THE SHARK RECTAL GLAND MODEL: A CHAMPION OF RECEPTOR MEDIATED CHLORIDE SECRETION THROUGH CFTR

Abstract

The dogfish shark salt gland was predicted by Smith and discovered by Burger at the Mount Desert Island Biological Laboratory in Salisbury Cove, Maine. It is an epithelial organ in the intestine composed of tubules that serve a single function: the secretion of hypertonic NaCl. Many G protein receptors are present on the basolateral surface of these tubules, including stimulatory receptors for vasoactive intestinal peptide, adenosine A₂, growth hormone releasing hormone, and inhibitory receptors for somatostatin and adenosine A₁. An entirely different class of stimulatory receptors is present as C-type natriuretic peptide receptors. Each stimulatory receptor evokes powerful NaCl secretion. G protein receptors bind to G_αs to activate the catalytic unit of adenylate cyclase to form cyclic adenosine monophosphate (cAMP) and protein kinase A that phosphorylates the regulatory domain of cystic fibrosis transmembrane conductance regulator, opening the channel. The C-type natriuretic peptide receptor stimulates by activating guanylate cyclase and endogenous cyclic guanosine monophosphate which inhibits type 3 phosphodiesterase, the enzyme that breaks down cAMP, thereby elevating cAMP and activating the protein kinase A pathway.

Fig. 1

(A) My father, John N. Forrest (1889−1982) in his World War I uniform. (B) By his dental chair.

Fig. 2.

(A) Paul B. Beeson, MD, Chairman of Internal Medicine at Yale, 1952−1965. (B) Paul Beeson (far right side) with his last group of medical interns at Yale.

Fig. 3.

(A) The dogfish shark, Squalus acanthias, a 400 million year old elasmobranch. (B) The digiform rectal gland of the dogfish shark, with a single artery (upper left), single vein (lower right) and single duct (not visible).

Fig. 4

Salt from the salt gland of the Galapagos marine iguana forms a crown of NaCl. The salt gland of reptiles and marine birds is located in the head and is composed of many arteries and veins making the organ nearly impossible to perfuse.

Fig. 5

In vitro perfusion of an isolated tubule of the rectal gland of Squalus acanthias (original magnification ×400) (Forrest, 1983 #628).

Fig. 6

Cover of The Journal of Clinical Investigation February 1998 issue first demonstrating hormonal trafficking of the CFTR chloride channel (red) to the apical membrane in a native epithelia.

Fig. 7

Phylogram of mammalian adenosine receptors (A₁, A_2a, A A_2b, and A₃) demonstrating that the shark A₀ receptor, cloned from the rectal gland (extreme right), roots the tree of all types of mammalian adenosine receptors and is likely the receptor from which all adenosine receptors evolved.

Fig. 8

Four possible pathways (circles) by which CNP could activate CFTR. Experiments in the SRG establish pathway 1 as dominant (23). CNP binds to and activates a CNP-selective receptor GC at the basolateral membrane (designated NPR-B), resulting in marked cGMP elevation. Endogenous cGMP, but not membrane-permeable and non-hydrolyzable cGMP analogs, compete with cAMP for degradation by a type 3 PDE (PDE3A). PDE 3A is the only PDE inhibited by cGMP resulting in a local rise of cAMP levels. cAMP in the apical compartment, through activation of cAMP-dependent PKA, causes phosphorylation of the R domain of CFTR and opening of the CFTR chloride channel (pathway 1, solid lines). Activation of CFTR by CNP is dependent on basal cAMP production by AC, which is inhibited by somatostatin through a specific receptor coupled to the inhibitory G protein Gαi. The three other potential cGMP signaling pathways (dotted lines) were excluded by the experiments, identifying pathway 1 as the predominant signaling pathway operating in the SRG epithelia and likely other epithelia. Abbreviations: CNP, C-type natriuretic peptide; CFTR, cystic fibrosis transmembrane conductance regulator; SRG, shark rectal gland; NPR-B, natriuretic peptide receptor B; cGMP, cyclic guanosine monophosphate; PDE, phosphodiesterase; PKA, protein kinase A; R, regulatory; AC, adenylyl cyclase; AMP, adenosine monophosphate; ATP, adenosine triphosphate; SSTR, somatostatin receptor; GC, guanylyl cyclase; ADP, adenosine diphosphate; cGK, cyclic G kinase; G_i, G- inhibitory.

Fig. 9

Comparison of the SRG (right side) to the TAL (left side), a secretory epithelia (SRG), and an absorptive epithelia (TAL). The Na/K ATPase pump is on the basolateral membrane in both tissues. The Na/K/2Cl cotransporter is on the basolateral membrane in the secretory SRG as is the dominant K exit channel. The Na/K/ 2Cl cotransporter and K channel are on the apical membrane in the absorptive TAL. Both have Na, K, and Cl entry into the cell; Na exits both cells via Na/K ATPase and K exits the cell through the dominant K exit channel. Both cells accumulate uphill Cl ions that leave the cell differently — through the CFTR channel in the secretory SRG and through a CLC-Kb/Barttin channel in the absorptive TAL. Abbreviations: SRG, shark rectal gland; TAL, thick ascending limb; ATPase, adenosine triphosphatase; CLC, chloride channel; BL, basolateral membrane; CLC-Kb, voltage gated basolateral chloride channel.

Fig. 10

(Left to right) The author, his wife Catherine Forrest, Suzane Forrest and her husband Dan Wong, Gwendolyn Forrest, Emily Forrest and her husband John K. Forrest. Two grandsons are in front, William Forrest and Andrew Forrest.