Beta-adrenergic stimulation of ion transport in primary cultures of avian salt glands

1987 ◽  
Vol 252 (6) ◽  
pp. C670-C676 ◽  
Author(s):  
R. J. Lowy ◽  
S. A. Ernst
Keyword(s):  
Ion Transport ◽  
Short Circuit ◽  
Primary Cultures ◽  
Short Circuit Current ◽  
Salt Gland ◽  
Adrenergic Stimulation ◽  
Salt Glands ◽  
Beta Adrenergic ◽  
Effector Pathway ◽  
Stimulation Of

Adrenergic stimulation of transmural ion transport was identified and characterized in primary cultures of avian salt gland. Adrenergic activation was mediated by beta-receptors since stimulation of the short-circuit current (Isc) was blocked by propranolol but not phentolamine. The Isc's elicited by isoproterenol, epinephrine, and norepinephrine were dose dependent, with respective EC50 values of 1.5 X 10(-8) M, 5.0 X 10(-6) M, and 1.1 X 10(-5) M. The apparent Ki for propranolol inhibition after isoproterenol stimulation was 7.5 X 10(-10) M. 8-Br cyclic AMP (8-Br cAMP) and forskolin-elicited Isc's that were insensitive to propranolol, were potentiated by theophylline, and inhibited by furosemide or ouabain. Isoproterenol also induced an increase in ouabain-sensitive respiration in acutely dispersed cells from salt-stressed juvenile or unstressed adult animals, but not in fully salt-stressed adults. The data indicate that, in addition to the well-established cholinergic receptors, beta-adrenergic receptors can control ion transport in these glands. Furthermore, the results suggest for the first time that an intracellular effector pathway involving cAMP is present.

Mechanism of ion transport by avian salt gland primary cell cultures

1989 ◽  
Vol 256 (6) ◽  
pp. R1184-R1191
Author(s):  
R. J. Lowy ◽  
D. C. Dawson ◽  
S. A. Ernst
Keyword(s):  
Ion Transport ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Salt Gland ◽  
Secretory Cells ◽  
Adrenergic Stimulation ◽  
Beta Adrenergic ◽  
Dose Dependent Fashion ◽  
Active Ion Transport ◽  
Net Flux

Confluent sheets formed from primary culture of avian salt gland secretory cells exhibit a short-circuit current (Isc) in response to cholinergic and beta-adrenergic stimulation [Lowy, R. J., D. C. Dawson, and S. A. Ernst. Am J. Physiol. 249 (Cell Physiol. 18): C41-C47, 1985]. To establish the ionic basis for the Isc, transmural fluxes of 22Na and 36Cl were measured. Under short-circuit conditions there was little net flux of either ion in the absence of agonists. Addition of carbachol elevated net serosal-to-mucosal Cl flux to 1.71 mu eq.h-1.cm-2, whereas a smaller increase to 0.85 mu eq.h-1.cm-2 occurred with isoproterenol. Neither agonist altered net Na flux. The stimulated Isc accounted for 70% of the net Cl flux induced by carbachol and nearly 100% of that induced by isoproterenol. Replacement of Cl by gluconate or Na by choline abolished (carbachol) or greatly reduced (isoproterenol) the Isc, which could be restored in a dose-dependent fashion by ion restitution. Active ion transport was preferentially inhibited by basal (vs. apical) addition of ouabain, furosemide, or barium. The results provide evidence that cholinergic and beta-adrenergic agonists elicit active transmural Cl secretion. They further suggest that transport is dependent on the Na+-K+-adenosine-triphosphatase, a Na-Cl cotransport process, and a basal K conductance, all features of a secondary active Cl secretory mechanism.

