In addition to amiloride-sensitive Na
+ channels, electrogenic Na
+ transport across the ocular surface, and thus fluid secretion, may also involve Na
+-glucose and Na
+-amino acid cotransport.
Figure 6A (left) shows that isosmolar addition of
d-glucose but not of
d-mannitol produced a small hyperpolarization that was reversed by the Na
+-glucose cotransporter inhibitor phloridzin.
l-Glucose (5 mM) produced no significant change in PD (data not shown,
n = 4 eyes). Under physiological conditions of high apical Na
+ concentration, extracellular
d-glucose saturability (
K m) was 2.5 mM as measured from PDs at increasing concentrations of
d-glucose (
Fig. 6A , middle and right). Hill analysis gave a
d-glucose-Na
+ coupling ratio of 0.89, consistent with 1:1 Na
+-glucose cotransport. The PD data in
Figure 6Awere modeled to determine the turnover rates of transporters 2 and 9 (see
Appendix for explanation of parameter selection). Modeling of the experimentally measured ∼4-mV hyperpolarization under conditions of saturated cotransport and tonic ENaC inhibition
(Fig. 6B)indicated a
\(J_{2}\) (SGLT-1) turnover equal to ∼75% of the
\(J_{1}\) (ENaC) basal activity (0.11 vs. 0.15 μeq/cm
2per hour). Because SGLT-1 likely transports two solutes per turnover, this implies similar osmolar absorptive capacities of amiloride-insensitive and amiloride-sensitive pathways. The basic and neutral amino acids
l-arginine and glycine also produced small, reversible hyperpolarizations in the presence of Na
+ (Fig. 6C) . Amino acid transport was saturated only at relatively high concentration (several mM) for both amino acids studied.
l-Arginine and glycine, added at 10 mM, yielded hyperpolarizations of 1.5 ± 0.9 mV (
n = 4, SE) and 2.1 ± 0.4 mV (
n = 5), respectively. PD analysis also revealed competitive substrate binding, where addition of
l-arginine to glycine-containing solution reproducibly caused a depolarization (
Fig. 6C , right). The Na
+ dependence of these electrogenic pathways of glucose and amino acid absorption was confirmed.
Figure 6D(left) shows hyperpolarizations produced by 5 mM
d-glucose and 1 mM
l-arginine (but not mannitol) in the presence of Na
+ and amiloride. The hyperpolarizations were abolished after Na
+ replacement by choline. Averaged results are summarized in
Figure 6D(right). Together, these results provide evidence for at least three distinct electrogenic Na
+ pathways at the ocular surface: amiloride-sensitive Na
+ channels, Na
+-glucose cotransport, and Na
+-amino acid cotransport.