scholarly journals Functional and molecular identification of a TASK-1 potassium channel regulating chloride secretion through CFTR channels in the shark rectal gland: implications for cystic fibrosis

2016 ◽  
Vol 311 (6) ◽  
pp. C884-C894 ◽  
Author(s):  
Connor J. Telles ◽  
Sarah E. Decker ◽  
William W. Motley ◽  
Alexander W. Peters ◽  
Ali Poyan Mehr ◽  
...  
Keyword(s):  
Potassium Channel ◽  
Reversal Potential ◽  
Chloride Secretion ◽  
Rectal Gland ◽  
Degenerate Primers ◽  
Reading Frame ◽  
Genomic Walking ◽  
Shark Rectal Gland ◽  
Mammalian Lung ◽  
Rapid Amplification

In the shark rectal gland (SRG), apical chloride secretion through CFTR channels is electrically coupled to a basolateral K+ conductance whose type and molecular identity are unknown. We performed studies in the perfused SRG with 17 K+ channel inhibitors to begin this search. Maximal chloride secretion was markedly inhibited by low-perfusate pH, bupivicaine, anandamide, zinc, quinidine, and quinine, consistent with the properties of an acid-sensitive, four-transmembrane, two-pore-domain K+ channel (4TM-K2P). Using PCR with degenerate primers to this family, we identified a TASK-1 fragment in shark rectal gland, brain, gill, and kidney. Using 5′ and 3′ rapid amplification of cDNA ends PCR and genomic walking, we cloned the full-length shark gene (1,282 bp), whose open reading frame encodes a protein of 375 amino acids that was 80% identical to the human TASK-1 protein. We expressed shark and human TASK-1 cRNA in Xenopus oocytes and characterized these channels using two-electrode voltage clamping. Both channels had identical current-voltage relationships (outward rectifying) and a reversal potential of −90 mV. Both were inhibited by quinine, bupivicaine, and acidic pH. The pKa for current inhibition was 7.75 for shark TASK-1 vs. 7.37 for human TASK-1, values similar to the arterial pH for each species. We identified this protein in SRG by Western blot and confocal immunofluorescent microscopy and detected the protein in SRG and human airway cells. Shark TASK-1 is the major K+ channel coupled to chloride secretion in the SRG, is the oldest 4TM 2P family member identified, and is the first TASK-1 channel identified to play a role in setting the driving force for chloride secretion in epithelia. The detection of this potassium channel in mammalian lung tissue has implications for human biology and disease.

Cyclooxygenase cloning in dogfish shark, Squalus acanthias, and its role in rectal gland Cl secretion

2002 ◽  
Vol 283 (3) ◽  
pp. R631-R637 ◽  
Author(s):  
T. Yang ◽  
S. J. Forrest ◽  
N. Stine ◽  
Y. Endo ◽  
A. Pasumarthy ◽  
...  
Keyword(s):  
Cdna Sequence ◽  
Chloride Secretion ◽  
Protein Product ◽  
Rectal Gland ◽  
Peak Response ◽  
Reading Frame ◽  
Acid Protein ◽  
Elasmobranch Species ◽  
Cox 2 ◽  
Cox 1

The present studies were carried out with the aims to determine the cDNA sequence for cyclooxygenase (COX) in an elasmobranch species and to study its role in regulation of chloride secretion in the perfused shark rectal gland (SRG). With the use of long primers (43 bp) derived from regions of homology between zebrafish and rainbow trout COX-2 genes, a 600-bp product was amplified from SRG and was found to be almost equally homologous to mammalian COX-1 and COX-2 (65%). The full-length cDNA sequence was obtained by 5′-RACE and by analyzing an EST clone generated by the EST Project of the Mt. Desert Island Biological Laboratory Marine DNA Sequencing Center. The longest open reading frame encodes a 593-amino acid protein that has 68 and 64% homology to mammalian COX-1 and COX-2, respectively. The gene and its protein product is designated as shark COX (sCOX). The key residues in the active site (Try385, His388, and Ser530) are conserved between the shark and mammalian COX. sCOX contains Val523 that has been shown to be a key residue determining the sensitivity to COX-2-specific inhibitors including NS-398. The mRNA of sCOX, detected by RT-PCR, was found in all tissues tested, including rectal gland, kidney, spleen, gill, liver, brain, and heart, but not in fin. In the perfused SRG, vasoactive intestinal peptide (VIP) at 5 nM induced rapid and marked Cl− secretion (basal: <250 μeq · h−1 · g−1; peak response: 3,108 ± 479 μeq · h−1 · g−1). In the presence of 50 μM NS-398, both the peak response (2,131 ± 307 μeq · h−1 · g−1) and the sustained response to VIP were significantly reduced. When NS-398 was removed, there was a prompt recovery of chloride secretion to control values. In conclusion, we have cloned the first COX in an elasmobranch species (sCOX) and shown that sCOX inhibition suppresses VIP-stimulated chloride secretion in the perfused SRG.

