Atriopeptin in cultured

stimulates chloride secretion shark rectal gland cells

KARL J. KARNAKY, JR., JOHN D. VALENTICH, MARK G. CURRIE, WILLIAM F. OEHLENSCHLAGER, AND MICHAEL P. KENNEDY. Departments of Anatomy and Cell Biology and of Pharmacology, Medical University of South Carolina, Charleston 29425; Grice Marine Biological Laboratory, Charleston, South Carolina 29412; Department of Physiology and Cell Biology, University of Texas Medical School, Houston, Texas 77025; and Mount Desert Island Biological Laboratory, Salsbury Cove, Maine 04672

KARNAKY, KARL J., JR., JOHN D. VALENTICH, MARK G. CURRIE, WILLIAM F. OEHLENSCHLAGER, AND MICHAEL P. KENNEDY. Atriopeptin stimulates chloride secretion in cultured shark rectal gland cells. Am. J. Physiol. 260 (Cell Physiol. 29): C1125-Cll30,1991.-Monolayer cultures of shark rectal gland (SRG) epithelial cells were treated with atriopeptin (AP), and the effects on Cl- secretion and intracellular guanosine 3’,5’cyclic monophosphate (cGMP) accumulation were examined. Basolateral or apical exposure to 10m7 M AP markedly stimulated @-fold) Cl-dependent, bumetanide-sensitive, short-circuit current (&). The AP-stimulated I,, exhibited transient oscillations before reaching a steady state. This behavior is not observed when I,, is activated by other secretagogues such as vasoactive intestinal peptide, %chloroadenosine, forskolin, or ionomycin. Intracellular cGMP was concomitantly elevated (lo-fold) by 10m7 M AP. Both I,, stimulation and cGMP accumulation responses exhibited a similar dose dependency beginning at an AP concentration of 1 nM. The bilateral response to AP suggests the presence of receptors on both apical and basolateral plasma membranes. These results are the first demonstration of a direct effect of AP on Cl-secreting epithelial cells. These data also suggest a role for cGMP in mediating Cl- secretion in these cells.

atria1 natriuretic peptide; second messengers epithelial transport; spiny dogfish shark

and cell signaling;

3’,5’-CYCLIC MONOPHOSPHATE (cGMP) regulates ion transport in both Cl-secreting and Na+-absorbing epithelia, yet the underlying signaling mechanisms involving this second messenger are poorly understood (14, 32). Increases in intracellular cGMP are frequently observed when cells are exposed to atriopeptin (AP) (14, ZO), a potent diuretic, natriuretic, and vasodilatory hormone having an important role in salt and fluid metabolism in species ranging from fish (8) to humans (9). Elasmobranchs utilize a special salt-secreting gland, the rectal gland, to regulate plasma ion concentrations and fluid volume (28). AP-II [rat atria1 natriuretic peptide (rANP) 5-271 stimulates chloride secretion in the intact perfused rectal gland, presumably by stimulating GUANOSINE

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the release of vasoactive intestinal peptide (VIP) from peritubular nerve terminals (26). We investigated possible direct effects of AP on cultured Cl-secreting cells from the spiny dogfish shark (Squalus acanthias) rectal gland (SRG). This preparation affords a simple model for investigating cGMP metabolism and its relation to ANP-mediated NaCl transport. Exposing SRG cultures to AP stimulates Cl-dependent, bumetanide-sensitive, short-circuit current (&), a direct measure of Cl- secretion by this preparation (33). In addition, intracellular cGMP is markedly elevated following exposure to AP. Increases in I,, are elicited by either apical or basolateral AP. These results are the first demonstration of a direct effect of AP on Cl-secreting epithelial cells. METHODS

Dogfish of either sex were either taken by gill nets from Frenchman’s Bay, ME, and kept in live cars until used, usually within 2 days of capture, or caught by hook and line off of Charleston Harbor, SC, and used the same day. Dogfish were pithed, and the rectal glands were removed under sterile conditions via an abdominal incision and transported on ice to the laboratory in sterile shark Ringer solution containing (in mM) 288 Na+, 289 Cl-, 6 K+, 20 HCO:, 5 Ca’+, 5 Mg2+, 350 urea, and 5 glucose. Tissue culture. SRG monolayer cultures were prepared as previously described (33). Briefly, minced rectal glands were digested using 2 mg/ml collagenase D (Boehringer Mannheim) for 90 min to give a suspension of isolated, intact tubules. Tubules were washed with shark Ringer solution and resuspended in culture medium consisting of Dulbecco’s modified Eagle’s medium/Ham’s F-12 medium (1:l) with 15 mM N-2-hydroxyethylpiperazine-N’2-ethanesulfonic acid (HEPES; Sigma) and (in mM) 100 NaCl, 21 NaHC03, 3.9 CaC12, 2.5 MgCl,, 300 urea, 150 trimethylamine oxide, and 2 L-glutamine. This nutrient medium was supplemented with 5% Nu-Serum and ITS (both from Collaborative Research) and penicillin (100

1991 the American

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U/ml)/streptomycin (100 pg/ml) (Sigma Chemical). Cultures were maintained at 20°C in 97% air-3% CO,. Electrophysiology. Tubules were inoculated into 35mm culture dishes containing type 1 collagen gels supported with Nitex mesh or Millipore CM Millicells coated with a dried film of type 1 collagen. Cultures were mounted in Ussing chambers, and I,, was measured using methods described previously (33). All shark Ringer solutions contained 1 mg/ml bovine serum albumin (BSA) to limit nonspecific AP binding to plastic surfaces. cGMP determination. SRG cells in 35mm culture dishes were equilibrated with low-bicarbonate shark Ringer solution containing (in mM) 288 Na+, 289 Cl-, 6 K’, 20 HEPES, 2 HCO:, 5 Ca’+, 5 Mg’+, 350 urea, 5 glucose, 1 mg/ml BSA, and lo-” M 3-isobutyl-l-methylxanthine (IBMX) for 20 min. Cells were then incubated for 10 min with varying concentrations of AP-III [rANP(5-28)]. Reactions were terminated and cGMP extracted with ice-cold 0.1 N HCl. Samples were centrifuged and the supernatants were lyophilized, resuspended in sodium acetate buffer (50 mM, pH 6.4), and assayed for cGMP by radioimmunoassay (29) using antiserum raised in our laboratory (cross-reactivity was

Atriopeptin stimulates chloride secretion in cultured shark rectal gland cells.

Monolayer cultures of shark rectal gland (SRG) epithelial cells were treated with atriopeptin (AP), and the effects on Cl- secretion and intracellular...
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