Vasoactive intestinal peptide stimulates ion transport in avian salt gland

1987 ◽  
Vol 253 (6) ◽  
pp. R801-R808 ◽  
Author(s):  
R. J. Lowy ◽  
J. H. Schreiber ◽  
S. A. Ernst
Keyword(s):  
Ion Transport ◽  
Vasoactive Intestinal Peptide ◽  
Short Circuit ◽  
Primary Cultures ◽  
Short Circuit Current ◽  
Salt Gland ◽  
Nerve Fibers ◽  
Secretory Cells ◽  
Isolated Cells ◽  
Salt Glands

Avian salt glands are considered to be under the control of cholinergic nerve fibers. Here we report evidence that vasoactive intestinal peptide (VIP) also regulates ion transport. Nerve fibers stained immunocytochemically with anti-VIP were distributed throughout the tissue within the peritubular connective tissue and were in close proximity to the secretory tubules. VIP applied to primary cultures of the secretory cells elicited active ion transport as assayed by short-circuit current (Isc) analysis. The mucosal-to-serosal positive Isc was produced in a dose-dependent fashion [(EC50) = 3.1 X 10(-9) M], was potentiated by theophylline, and was inhibited by either ouabain or furosemide. This Isc was independent of activation by cholinergic agonists. VIP also increased ouabain-sensitive respiration 14-18% in acutely isolated cells from salt-stressed and unstressed animals. These data demonstrate for the first time that VIP is present in the avian salt gland and can act as a secretagogue by directly affecting the secretory cells. In addition, the results provide evidence for direct control of ion transport by an adenosine 3',5'-cyclic monophosphate-linked neurohormone in both adult unstressed and fully salt-stressed animals.

Primary culture of duck salt gland. II. Neurohormonal stimulation of active transport

1985 ◽  
Vol 249 (1) ◽  
pp. C41-C47 ◽  
Author(s):  
R. J. Lowy ◽  
D. C. Dawson ◽  
S. A. Ernst
Keyword(s):  
Ion Transport ◽  
Short Circuit ◽  
Primary Cultures ◽  
Short Circuit Current ◽  
Salt Gland ◽  
Hormonal Control ◽  
Regulatory Control ◽  
Secretory Cells ◽  
Active Ion Transport ◽  
Secretory Transport

Primary cultures of structurally polarized sheets of avian salt gland secretory cells were mounted in Lucite chambers for transmural electrophysiological analysis. Transmural resistance values increased during the first 3 days of culture to 293 +/- 35 omega X cm2 and then decreased slowly thereafter. There was little short-circuit current (Isc) in the absence of secretagogues. Serosal addition of either carbachol or epinephrine resulted in a Isc consistent with positive charge flow from mucosa to serosa, thus demonstrating that these cell layers were capable of active ion transport in response to either cholinergic or adrenergic neurohormonal stimulation. Serosal ouabain or furosemide abolished the response to either agonist, while theophylline enhanced the response. Receptor specificity for the electrical responses was shown by selective inhibition of carbachol- and epinephrine-induced Isc by atropine and propranolol, respectively. The results demonstrate that these primary epithelial cell cultures are capable of active ion transport and are sensitive to known inhibitors of secretory transport, and suggest that intracellular coupling mechanisms for hormonal control are retained in culture. These cultures should be useful for studying mechanisms of ion secretory transport and their regulatory control.

Pharmacological modulation of ion transport across wild-type and ΔF508 CFTR-expressing human bronchial epithelia

2000 ◽  
Vol 279 (2) ◽  
pp. C461-C479 ◽  
Author(s):  
Daniel C. Devor ◽  
Robert J. Bridges ◽  
Joseph M. Pilewski
Keyword(s):  
Cystic Fibrosis ◽  
Ion Transport ◽  
Short Circuit ◽  
Primary Cultures ◽  
Short Circuit Current ◽  
Bronchial Epithelium ◽  
Disulfonic Acid ◽  
Wild Type ◽  
Pharmacological Agents ◽  
Transport Defect

