified a novel 132-bp exon of the maxi-K channel from human myometrial cells that encodes 44 amino acids within the first intracellular loop of the channel protein. Distribution analysis reveals that this exon is expressed predominantly in human smooth muscle tissues with the highest abundance in the uterus and aorta and resembles the previously reported distribution of the total maxi-K channel transcript. Single-channel K+ current measurements in fibroblasts transfected with the maxi-K channel containing this novel 132-bp exon demonstrate that the presence of this insert attenuates the sensitivity to voltage and intracellular Ca2+. Alternative splicing to introduce this 132-bp exon into the maxi-K channel may elicit another mode to modulate cell excitability.

Targeted disruption of the Nhe1 gene fails to inhibit β1-adrenergic receptor-induced parotid gland hypertrophy

2001 ◽  
Vol 280 (4) ◽  
pp. G694-G700
Author(s):  
James E. Melvin ◽  
Ha-Van Nguyen ◽  
Keith Nehrke ◽  
Claire M. Schreiner ◽  
Kelly G. Ten Hagen ◽  
...  
Keyword(s):  
Salivary Gland ◽  
Adrenergic Receptor ◽  
Acinar Cells ◽  
Receptor Activation ◽  
Parotid Glands ◽  
Targeted Disruption ◽  
Nhe1 Activity ◽  
Salivary Gland Hypertrophy ◽  
The Relationship

Chronic β1-adrenergic receptor activation results in hypertrophy and hyperplasia of rodent salivary gland acinar cells. Na+/H+ exchanger isoform 1 (NHE1) regulates cell volume and the induction of cell proliferation in many tissues. To investigate the relationship between NHE1 and the response of parotid glands to β1-adrenergic agonists, we examined by Northern blot analysis NHE1 expression in saline-treated mice and mice 30 min and 2, 6, and 24 h after isoproterenol injection. NHE1 transcripts increased ∼50% by 2 h, and a more than twofold increase was noted at 24 h. Isoproterenol did not acutely increase Na+/H+ exchanger activity; however, exchanger activity was significantly elevated by 24 h. To test whether NHE1 activity is essential for inducing salivary gland hypertrophy in vivo, mice with targeted disruption of Nhe1 were treated with isoproterenol. Na+/H+ exchanger activity was absent in acinar cells from Nhe1−/− mice, nevertheless, the lack of NHE1 failed to inhibit isoproterenol-induced hypertrophy. These data directly demonstrate that acinar cell hypertrophy induced by chronic β1-adrenergic receptor stimulation occurs independently of NHE1 activity.

NO causes perinatal pulmonary vasodilation through K+-channel activation and intracellular Ca2+release

1999 ◽  
Vol 276 (6) ◽  
pp. L925-L932 ◽  
Author(s):  
Connie B. Saqueton ◽  
Robert B. Miller ◽  
Valerie A. Porter ◽  
Carlos E. Milla ◽  
David N. Cornfield
Keyword(s):  
Channel Blocker ◽  
Control Period ◽  
Left Pulmonary Artery ◽  
Competitive Inhibitor ◽  
Late Gestation ◽  
K Channel ◽  
Channel Activation ◽  
Pulmonary Vasodilation ◽  
Voltage Dependent ◽  
Intracellular Ca

Evidence suggests that nitric oxide (NO) causes perinatal pulmonary vasodilation through K+-channel activation. We hypothesized that this effect worked through cGMP-dependent kinase-mediated activation of Ca2+-activated K+ channel that requires release of intracellular Ca2+ from a ryanodine-sensitive store. We studied the effects of 1) K+-channel blockade with tetraethylammonium, 4-aminopyridine, a voltage-dependent K+-channel blocker, or glibenclamide, an ATP-sensitive K+-channel blocker; 2) cyclic nucleotide-sensitive kinase blockade with either KT-5823, a guanylate-sensitive kinase blocker, or H-89, an adenylate-sensitive kinase blocker; and 3) blockade of intracellular Ca2+ release with ryanodine on NO-induced pulmonary vasodilation in acutely prepared late-gestation fetal lambs. N-nitro-l-arginine, a competitive inhibitor of endothelium-derived NO synthase, was infused into the left pulmonary artery, and tracheotomy was placed. The animals were ventilated with 100% oxygen for 20 min, followed by ventilation with 100% oxygen and inhaled NO at 20 parts/million (ppm) for 20 min. This represents the control period. In separate protocols, the animals received an intrapulmonary infusion of the different blockers and were ventilated as above. Tetraethylammonium ( n = 6 animals) and KT-5823 ( n = 4 animals) attenuated the response, whereas ryanodine ( n = 5 animals) blocked NO-induced perinatal pulmonary vasodilation. 4-Aminopyridine ( n = 5 animals), glibenclamide ( n = 5 animals), and H-89 ( n = 4 animals) did not affect NO-induced pulmonary vasodilation. We conclude that NO causes perinatal pulmonary vasodilation through cGMP-dependent kinase-mediated activation of Ca2+-activated K+ channels and release of Ca2+ from ryanodine-sensitive stores.

scholarly journals Effects of muscarinic, alpha-adrenergic, and substance P agonists and ionomycin on ion transport mechanisms in the rat parotid acinar cell. The dependence of ion transport on intracellular calcium.

