Lateral intercellular space volume as a determinant of CFTR-mediated anion secretion across small intestinal mucosa

Studies of full-thickness, small intestinal preparations have shown that maximal anion secretion [indexed by short-circuit current ( Isc)] during intracellular cAMP (cAMPi) stimulation is transient and followed by a decline toward baseline. Declining Isc is preceded by decreases in transepithelial conductance (Gt), which in the small intestine reflects the lateral intercellular space (LIS) volume of the paracellular pathway. We hypothesized that decreases in LIS volume limit the magnitude and duration of cAMPi-stimulated anion secretion. Experimental manipulations to increase the patency of the LIS (assessed by Gt and electron microscopy) were investigated for an effect on the magnitude of cAMPi-stimulated anion secretion (assessed by the Isc and isotopic fluxes) across murine small intestine. In control studies, changes of Gt after cAMPi stimulation were associated with a morphological “collapse” of the LIS, which did not occur in intestine of CFTR-null mice. Removal of the outer intestinal musculature, exposure to a serosal hypertonic solution, or increased serosal hydrostatic pressure minimized reductions in Gt and increased the cAMPi-stimulated Isc response. Increased Isc primarily resulted from increased Cl− secretion that was largely bumetanide sensitive. However, bumetanide-insensitive Isc was also increased, and similar increases occurred in the Na+-K+-2Cl− cotransporter (NKCC1)-null intestine, indicating that activities of non-NKCC1 anion uptake proteins are also affected by LIS volume. Thus LIS patency is an important determinant of the magnitude and duration of CFTR-mediated anion secretion in murine small intestine. Decreases in LIS volume may limit the pool of available anions to basolateral transporters involved in transepithelial secretion.

NO increases permeability of cultured human cervical epithelia by cGMP-mediated increase in G-actin

Human cervical epithelial cells express mRNA for the nitric oxide (NO) synthase (NOS) isoforms ecNOS, bNOS, and iNOS and release NO into the extracellular medium. NG-nitro-l-arginine methyl ester (l-NAME), an NOS inhibitor, and Hb, an NO scavenger, decreased paracellular permeability; in contrast, the NO donors sodium nitroprusside (SNP) and N-(ethoxycarbonyl)-3-(4-morpholinyl)sydnonimine increased paracellular permeability across cultured human cervical epithelia on filters, suggesting that NO increases cervical paracellular permeability. The objective of the study was to understand the mechanisms of NO action on cervical paracellular permeability. 8-Bromo-cGMP (8-BrcGMP) also increased permeability, and the effect was blocked by KT-5823 (a blocker of cGMP-dependent protein kinase), but not by LY-83583 (a blocker of guanylate cyclase). In contrast, LY-83583 and KT-5823 blocked the SNP-induced increase in permeability. Treatment with SNP increased cellular cGMP, and the effect was blocked by Hb and LY-83583, but not by KT-5823. Neither SNP nor 8-BrcGMP had modulated cervical cation selectivity. In contrast, both agents increased fluorescence from fura 2-loaded cells in the Ca2+-insensitive wavelengths, indicating that SNP and 8-BrcGMP stimulate a decrease in cell size and in the resistance of the lateral intercellular space. Neither SNP nor 8-BrcGMP had an effect on total cellular actin, but both agents increased the fraction of G-actin. Hb blocked the SNP-induced increase in G-actin, and KT-5823 blocked the 8-BrcGMP-induced increase in G-actin. On the basis of these results, it is suggested that NO acts on guanylate cyclase and stimulates an increase in cGMP; cGMP, acting via cGMP-dependent protein kinase, shifts actin steady-state toward G-actin; this fragments the cytoskeleton and renders cells more sensitive to decreases in cell size and resistance of the lateral intercellular space and, hence, to increases in permeability. These results may be important for understanding NO regulation of transcervical paracellular permeability and secretion of cervical mucus in the woman.

Water permeability and pathways in the proximal tubule

The route of water transport in the proximal tubule could be either transjunctional or transcellular. A transjunctional route is supported by data showing high osmotic-to-diffusive water permeability ratios, the possible correlation of junctional leakiness to ions and nonelectrolytes with water permeability, and solvent drag of nonelectrolytes and ions. These data, however, are not convincing. A transcellular route of water transport is supported by data showing that the osmotic water permeability (Pf) for apical and/or basolateral cell membranes is sufficiently high to account for the transepithelial Pf, making a tentative conclusion for a transcellular route of water transport possible. In addition, measurements of Pf have yielded insights into the mechanism of solute-solvent coupling. Pf has been reported to be mostly between 0.1 and 0.3 cm/s. In the rabbit proximal straight and the Necturus proximal convoluted tubule, in which water transport rates are low, this range of Pf will account for volume absorption with only small osmotic gradients (less than 6 mosmol). Higher osmotic gradients are required in the rat and possibly the rabbit proximal convoluted tubule, where water transport rates are higher. Solute-solvent coupling in all species is probably due to both luminal hypotonicity and lateral intercellular space hypertonicity. These two processes are directly linked. Mass balance requires that generation of luminal hypotonicity also generates a hypertonic absorbate and, thus, some degree of lateral intercellular space hypertonicity. It is likely that, in the rabbit at least, effective osmotic pressure gradients due to differences in solute reflection coefficients play little role in solute-solvent coupling.