Forskolin, UTP, 1-ethyl-2-benzimidazolinone (1-EBIO), NS004, 8-methoxypsoralen (Methoxsalen; 8-MOP), and genistein were evaluated for their effects on ion transport across primary cultures of human bronchial epithelium (HBE) expressing wild-type (wt HBE) and ΔF508 (ΔF-HBE) cystic fibrosis transmembrane conductance regulator. In wt HBE, the baseline short-circuit current ( I sc) averaged 27.0 ± 0.6 μA/cm2 ( n = 350). Amiloride reduced this I sc by 13.5 ± 0.5 μA/cm2 ( n = 317). In ΔF-HBE, baseline I sc was 33.8 ± 1.2 μA/cm2 ( n = 200), and amiloride reduced this by 29.6 ± 1.5 μA/cm2 ( n = 116), demonstrating the characteristic hyperabsorption of Na+ associated with cystic fibrosis (CF). In wt HBE, subsequent to amiloride, forskolin induced a sustained, bumetanide-sensitive I sc(Δ I sc = 8.4 ± 0.8 μA/cm2; n = 119). Addition of acetazolamide, 5-( N-ethyl- N-isopropyl)-amiloride, and serosal 4,4′-dinitrostilben-2,2′-disulfonic acid further reduced I sc, suggesting forskolin also stimulates HCO3 − secretion. This was confirmed by ion substitution studies. The forskolin-induced I scwas inhibited by 293B, Ba2+, clofilium, and quinine, whereas charybdotoxin was without effect. In ΔF-HBE the forskolin I sc response was reduced to 1.2 ± 0.3 μA/cm2 ( n = 30). In wt HBE, mucosal UTP induced a transient increase in I sc (Δ I sc = 15.5 ± 1.1 μA/cm2; n = 44) followed by a sustained plateau, whereas in ΔF-HBE the increase in I sc was reduced to 5.8 ± 0.7 μA/cm2 ( n = 13). In wt HBE, 1-EBIO, NS004, 8-MOP, and genistein increased I sc by 11.6 ± 0.9 ( n = 20), 10.8 ± 1.7 ( n = 18), 10.0 ± 1.6 ( n = 5), and 7.9 ± 0.8 μA/cm2( n = 17), respectively. In ΔF-HBE, 1-EBIO, NS004, and 8-MOP failed to stimulate Cl− secretion. However, addition of NS004 subsequent to forskolin induced a sustained Cl−secretory response (2.1 ± 0.3 μA/cm2, n = 21). In ΔF-HBE, genistein alone stimulated Cl− secretion (2.5 ± 0.5 μA/cm2, n = 11). After incubation of ΔF-HBE at 26°C for 24 h, the responses to 1-EBIO, NS004, and genistein were all potentiated. 1-EBIO and genistein increased Na+ absorption across ΔF-HBE, whereas NS004 and 8-MOP had no effect. Finally, Ca2+-, but not cAMP-mediated agonists, stimulated K+ secretion across both wt HBE and ΔF-HBE in a glibenclamide-dependent fashion. Our results demonstrate that pharmacological agents directed at both basolateral K+ and apical Cl− conductances directly modulate Cl−secretion across HBE, indicating they may be useful in ameliorating the ion transport defect associated with CF.

Modulation of aldosterone-induced stimulation of ENaC synthesis by changing the rate of apical Na+ entry

2001 ◽  
Vol 281 (4) ◽  
pp. F687-F692 ◽  
Author(s):  
Lisette Dijkink ◽  
Anita Hartog ◽  
Carel H. Van Os ◽  
René J. M. Bindels
Keyword(s):  
Feedback Inhibition ◽  
Collecting Duct ◽  
Short Circuit ◽  
Primary Cultures ◽  
Short Circuit Current ◽  
Inhibitory Effect ◽  
Cortical Collecting Duct ◽  
Entry Rate ◽  
Protein Levels ◽  
Stimulation Of

Primary cultures of immunodissected rabbit connecting tubule and cortical collecting duct cells were used to investigate the effect of apical Na+ entry rate on aldosterone-induced transepithelial Na+ transport, which was measured as benzamil-sensitive short-circuit current ( I sc). Stimulation of the apical Na+ entry, by long-term short-circuiting of the monolayers, suppressed the aldosterone-stimulated benzamil-sensitive I sc from 320 ± 49 to 117 ± 14%, whereas in the presence of benzamil this inhibitory effect was not observed (335 ± 74%). Immunoprecipitation of [35S]methionine-labeled β-rabbit epithelial Na+ channel (rbENaC) revealed that the effects of modulation of apical Na+ entry on transepithelial Na+ transport are exactly mirrored by β-rbENaC protein levels, because short-circuiting the monolayers decreased aldosterone-induced β-rbENaC protein synthesis from 310 ± 51 to 56 ± 17%. Exposure to benzamil doubled the β-rbENaC protein level to 281 ± 68% in control cells but had no significant effect on aldosterone-stimulated β-rbENaC levels (282 ± 68%). In conclusion, stimulation of apical Na+ entry suppresses the aldosterone-induced increase in transepithelial Na+transport. This negative-feedback inhibition is reflected in a decrease in β-rbENaC synthesis or in an increase in β-rbENaC degradation.

