Modulation of salt permeabilities of intestinal brush-border membrane vesicles by micromolar levels of internal calcium.

A possible modulation of ion permeabilities of rat intestinal brush-border membrane vesicles by Ca2+, a putative second messenger of salt secretion, was explored by three independent methods: (1) measurements of [3H]glucose accumulation driven by a Na+ gradient; (2) stopped-flow spectrophotometry of salt-induced osmotic swelling; (3) 86Rb+, 22Na+ and 36Cl- flux measurements. Cytoskeleton-deprived membrane vesicles were prepared from isolated brushborders by thiocyanate treatment. Intravescicular Ca2+ levels were varied by preincubating vesicles in Ca-EGTA buffers in the presence of the Ca2+-ionophore A23187. At Ca2+free greater than 10(-5) M, initial Na+-dependent glucose uptake in the presence of a 0.1 M NaSCN gradient (but not in its absence) was inhibited by about 50 per cent as compared to EGTA alone (ED50 approximately equal to 10(-6) M Ca2+). By contrast, initial rates of 22Na+ uptake and reswelling rates of vesicles exposed to a NaSCN gradient were increased at least 2-fold by 10(-5) M Ca2+free. Both observations are compatible with a Ca2+-induced increase of the Na+-permeability of the vesicle membrane. The modulation of ion transport was fully reversible and critically dependent on internal Ca2+, suggesting a localization of Ca2+-sensor sites at the inner surface of the microvillous membrane. As shown by radiotracer and osmotic swelling measurements, micromolar Ca2+ additionally increased the flux rate of K+, Rb+, Cl- and NO-3 but did not change the membrane permeability for small uncharged molecules, including glucose and mannitol. The effect of Ca2+ on ion permeabilities could be blocked by Ba2+ (10(-3) M) or Mg2+ (10(-2) M), but not by amiloride (10(-3) M), apamin (2 X 10(-7) M), trifluoperazine (10(-4) M) or quinine (5 X 10(-4) M). At present it is unclear whether Ca2+ activates a nonselective cation and anion channel or multiple highly selective channels in the vesicle membrane.

Phosphoinositide metabolism in intestinal brush borders: stimulation of IP3 formation by guanine nucleotides and Ca2+.

The potential role of polyphosphoinositide (PPI) metabolism as a signal-transduction mechanism in apical membranes of polarized epithelial cells was evaluated by examining the formation and breakdown of PPI in rat intestinal brush-border membranes (BBM) prelabeled by intraluminal injection of [3H]inositol in vivo or by [gamma-32P]ATP in vitro. Freshly isolated BBM prelabeled with [3H]inositol contained higher amounts of [3H]phosphatidylinositol 4,5-diphosphate compared with a basolateral membrane (BLM) preparation (approximately 14 and 6.8% of total [3H]PPIs, respectively) and were enriched in inositol lipid kinases, diacylglycerol (DAG) kinase, and phosphomonoesterases degrading PPI, inositol bisphosphate, and inositol triphosphate (IP3). In the presence of nonhydrolysable GTP analogues and low Ca2+ (pCa 6-8) or at high Ca2+ alone (pCa 4) endogenous pools of PPI were rapidly depleted by an intrinsic PPI-specific phospholipase C apparently coupled to a GTP-binding protein (G protein). Surprisingly, despite the assignment of most G protein-coupled hormone receptors to the BLM, the capacity of isolated BBM to release [3H]IP3 in response to Ca2+ or GTP gamma S appeared comparable to that in a BLM preparation. Intestinal secretagogues acting through apical membrane receptors (adenosine, heat-stable Escherichia coli toxin), however, were unable to promote [3H]IP3 release in isolated BBM in the presence of GTP. PPI metabolism in BBM may be coupled to receptors for as yet unidentified secretagogues or may serve as an amplification mechanism for hormone-stimulated PPI breakdown in BLM. The local release of DAG and IP3 at the interior of the intestinal microvilli likely plays a role in the regulation of ion transport systems in microvillar membranes.