scholarly journals Gastric inhibitory peptide, serotonin, and glucagon are unexpected chloride secretagogues in the rectal gland of the skate (Leucoraja erinacea)

2014 ◽  
Vol 306 (9) ◽  
pp. R674-R680 ◽  
Author(s):  
Catherine A. Kelley ◽  
Sarah E. Decker ◽  
Patricio Silva ◽  
John N. Forrest
Keyword(s):  
Gastric Inhibitory Polypeptide ◽  
Chloride Secretion ◽  
Mean Value ◽  
Rectal Gland ◽  
Inhibitory Peptide ◽  
Camp Content ◽  
Shark Rectal Gland ◽  
Dogfish Shark ◽  
Insulinotropic Peptide ◽  
G Protein Coupled

Since the discovery of the rectal gland of the dogfish shark 50 years ago, experiments with this tissue have greatly aided our understanding of secondary active chloride secretion and the secretagogues responsible for this function. In contrast, very little is known about the rectal gland of skates. In the present experiments, we performed the first studies in the perfused rectal gland of the little skate ( Leucoraja erinacea), an organ weighing less than one-tenth of the shark rectal gland. Our results indicate that the skate gland can be studied by modified perfusion techniques and in primary culture monolayers, and that secretion is blocked by the inhibitors of membrane proteins required for secondary active chloride secretion. Our major finding is that three G protein-coupled receptor agonists, the incretin gastric inhibitory polypeptide (GIP), also known as glucose-dependent insulinotropic peptide, as well as glucagon and serotonin, are unexpected potent chloride secretagogues in the skate but not the shark. Glucagon stimulated chloride secretion to a mean value of 1,661 ± 587 μeq·h−1·g−1 and serotonin stimulated to 2,893 ± 699 μeq·h−1·g−1. GIP stimulated chloride secretion to 3,733 ± 679 μeq·h−1·g−1 and significantly increased tissue cAMP content compared with basal conditions. This is the first report of GIP functioning as a chloride secretagogue in any species or tissue.

Mechanism of active chloride secretion by shark rectal gland: role of Na-K-ATPase in chloride transport

1977 ◽  
Vol 233 (4) ◽  
pp. F298-F306 ◽  
Author(s):  
P. Silva ◽  
J. Stoff ◽  
M. Field ◽  
L. Fine ◽  
J. N. Forrest ◽  
...  
Keyword(s):  
Chloride Transport ◽  
Sodium Concentration ◽  
Chloride Secretion ◽  
Squalus Acanthias ◽  
Rectal Gland ◽  
Tentative Hypothesis ◽  
Shark Rectal Gland ◽  
Electrochemical Equilibrium

The isolated rectal gland of Squalus acanthias was stimulated to secrete chloride against an electrical and a chemical gradient when perfused in vitro by theophylline and/or dibutyryl cyclic AMP. Chloride secretion was depressed by ouabain which inhibits Na-K-ATPase. Thiocyanate and furosemide also inhibited chloride secretion but ethoxzolamide, a carbonic anhydrase inhibitor, did not. Chloride transport was highly dependent on sodium concentration in the perfusate. The intracellular concentration of chloride averaged 70-80 meq/liter in intact glands, exceeding the level expected at electrochemical equilibrium and suggesting active transport of chloride into the cell. These features suggest a tentative hypothesis for chloride secretion by the rectal gland in which the uphill transport of chloride into the cytoplasm is coupled through a membrane carrier to the downhill movement of sodium along its electrochemical gradient. The latter is maintained by the Na-K-ATPase pump while chloride is extruded into the duct by electrical forces.

scholarly journals AMP-activated protein kinase and adenosine are both metabolic modulators that regulate chloride secretion in the shark rectal gland (Squalus acanthias)

2018 ◽  
Vol 314 (4) ◽  
pp. C473-C482
Author(s):  
Rugina I. Neuman ◽  
Juliette A. M. van Kalmthout ◽  
Daniel J. Pfau ◽  
Dhariyat M. Menendez ◽  
Lawrence H. Young ◽  
...  
Keyword(s):  
Feedback Inhibition ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Chloride Secretion ◽  
Squalus Acanthias ◽  
Catalytic Subunit ◽  
Rectal Gland ◽  
Apical Side ◽  
Shark Rectal Gland ◽  
Amp Activated Protein Kinase

