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The epithelial tight junction (TJ) was first described ultrastructurally as a fusion of the outer lipid leaflets of the adjoining cell membrane bilayers (hemifusion). The discovery of an increasing number of integral TJ and TJ-associated proteins has eclipsed the original lipid-based model with the wide acceptance of a protein-centric model for the TJ. In this review, we stress the importance of lipids in TJ structure and function. A lipid-protein hybrid model accommodates a large body of information supporting the lipidic characteristics of the TJ, harmonizes with the accumulating evidence supporting the TJ as an assembly of lipid rafts, and focuses on an important, but relatively unexplored, field of lipid-protein interactions in the morphology, physiology, and pathophysiology of the TJ.

The tight junction has been characterized as a domain of focal fusions of the exoplasmic leaflets of the lipid bilayers from adjacent epithelial cells. Approximating membranes to within fusion distance is a thermodynamically unfavorable process and requires the participation of membrane-bridging or -fusion proteins. No known tight junction protein exhibits such activities. Annexin A2 (A2), in particular its heterotetramer (A2t), is known to form junctions between lipid bilayer structures through molecular bridging of their external leaflets. We demonstrate abundant A2 expression in Madin-Darby canine kidney II monolayers by two-dimensional gel electrophoresis. Confocal immunofluorescence microscopic analysis suggests the bulk of A2 is located along the apical and lateral plasma membrane in its tetrameric configuration, consisting of two A2 and two p11 (an 11-kDa calmodulin-related protein, S100A10) subunits. Immunocytochemistry and ultrastructural immunogold labeling demonstrate colocalization of the A2 subunit with bona fide tight junction proteins, zonula occludens-1, occludin, and claudin-1, at cell-cell contacts. The extracellular addition of a synthetic peptide, targeted to disrupt the binding between A2 and p11, completely aborts tight junction assembly in calcium chelation studies. We propose A2t as a member of a new class of tight junction proteins responsible for the long-observed convergence of adjacent exoplasmic lipid leaflets in tight junction assembly.

Ileum displays little active transcellular calcium (Ca2+) absorption but is credited with the bulk of Ca2+ absorbed in vivo. We examined the effect of taurodeoxycholic acid (TDC, 2 mM), a bile salt, on mannitol (MN, a marker of intercellular solute traffic) and Ca2+ fluxes in rat ileum. In the absence of electrochemical gradients between the mucosal (M) and serosal (S) bathing media in an Ussing chamber, net flux (Jnet) was observed in the S-to-M direction for both MN and Ca2+, i.e., the unidirectional secretory S-to-M flux (Js-->m) exceeded the absorptive M-to-S flux (Jm-->s). Mucosal TDC caused simultaneous increase in transepithelial conductance and Js-->m for both MN and Ca2+. This was followed by even greater increases in MN and Ca2+ Jm-->s, so that ultimately Jm-->s equaled Js-->m in each case. In control tissue, Js-->m for Ca2+ appeared to permeate exclusively through the intercellular MN pathway while part of Jm-->s for Ca2+ appeared to traverse through a non-MN route. After the TDC-induced increase in intercellular solute permeability, both Ca2+ fluxes appeared to traverse through the aqueous MN conduit. During the postprandial state, the presence of bile salts and the relative abundance of Ca2+ in ileal lumen can cause bulk Ca2+ absorption through the intercellular pathway.

Transport characteristics of Ca and Mg were compared at three different levels of the absorptive process in male (120-170 g) Wistar rats. Balance studies in intact rats revealed that fractional Ca absorption decreased with increased Ca intake so that net Ca absorption remained constant. Fractional Mg absorption decreased modestly with increased Mg intake so that net Mg absorption increased proportionately with increased dietary Mg. Everted duodenal sacs demonstrated the presence of active Ca absorption with serosal-to-mucosal (S/M) ratio of 2.65 +/- 0.20 (n = 6), which was greater than unity (P less than 0.001). In contrast, the S/M for Mg did not exceed unity. Mucosal duodenal Ca uptake exhibited a large saturable (Michaelis constant of 4.80 +/- 0.34 mM, maximal velocity of 4.71 +/- 0.13 nmol.min-1.mg-1) and a small nonsaturable component (0.12 +/- 0.01 nmol.min-1.mg-1) in 5-wk-old rats (120 g). In 72-wk-old rats (600 g) the diffusional component of Ca uptake became predominant, and the slope increased significantly to 0.32 +/- 0.01 (P less than 0.05). Duodenal Mg uptake was completely concentration dependent and exhibited no age-related changes.

Some proposed mechanisms for the hypotensive effect of high calcium intake involve reduction in vascular responsivity. To assess the effect of dietary calcium on vascular responsivity, spontaneously hypertensive rats (SHR) were placed on normal (N-Ca; 0.4%) or high (H-Ca; 2.8%) casein-based synthetic diet for 4 wk. Intraarterial pressure, pressor response to graded intravenous infusion of norepinephrine (NE) and angiotensin II (ANG II), and in vitro vascular reactivity of tail artery segments to NE and transmural nerve stimulation (TNS) were studied. Urinary electrolyte excretion, plasma renin activity (PRA), aldosterone, NE, and epinephrine (EPI) were also determined. H-Ca SHR had a lower intraarterial systolic and diastolic pressure. However, H-Ca SHR had greater in vivo pressor response to both ANG II and NE. Maximal contractile force developed by tail artery segments in vitro in response to NE and TNS was slightly, but not significantly, higher in H-Ca SHR. In vitro dose-response curves to NE and TNS were not significantly different. Although H-Ca SHR had increased urinary excretion of sodium throughout the study period, PRA and aldosterone levels were similar in both groups. Plasma NE and EPI levels in the two groups were also not different. Despite lowered intra-arterial blood pressure, H-Ca SHR exhibited enhanced pressor response to ANG II and NE in vivo and a similar in vitro vascular reactivity to NE and TNS when compared with N-Ca SHR. Our results do not support a role for alterations in vascular reactivity to NE or ANG II in the hypotensive effect of high calcium intake in SHR.(ABSTRACT TRUNCATED AT 250 WORDS)

Intestinal calcium (Ca) hyperabsorption is a well-documented feature of experimental phosphorus depletion (PD). To further evaluate the effect of PD on Ca absorption we studied metabolic balance and in vitro everted duodenal sac uptake of Ca and phosphorus (P) in weanling male rats. Animals were assigned to three dietary groups: normal, 0.3% P ad libitum (NP); low, 0.03% P ad libitum (LP); and normal, 0.3% P but pair-fed with assigned LP mates (NP-PF). Results indicate that although PD led to an early but unsustained increase in 45Ca uptake by the everted duodenal sac in vitro, net intestinal Ca retention is consistently decreased in rats on the LP diet compared with rats eating either the NP or NP-PF diet. The reduction in net intestinal Ca absorption is reflected by an increase in fecal Ca, both in absolute quantities and in proportion to dietary Ca intake. The initial negative P balance after the initiation of the LP diet was promptly, albeit precariously, corrected. This was associated with a sustained increase in duodenal 32P uptake in vitro and virtual cessation of growth. Because the biosynthesis of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and its accumulation in intestinal mucosa have been reported to increase with PD, our study represents an example in which the physiological interrelationship between the activity of 1,25(OH)2D3 and intestinal Ca absorption may be dissociated.

Page G480: U. Gafter, J. A. Kraut, D. B. N. Lee, V. Silis, M. W. Walling, K. Kurokawa, M. R. Haussler, and J. W. Coburn. “Effect of metabolic acidosis on intestinal absorption of calcium and phosphorus.” Page G480: M. W. Walling deceased 30 November 1979.