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Development of human fetal airways requires interaction of the respiratory epithelium and the extracellular matrix through integrins. Nevertheless, the specific roles of β1-integrins during development and tubular morphogenesis are still unknown. To analyze β1-integrin localization and influence during migration, we developed a model of human fetal tracheal explants growing on collagen and overlaid with a second layer of collagen to form a sandwich. In this configuration, cord and tubule formation proceeded normally but were inhibited by incubation with anti-β1-integrin subunit antibodies. On a collagen matrix, β1-integrins were immunolocalized on the entire plasma membrane of migrating epithelial cells and almost exclusively on the basal plasma membrane of nonmigratory epithelial cells. In a sandwich configuration, β1-integrins became detectable in the cytoplasm of epithelial cells. Coating cultures with collagen transiently altered the morphology of migrating cells and their speed and direction of migration, whereas incubation with anti-β1-integrin subunit antibodies irreversibly altered these parameters. These observations suggest that the matrix environment, by modulating β1-integrin expression patterns, plays a key role during tubular morphogenesis of human fetal tracheal epithelium, principally by modulating epithelial cell migration.

Steady-state cytosolic calcium (Ca2+i) concentration in a vascular smooth muscle cell is determined by Ca2+ influx and Ca2+ extrusion across the plasma membrane, yet no means for determining the absolute magnitude of these transmembrane Ca2+ fluxes in the basal state of the resting cell has been devised. We now report a method that combines fluorescence measurement of Ca2+i, 45Ca kinetics, and computer modeling to yield the basal plasma membrane Ca2+ flux in A7r5 vascular smooth muscle cells. Kinetic analysis of basal Ca2+i and Ca2+i transients following chelation of extracellular Ca2+ yields a unique value for the ratio of the rate constant governing Ca2+ pumping into the sarcoplasmic reticulum (SR) to that for plasma membrane Ca2+ extrusion (1.12 +/- 0.06). When this ratio was used to constrain the least-squares fitting of 45Ca efflux data from A7r5 cells, it was possible to determine unique values for the unidirectional, steady-state Ca2+ fluxes across both SR and plasma membranes. The basal unidirectional plasma membrane Ca2+ flux was 0.062 +/- 0.018 fmol . min-1 . cell, and the basal SR Ca2+ flux was 0.069 +/- 0.019 fmol . min-1 . cell-1. These results demonstrate, within the limitations of measuring the absolute value of Ca2+i, the feasibility of measuring previously unresolvable subpicoamp basal Ca2+ fluxes in intact cells under normal physiological conditions.

The rates of muscle glucose uptake of trained (TR) and untrained (UT) obese Zucker rats were assessed by hindlimb perfusion under basal conditions (no insulin) in the presence of a maximally stimulating concentration of insulin (10 mU/ml) and after muscle contraction elicited by electrical stimulation of the sciatic nerve. Perfusate contained 28 mM glucose and 7.5 microCi/mmol of 2-deoxy-D-[3H]glucose. Muscle GLUT-4 concentration was determined by Western blot analysis and expressed as a percentage of a heart standard. The rates of insulin-stimulated glucose uptake were significantly higher in the plantaris, red gastrocnemius (RG), and white gastrocnemius (WG), but not the soleus or extensor digatorum longus (EDL) of TR compared with UT rats. After muscle contraction the rates of glucose uptake in the TR rats were significantly higher in the soleus, plantaris, and RG. TR rats had significantly higher GLUT-4 protein concentration and citrate synthase activity than the UT rats in the soleus, plantaris, RG, and WG. Basal plasma membrane GLUT-4 protein concentration of TR rats was 144% above UT rats (P < 0.01). Stimulation by insulin and contraction resulted in a significant increase in plasma membrane GLUT-4 protein concentration in UT rats only. However, plasma membrane GLUT-4 protein concentration in insulin- and contraction-stimulated TR rats remained 53% and 30% greater than that of UT rats, respectively (P < 0.05). Exercise training did not alter basal, insulin-, or contraction-stimulated GLUT-4 functional activity.(ABSTRACT TRUNCATED AT 250 WORDS)

The placenta absorbs anionic amino acids from the maternal and fetal circulations but does not significantly transfer these amino acids from mother to fetus. Uptake of L-aspartate and L-glutamate by basal (fetal-facing) plasma membrane vesicles from placental syncytiotrophoblast was stimulated by an inward sodium and an outward potassium gradient. Measurable saturable uptake was entirely sodium dependent and electrogenic. Studies of concentration dependence resolved a high-affinity (microM) system that has characteristics of the X-AG system found in other tissues including the placental microvillous plasma membrane. Uptake of 0.2 microM L-glutamate was inhibited by 2 mM L-glutamate, L-aspartate, D-aspartate, L-cysteate, and L-cysteinesulfinic acid and was uninhibited by 2 mM D-glutamate, L-glutamine, L-alanine, L-serine, L-asparagine, and taurine or by 1 mM methylaminoisobutyric acid. The X-AG system in the two membranes of the placental syncytiotrophoblast may mediate the concentrative uptake of anionic amino acids from the maternal and fetal circulations into the placenta.

As a first step in understanding the cellular basis of maternal-fetal calcium transfer, we examined the characteristics of calcium uptake by a highly purified preparation of the syncytiotrophoblast basal (fetal facing) plasma membrane. In the presence of nanomolar concentrations of free calcium, basal membranes demonstrated substantial ATP-dependent calcium uptake [K0.5 = 119 nM, maximum velocity (Vmax) = 2 nM X min-1 X mg-1]. This uptake required magnesium, was not significantly affected by Na+ or K+ (50 mM), or sodium azide (10 mM). Intravesicular calcium was rapidly and completely released by the calcium ionophore A23187. Calcium transport was significantly stimulated by the calcium-dependent regulatory protein calmodulin. Placental membrane fractions enriched in endoplasmic reticulum (ER) and mitochondria also demonstrated ATP-dependent calcium uptake. In contrast to basal membrane, mitochondrial calcium uptake was completely inhibited by azide. The rate of calcium uptake by the ER was only 20% of that of basal membranes. We conclude that the placental basal plasma membrane possesses a high-affinity calcium transport system similar to that found in plasma membranes of a variety of cell types. This transporter is situated to permit it to function in vivo in maternal-fetal calcium transfer.