Effects of tumor promoters on sodium ion transport across frog skin

1985 ◽  
Vol 248 (5) ◽  
pp. C457-C465 ◽  
Author(s):  
M. M. Civan ◽  
D. Rubenstein ◽  
T. Mauro ◽  
T. G. O'Brien
Keyword(s):  
Ion Transport ◽  
Frog Skin ◽  
Phorbol Esters ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Tumor Promoter ◽  
Tumor Promoters ◽  
Transport Effects ◽  
Preliminary Study ◽  
Stimulation Of

Phorbol esters are tumor promoters and mitogens whose effects may be mediated by changes in ion transport across membranes. Clarification of the transport effects of these agents should be facilitated by using a well-characterized model epithelial system whose intracellular and transmural parameters are readily measurable. The current results constitute a preliminary study of the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), phorbol-12,13-dibutyrate (PDBU), and phorbol on the short-circuit current (Isc) across frog skin. TPA produced two effects: a stimulation of Isc of variable magnitude and a far more constant inhibition of the natriferic action of vasopressin. These effects appear related to the action of TPA as a tumor promoter insofar as PDBU (an active ester) also inhibited the natriferic response to vasopressin, whereas phorbol (inactive as a tumor promoter) had no significant effect. TPA is largely active from the mucosal medium, inhibits the natriferic response to adenosine 3',5'-cyclic monophosphate (cAMP) as well as that to vasopressin, and does not stimulate Isc in the presence of 10(-4) M mucosal amiloride. Inhibition of prostaglandin E1 production by indomethacin had no effect on the actions of TPA. The results indicate that frog skin is a promising model for studying the transport effects of the phorbol esters. The data further suggest that TPA acts on frog skin by activating the physiological amiloride- and cAMP-sensitive channels gating apical Na+ entry from the mucosal medium into the epithelial cells.

Chloride transport in glands of frog skin

1983 ◽  
Vol 244 (3) ◽  
pp. C221-C226 ◽  
Author(s):  
I. G. Thompson ◽  
J. W. Mills
Keyword(s):  
Frog Skin ◽  
Chloride Transport ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Serosal Side ◽  
Adrenergic Stimulation ◽  
Beta Adrenergic ◽  
Skin Glands ◽  
Frog Skin Glands ◽  
Beta Adrenergic Agonist

The effects of beta-adrenergic stimulation on the bidirectional fluxes of Na+ and Cl- across the frog skin glands were determined. Isoproterenol elicited net serosal-to-mucosal fluxes of both Na+ (JNanet) and Cl- (JClnet) equal to 0.19 +/- 0.05 (SE) and 0.57 +/- 0.05 mueq X cm-2 X h-1, respectively. The residual current (JClnet - JNanet) of 0.38 +/- 0.05 mueq X cm-2 X h-1 closely approximates the isoproterenol-induced short-circuit current of 0.30 +/- 0.04 mueq X cm-2 X h-1. Furosemide added to the serosal side prior to isoproterenol inhibited the isoproterenol-induced net fluxes of both Na+ and Cl-. The addition of dibutyryl cAMP and 3-isobutyl-1-methylxanthine to the serosal side mimicked the action of isoproterenol by stimulating glandular short-circuit current. We conclude that an active Cl(-)-transport mechanism resides in the frog skin glands and is 1) stimulated by a beta-adrenergic agonist (its action is mimicked by cAMP) and 2) inhibited by the loop diuretic furosemide.