The production of endogenous adenosine during secretagogue stimulation of CFTR leads to feedback inhibition limiting further chloride secretion in the rectal gland of the dogfish shark (Squalus acanthias). In the present study, we examined the role of AMP-kinase (AMPK) as an energy sensor also modulating chloride secretion through CFTR. We found that glands perfused with forskolin and isobutylmethylxanthine (F + I), potent stimulators of chloride secretion in this ancient model, caused significant phosphorylation of the catalytic subunit Thr172 of AMPK. These findings indicate that AMPK is activated during energy-requiring stimulated chloride secretion. In molecular studies, we confirmed that the activating Thr172 site is indeed present in the α-catalytic subunit of AMPK in this ancient gland, which reveals striking homology to AMPKα subunits sequenced in other vertebrates. When perfused rectal glands stimulated with F + I were subjected to severe hypoxic stress or perfused with pharmacologic inhibitors of metabolism (FCCP or oligomycin), phosphorylation of AMPK Thr172 was further increased and chloride secretion was dramatically diminished. The pharmacologic activation of AMPK with AICAR-inhibited chloride secretion, as measured by short-circuit current, when applied to the apical side of shark rectal gland monolayers in primary culture. These results indicate that that activated AMPK, similar to adenosine, transmits an inhibitory signal from metabolism, that limits chloride secretion in the shark rectal gland.

Mode of activation of salt secretion by C-type natriuretic peptide in the shark rectal gland

1999 ◽  
Vol 277 (6) ◽  
pp. R1725-R1732 ◽  
Author(s):  
Patricio Silva ◽  
Richard J. Solomon ◽  
Franklin H. Epstein
Keyword(s):  
Short Circuit ◽  
Short Circuit Current ◽  
Chloride Secretion ◽  
Squalus Acanthias ◽  
Rectal Gland ◽  
Stimulatory Action ◽  
Kinase C ◽  
Shark Rectal Gland ◽  
Pkc Activation ◽  
Stimulation Of

We studied the modes of activation of the salt-secreting rectal gland of the spiny dogfish, Squalus acanthias, by the native cardiac peptide CNP. The stimulatory action of CNP in isolated perfused glands is inhibited by 10 mM procaine, presumably by blocking release of vasoactive intestinal peptide (VIP) from nerves. Procaine reduces the slope of the dose-response curve of human CNP and that of shark CNP (each P < 0.0001). CNP increases short-circuit current in cultured rectal gland cells from 4.8 ± 1.6 to 27.0 ± 7.8 μA/cm2. It also stimulates the secretion of chloride in isolated perfused glands in the presence of 10 mM procaine from 72 ± 31 to 652 ± 173 μeq ⋅ h−1 ⋅ g−1. These results suggest that CNP has a direct cellular action not mediated by the neural release of VIP. The residual stimulation of perfused glands in the presence of procaine was almost completely inhibited by staurosporine [10 nM; an inhibitor of protein kinase C (PKC)] from 652 ± 173 to 237 ± 61 μeq ⋅ h−1 ⋅ g−1. Although CNP stimulates guanylyl cyclase in shark rectal gland, chloride secretion of perfused glands was not elicited by 8-bromoadenosine-cGMP (8-BrcGMP) alone nor by the activator of PKC phorbol ester. The combination of PKC activation and 8-BrcGMP infusion, however, stimulated chloride secretion in perfused glands from 94 ± 30 to 506 ± 61 μeq ⋅ h−1 ⋅ g−1, a level comparable to that observed in glands blocked with procaine. Several parallel pathways appear to be synergistic in activating chloride secretion stimulated by CNP in the rectal gland.

Neuropeptide Y inhibits chloride secretion in the shark rectal gland

1993 ◽  
Vol 265 (2) ◽  
pp. R439-R446 ◽  
Author(s):  
P. Silva ◽  
F. H. Epstein ◽  
K. J. Karnaky ◽  
S. Reichlin ◽  
J. N. Forrest
Keyword(s):  
Neuropeptide Y ◽  
Direct Action ◽  
Short Circuit ◽  
Primary Cultures ◽  
Short Circuit Current ◽  
Chloride Secretion ◽  
Rectal Gland ◽  
Maximal Effect ◽  
Gland Cells ◽  
Shark Rectal Gland

