Many epithelia, including the superficial epithelia of the airways, are thought to secrete “volume sensors,” which regulate the volume of the mucosal lining fluid. The epithelial Na + channel (ENaC) is often the rate limiting factor in fluid absorption, and must be cleaved by extracellular and/or intracellular proteases before it can conduct Na + and absorb excess mucosal liquid, a process that can be blocked by proteases inhibitors. In the airways, airway surface liquid dilution or removal activates ENaC. Therefore, we hypothesized that endogenous proteases are membrane-anchored, whereas endogenous proteolysis inhibitors are soluble and can function as airway surface liquid volume sensors to inhibit ENaC activity. Using a proteomic approach, we identified short palate, lung, and nasal epithelial clone (SPLUNC)1 as a candidate volume sensor. Recombinant SPLUNC1 inhibited ENaC activity in both human bronchial epithelial cultures and Xenopus oocytes. Knockdown of SPLUNC1 by shRNA resulted in a failure of bronchial epithelial cultures to regulate ENaC activity and airway surface liquid volume, which was restored by adding recombinant SPLUNC1 to the airway surface liquid. Despite being able to inhibit ENaC, recombinant SPLUNC1 had little effect on extracellular serine protease activity. However, SPLUNC1 specifically bound to ENaC, preventing its cleavage and activation by serine proteases. SPLUNC1 is highly expressed in the airways, as well as in colon and kidney. Thus, we propose that SPLUNC1 is secreted onto mucosal surfaces as a soluble volume sensor whose concentration and dilution can regulate ENaC activity and mucosal volumes, including that of airway surface liquid.

We studied the in vitro effects of omega-3 fish oils and other fatty acids on the activity of crude protein kinase C from S49 lymphoma cells, on partially purified enzyme from rat cerebrum, on homogeneous protein kinase C from bovine brain, and, for comparison, on type I adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase. In the absence of exogenous phospholipid, the fish oils cis-5,8,11,14,17-eicosapentaenoic acid (EPA) and acid (DCHA) enhance the catalytic cis-4,7,10,13,16,19-docosahexaenoic activity of protein kinase C and support the binding of [3H]phorbol 12,13-dibutyrate, both to approximately 50% of the level supported by phosphatidylserine. In the presence of phosphatidylserine, the omega-3 fatty acids reduce catalytic activity and [3H]phorbol 12,13-dibutyrate binding by about one-half. The effects of the omega-3 fatty acids on enzyme activity suggest that fish oils act as partial agonists competitively with phosphatidylserine. EPA, DCHA, and arachidonate (but not a variety of saturated fatty acids) inhibit the cAMP-dependent protein kinase. Thus dietary fish oils and cellular fatty acids mobilized by the action of phospholipase A2 may differentially modulate the activities of protein kinase C and cAMP-dependent protein kinase. These data suggest means by which unsaturated fatty acids mobilized within cells may act as second messengers.

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The deficiency of dystrophin, a critical membrane stabilizing protein, in the mdx mouse causes an elevation in intracellular calcium in myocytes. One mechanism that could elicit increases in intracellular calcium is enhanced influx via the L-type calcium channels. This study investigated the effects of the dihydropyridines BAY K 8644 and nifedipine and alterations in dihydropyridine receptors in dystrophin-deficient mdx hearts. A lower force of contraction and a reduced potency of extracellular calcium (P < 0.05) were evident in mdx left atria. The dihydropyridine agonist BAY K 8644 and antagonist nifedipine had 2.7- and 1.9-fold lower potencies in contracting left atria (P < 0.05). This corresponded with a 2.0-fold reduction in dihydropyridine receptor affinity evident from radioligand binding studies of mdx ventricular homogenates (P < 0.05). Increased ventricular dihydropyridine receptor protein was evident from both radioligand binding studies and Western blot analysis and was accompanied by increased mRNA levels (P < 0.05). Patch-clamp studies in isolated ventricular myocytes showed no change in L-type calcium current density but revealed delayed channel inactivation (P < 0.05). This study indicates that a deficiency of dystrophin leads to changes in dihydropyridine receptors and L-type calcium channel properties that may contribute to enhanced calcium influx. Increased influx is a potential mechanism for the calcium overload observed in dystrophin-deficient cardiac muscle.

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