Adrenergic stimulation of Na+transport across alveolar epithelial cells involves activation of apical Cl−channels

1998 ◽  
Vol 275 (6) ◽  
pp. C1610-C1620 ◽  
Author(s):  
Xinpo Jiang ◽  
David H. Ingbar ◽  
Scott M. O’Grady
Keyword(s):  
Epithelial Cells ◽  
Short Circuit ◽  
Methanesulfonic Acid ◽  
Reversal Potential ◽  
Short Circuit Current ◽  
Alveolar Epithelial Cells ◽  
Sprague Dawley ◽  
Adrenergic Stimulation ◽  
Alveolar Epithelial ◽  
Stimulation Of

Alveolar epithelial cells were isolated from adult Sprague-Dawley rats and grown to confluence on membrane filters. Most of the basal short-circuit current ( I sc; 60%) was inhibited by amiloride (IC50 0.96 μM) or benzamil (IC50 0.5 μM). Basolateral addition of terbutaline (2 μM) produced a rapid decrease in I sc, followed by a slow recovery back to its initial amplitude. When Cl− was replaced with methanesulfonic acid, the basal I sc was reduced and the response to terbutaline was inhibited. In permeabilized monolayer experiments, both terbutaline and amiloride produced sustained decreases in current. The current-voltage relationship of the terbutaline-sensitive current had a reversal potential of −28 mV. Increasing Cl− concentration in the basolateral solution shifted the reversal potential to more depolarized voltages. These results were consistent with the existence of a terbutaline-activated Cl− conductance in the apical membrane. Terbutaline did not increase the amiloride-sensitive Na+ conductance. We conclude that β-adrenergic stimulation of adult alveolar epithelial cells results in an increase in apical Cl− permeability and that amiloride-sensitive Na+ channels are not directly affected by this stimulation.

Na+/Ca2+ exchange regulates Ca2+-dependent duodenal mucosal ion transport and HCO3− secretion in mice

2005 ◽  
Vol 288 (3) ◽  
pp. G457-G465 ◽  
Author(s):  
Hui Dong ◽  
Zachary M. Sellers ◽  
Anders Smith ◽  
Jimmy Y. C. Chow ◽  
Kim E. Barrett
Keyword(s):  
Western Blot ◽  
Ion Transport ◽  
Muscarinic Receptors ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Duodenal Mucosa ◽  
Ussing Chambers ◽  
Intracellular Ca ◽  
Ph Stat ◽  
Stimulation Of

Stimulation of muscarinic receptors in duodenal mucosa raises intracellular Ca2+, which regulates ion transport, including HCO3− secretion. However, the underlying Ca2+ handling mechanisms are poorly understood. The aim of the present study was to determine whether Na+/Ca2+ exchanger (NCX) plays a role in the regulation of duodenal mucosal ion transport and HCO3− secretion by controlling Ca2+ homeostasis. Mouse duodenal mucosa was mounted in Ussing chambers. Net ion transport was assessed as short-circuit current ( Isc), and HCO3− secretion was determined by pH-stat. Expression of NCX in duodenal mucosae was analyzed by Western blot, and cytosolic Ca2+ in duodenocytes was measured by fura 2. Carbachol (100 μM) increased Isc in a biphasic manner: an initial transient peak within 2 min and a later sustained plateau starting at 10 min. Carbachol-induced HCO3− secretion peaked at 10 min. 2-Aminoethoxydiphenylborate (2-APB, 100 μM) or LiCl (30 mM) significantly reduced the initial peak in Isc by 51 or 47%, respectively, and abolished the plateau phase of Isc without affecting HCO3− secretion induced by carbachol. Ryanodine (100 μM), caffeine (10 mM), and nifedipine (10 μM) had no effect on either response to carbachol. In contrast, nickel (5 mM) and KB-R7943 (10–30 μM) significantly inhibited carbachol-induced increases in duodenal mucosal Isc and HCO3− secretion. Western blot analysis showed expression of NCX1 proteins in duodenal mucosae, and functional NCX in duodenocytes was demonstrated in Ca2+ imaging experiments where Na+ depletion elicited Ca2+ entry via the reversed mode of NCX. These results indicate that NCX contributes to the regulation of Ca2+-dependent duodenal mucosal ion transport and HCO3− secretion that results from stimulation of muscarinic receptors.

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