We studied the effects of the 36-amino acid peptide, neuropeptide Y (NPY), on salt secretion by the rectal gland of Squalus acanthias. We used three preparations: whole isolated perfused glands, freshly prepared separated rectal gland tubules, and confluent monolayers of cultured rectal gland cells. In perfused glands NPY inhibited secretion stimulated by vasoactive intestinal peptide (VIP), forskolin, or adenosine 3',5'-cyclic monophosphate (cAMP) and theophylline. Maximal inhibition of 63 +/- 3.4% was seen at 3 x 10(-8) M NPY, with half-maximal effect at 3 x 10(-9) M. NPY did not inhibit the basal activity of rectal gland adenylate cyclase or that stimulated by VIP. The inhibitory action of NPY was not prevented by procaine, nifedipine, or diltiazem, suggesting that it was not secondary to the release of somatostatin or other unknown neurotransmitters from rectal gland nerves. In confirmation, somatostatin was not detected in the venous effluent after administration of NPY. NPY also inhibited transport-related oxygen consumption in separated rectal gland tubules and inhibited short-circuit current generated by confluent monolayers of primary cultures of rectal gland cells. The results indicate that NPY inhibits chloride secretion by a direct action on cells of the shark rectal gland at a site distal to the generation of cAMP.

Ultrastructural and cytochemical characterization of cultured dogfish shark rectal gland cells

1996 ◽  
Vol 271 (6) ◽  
pp. C1993-C2003 ◽  
Author(s):  
J. D. Valentich ◽  
K. J. Karnaky ◽  
T. W. Ecay
Keyword(s):  
Smooth Muscle ◽  
Epithelial Cells ◽  
Indirect Effects ◽  
Chloride Secretion ◽  
Cellular Heterogeneity ◽  
Rectal Gland ◽  
Tubular Epithelial Cells ◽  
Shark Rectal Gland ◽  
Dogfish Shark

The dogfish shark rectal gland (SRG) is histologically complex, containing connective, nerve, and smooth muscle tissue and at least three types of epithelial cells: secretory tubule, central duct, and endothelial. This cellular heterogeneity precludes studies of the intact SRG from distinguishing direct tubular effects of mediators that modulate chloride secretion from their indirect effects on nonepithelial cells such as neurons. Primary SRG cultures express high levels of secretagogue-stimulated chloride secretion, suggesting that SRG cells retain a significant level of cytodifferentiation in vitro. However, because nontubular cells could contaminate these cultures, the question of whether secretagogues activate chloride secretion through direct or indirect effects on tubular epithelial cells remains unresolved. To address this issue, detailed ultrastructural and cytochemical analyses of SRG cultures were carried out to assess the level of cellular heterogeneity and the degree of cytodifferentiation expressed by SRG cells in vitro. The results demonstrate that, after 15 days, primary SRG monolayer cultures are composed exclusively of tubular epithelial cells with no detectable contamination by central duct cells, fibroblasts, smooth muscle cells, endothelial cells, or neurons. Tubular epithelial cells express most of the structural features of native SRG cells, including numerous mitochondria, massive basolateral surface amplification, complex tight junctions, and an extensive tubulovesicular system in the apical cytoplasm. Cultured SRG cells also display a striking level of polarization of cytoplasmic organelles and plasma membrane secretagogue receptors. These results account for the exceptionally high rates of electrogenic chloride secretion by SRG tubular epithelial cells in vitro and confirm that the effects of secretagogues on transport activity reflect their direct interaction with tubular epithelial cells.

Cloning and functional characterization of a monoterpene synthase gene from Eleutherococcus trifoliatus

Holzforschung ◽  
2015 ◽  
Vol 69 (2) ◽  
pp. 163-171 ◽  
Author(s):  
Kuan-Feng Huang ◽  
Yi-Ru Lee ◽  
Yen-Hsueh Tseng ◽  
Sheng-Yang Wang ◽  
Fang-Hua Chu
Keyword(s):  
Functional Characterization ◽  
Open Reading Frame ◽  
Terpene Synthase ◽  
Gas Chromatography Mass Spectrometry ◽  
Degenerate Primers ◽  
Reading Frame ◽  
Monoterpene Synthase ◽  
Young Leaves ◽  
Rapid Amplification

AbstractEleutherococcus trifoliatusalso known as the three-leavedEleutherococcus, a member of the Araliaceae (ginseng) family, is commonly used in traditional Chinese medicine. Recently, many studies have demonstrated the bioactivities of the secondary metabolites inE. trifoliatus. In this study, a monoterpene synthase fromE. trifoliatushas been characterized. A pair of degenerate primers was designed and a fragment with conserved region of terpene synthase (TPS) was obtained. After 5′- and 3′-rapid amplification of cDNA ends (RACE), the full-length cDNA was obtained. The gene designatedEtLIMcontains an open reading frame of 1752 bp with a predicated molecular mass of 67.3 kDa. It was expressed in young leaves, stems, and drupes. The product ofEtLIMhas been identified by gas chromatography/mass spectrometry (GC/MS) as limonene.

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