1989 ◽  
Vol 93 (2) ◽  
pp. 285-319 ◽  
Author(s):  
S P Soltoff ◽  
M K McMillian ◽  
L C Cantley ◽  
E J Cragoe ◽  
B R Talamo
Keyword(s):  
Substance P ◽  
Ion Transport ◽  
Acinar Cell ◽  
Fluid Secretion ◽  
Ion Fluxes ◽  
Transport Systems ◽  
K Channel ◽  
Muscarinic Agonist ◽  
Parotid Acinar Cell ◽  
Rat Parotid

The relationship between receptor-mediated increases in the intracellular free calcium concentration [( Ca]i) and the stimulation of ion fluxes involved in fluid secretion was examined in the rat parotid acinar cell. Agonist-induced increases in [Ca]i caused the rapid net loss of up to 50-60% of the total content of intracellular chloride (Cli) and potassium (Ki), which is consistent with the activation of calcium-sensitive chloride and potassium channels. These ion movements were accompanied by a 25% reduction in the intracellular volume. The relative magnitudes of the losses of Ki and the net potassium fluxes promoted by carbachol (a muscarinic agonist), phenylephrine (an alpha-adrenergic agonist), and substance P were very similar to their characteristic effects on elevating [Ca]i. Carbachol stimulated the loss of Ki through multiple efflux pathways, including the large-conductance Ca-activated K channel. Carbachol and substance P increased the levels of intracellular sodium (Nai) to more than 2.5 times the normal level by stimulating the net uptake of sodium through multiple pathways; Na-K-2Cl cotransport accounted for greater than 50% of the influx, and approximately 20% was via Na-H exchange, which led to a net alkalinization of the cells. Ionomycin stimulated similar fluxes through these two pathways, but also promoted sodium influx through an additional pathway which was nearly equivalent in magnitude to the combined uptake through the other two pathways. The carbachol-induced increase in Nai and decrease in Ki stimulated the activity of the sodium pump, measured by the ouabain-sensitive rate of oxygen consumption, to nearly maximal levels. In the absence of extracellular calcium or in cells loaded with the calcium chelator BAPTA (bis[o-aminophenoxy]ethane-N,N,N',N'-tetraacetic acid) the magnitudes of agonist- or ionomycin-stimulated ion fluxes were greatly reduced. The parotid cells displayed a marked desensitization to substance P; within 10 min the elevation of [Ca]i and alterations in Ki, Nai, and cell volume spontaneously returned to near baseline levels. In addition to quantitating the activation of various ion flux pathways in the rat parotid acinar cell, these results demonstrate that the activation of ion transport systems responsible for fluid secretion in this tissue is closely linked to the elevation of [Ca]i.

A maturational shift in pulmonary K+ channels, from Ca2+ sensitive to voltage dependent

1998 ◽  
Vol 275 (6) ◽  
pp. L1019-L1025 ◽  
Author(s):  
Helen L. Reeve ◽  
E. Kenneth Weir ◽  
Stephen L. Archer ◽  
David N. Cornfield
Keyword(s):  
Pulmonary Circulation ◽  
Acute Hypoxia ◽  
Outward Current ◽  
Resting Membrane Potential ◽  
Transient Outward Current ◽  
Abrupt Decrease ◽  
Whole Cell ◽  
Electrophysiological Properties ◽  
Voltage Dependent ◽  
Intracellular Ca

The mechanism responsible for the abrupt decrease in resistance of the pulmonary circulation at birth may include changes in the activity of O2-sensitive K+ channels. We characterized the electrophysiological properties of fetal and adult ovine pulmonary arterial (PA) smooth muscle cells (SMCs) using conventional and amphotericin B-perforated patch-clamp techniques. Whole cell K+ currents of fetal PASMCs in hypoxia were small and characteristic of spontaneously transient outward currents. The average resting membrane potential (RMP) was −36 ± 3 mV and could be depolarized by charybdotoxin (100 nM) or tetraethylammonium chloride (5 mM; both blockers of Ca2+-dependent K+ channels) but not by 4-aminopyridine (4-AP; 1 mM; blocker of voltage-gated K+ channels) or glibenclamide (10 μM; blocker of ATP-dependent K+channels). In hypoxia, chelation of intracellular Ca2+ by 5 mM 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid further reduced the amplitude of the whole cell K+ current and prevented spontaneously transient outward current activity. Under these conditions, the remaining current was partially inhibited by 1 mM 4-AP. K+ currents of fetal PASMCs maintained in normoxia were not significantly reduced by acute hypoxia. In normoxic adult PASMCs, whole cell K+ currents were large and RMP was −49 ± 3 mV. These 4-AP-sensitive K+ currents were partially inhibited by exposure to acute hypoxia. We conclude that the K+ channel regulating RMP in the ovine pulmonary circulation changes after birth from a Ca2+-dependent K+ channel to a voltage-dependent K+ channel. The maturational-dependent differences in the mechanism of the response to acute hypoxia may be due to this difference in K+ channels.

Mechanisms of Ca2+-stimulated fluid secretion by porcine bronchial submucosal gland serous acinar cells

2010 ◽  
Vol 298 (2) ◽  
pp. L210-L231 ◽  
Author(s):  
Robert J. Lee ◽  
J. Kevin Foskett
Keyword(s):  
Basolateral Membrane ◽  
Acinar Cells ◽  
Fluid Secretion ◽  
Cell Swelling ◽  
Solute Content ◽  
Intracellular Ca ◽  
Shrinkage And Swelling ◽  
Serous Cell ◽  
Rat Parotid ◽  
Necessary And Sufficient

The serous acini of airway submucosal glands are important for fluid secretion in the lung. Serous cells are also sites of expression of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel. However, the mechanisms of serous cell fluid secretion remain poorly defined. In this study, serous acinar cells were isolated from porcine bronchi and studied using optical techniques previously used to examine fluid secretion in rat parotid and murine nasal acinar cells. When stimulated with the cholinergic agonist carbachol, porcine serous cells shrank by ∼20% (observed via DIC microscopy) after a profound elevation of intracellular [Ca2+] ([Ca2+]i; measured by simultaneous fura 2 fluorescence imaging). Upon removal of agonist and relaxation of [Ca2+]i to resting levels, cells swelled back to resting volume. Similar results were observed during stimulation with histamine and ATP, and elevation of [Ca2+]i was found to be necessary and sufficient to activate shrinkage. Cell volume changes were associated with changes in [Cl−]i (measured using SPQ fluorescence), suggesting that shrinkage and swelling are caused by loss and gain of intracellular solute content, respectively, likely reflecting changes in the secretory state of the cells. Shrinkage was inhibited by niflumic acid but not by GlyH-101, suggesting Ca2+-activated secretion is mediated by alternative non-CFTR Cl− channels, possibly including Ano1 (TMEM16A), expressed on the apical membrane of porcine serous cells. Optimal cell swelling/solute uptake required activity of the Na+K+2Cl− cotransporter and Na+/H+ exchanger, both of which are expressed on the basolateral membrane of serous acini and likely contribute to sustaining transepithelial secretion.

Effect of chronic hypoxia on K+ channels: regulation in human pulmonary vascular smooth muscle cells

1997 ◽  
Vol 272 (4) ◽  
pp. C1271-C1278 ◽  
Author(s):  
W. Peng ◽  
J. R. Hoidal ◽  
S. V. Karwande ◽  
I. S. Farrukh
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Chronic Hypoxia ◽  
Muscle Cells ◽  
K Channel ◽  
Resting Membrane Potential ◽  
Delayed Rectifier ◽  
Voltage Dependent ◽  
Dependent Protein ◽  
Pulmonary Arterial

We investigated the effects of chronic hypoxia on the major outward K+ currents in early cultured human main pulmonary arterial smooth muscle cells (HPSMC). Unitary currents were measured from inside-out, outside-out, and cell-attached patches of HPSMC. Chronic hypoxia depolarized resting membrane potential (Em) and reduced the activity of a charybdotoxin (CTX)- and iberiotoxin-sensitive, Ca2+-dependent K+ channel (KCa). The 4-aminopyridine-sensitive and CTX-insensitive channel or the delayed rectifier K+ channel was unaffected by chronic hypoxia. Chronic hypoxia caused a +33- to +53-mV right shift in voltage-dependent activation of K(Ca) and a decrease in K(Ca) activity at all cytosolic Ca2+ concentrations ([Ca2+]i) in the range of 0.1-10 microM. Thus the hypoxia-induced decrease in K(Ca) activity was most likely due to a decrease in K(Ca) sensitivity to Em and [Ca2+]i. Chronic hypoxia reduced the ability of nitric oxide (NO.) and guanosine 3',5'-cyclic monophosphate (cGMP) to activate K(Ca). The cGMP-dependent protein kinase-induced activation of K(Ca) was also significantly inhibited by chronic hypoxia. In addition, inhibiting channel dephosphorylation with calyculin A caused significantly less increase in K(Ca) activity in membrane patches excised from chronically hypoxic HPSMC compared with normoxic controls. This suggests that the mechanism by which hypoxia modulates NO.-induced K(Ca) activation is by decreasing the NO./cGMP-mediated phosphorylation of the channel.

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