Skeletal unloading induces resistance to insulin-like growth factor I on bone formation.

Skeletal unloading results in an inhibition of bone formation associated with a decrease in osteoblast number, impaired mineralization of bone, and altered proliferation and differentiation of osteoprogenitor cells. Although such changes are likely to be mediated by multiple factors, resistance to the growth-promoting action of insulin-like growth factor I (IGF-I) has been hypothesized to play an important role. To determine whether skeletal unloading induces resistance to IGF-I on bone formation, we examined the response of unloaded (hindlimb elevation) and normally loaded tibia and femur to IGF-I administration. To eliminate the variable of endogenous growth hormone production and secretion during exogenous IGF-I administration, we used growth hormone-deficient dwarf rats (dw-4). The rats were given IGF-I (2.5 mg/kg/day) or vehicle during 7 and 14 days of unloading or normal loading. This significantly increased the serum level of IGF-I in both the normally loaded and unloaded rats. Unloading did not affect the serum level of IGF-I in the vehicle-treated rats. IGF-I markedly increased periosteal bone formation at the tibiofibular junction of normally loaded rats. Unloading decreased bone formation in the vehicle-treated rats, and blocked the ability of IGF-I to increase bone formation. On the other hand, IGF-I increased periosteal bone formation at the midpoint of the humerus (normally loaded in this model) in both hindlimb-elevated and normally loaded rats. IGF-I significantly increased osteogenic colony number, total ALP activity, and total mineralization in bone marrow osteoprogenitor (BMOp) cells of normally loaded rats. Unloading reduced these parameters in the vehicle-treated rats, and blocked the stimulation by IGF-I. Furthermore, IGF-I administration (10 ng/ml) in vitro significantly increased cell proliferation of the BMOp cells isolated from normally loaded bone, but not that of cells from unloaded bone. These results indicate that skeletal unloading induces resistance to IGF-I on bone formation.

Six putative IQ motifs of the recombinant chicken intestinal brush border myosin I are involved in calmodulin binding.

Chicken brush border myosin I has up to six IQ sequence motifs at which it may bind calmodulin. To determine the relative contributions of these motifs to calmodulin binding, fusion deletion fragments were expressed in Escherichia coli and their ability to bind calmodulin was assessed by the gel overlay technique. The first three N-terminal IQ sites showed strong binding with calmodulin. Surprisingly, the last three incomplete IQ motifs also contributed substantial calmodulin binding. The first and fourth IQ sites bound calmodulin but tended to reduce binding in combination with other sites. The data indicate that interactions among all six IQ motifs contribute to the ability of myosin I to bind calmodulin.

Stimulation by 1,25-dihydroxyvitamin D3 of adenylate cyclase along the villus of chick duodenum.

Administration of 650 pmol 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] to vitamin D-deficient chicks increased adenylate cyclase activity in the basolateral membrane of duodenal epithelial cells within 24 h. This increase in enzymatic activity was accompanied by an increase in calmodulin content of the basolateral membrane. Although neither exogenously added calmodulin (up to 10 micrograms/ml) nor calcium (from 10(-7)-10(-5) M) stimulated enzyme activity, calmodulin antagonists trifluoperazine, W7, and W13 inhibited it. When calmodulin content, adenylate cyclase activity, and alkaline phosphatase activity were measured in cells sequentially eluted from the tip to the base of the villus, cells from the midregion and base had the highest calmodulin content and adenylate cyclase activity, whereas alkaline phosphatase activity (a brush border membrane enzyme) was highest in cells eluted from the tip. Adenylate cyclase activity was increased by 1,25-(OH)2D3, particularly in cells from the midvillus. Our results indicate that the response of adenylate cyclase activity to 1,25-(OH)2D3 varies along the villus and suggest that calmodulin may be involved.

Effect of ethanol on intestinal calcium transport in chicks.

To test the hypothesis that chronic ethanol exposure would alter the membrane fluidity of the intestinal brush-border membrane, which would lead to calcium malabsorption, we gave chicks 15% ethanol in their drinking water from hatching to 3 or 4 wk of age. Although such chicks grew less quickly than their hatchmates not ingesting ethanol, their ability to absorb calcium from the duodenum in vivo was unimpaired. However, when calcium accumulation by isolated duodenal brush-border membrane vesicles (BBMVs) was assessed, the BBMVs from chicks ingesting ethanol for 1-3 wk had a lower rate of uptake than the BBMVs from the controls at all calcium concentrations evaluated (0.1-25 mM). This difference could not be explained by differences in membrane fluidity as assessed by fluorescence depolarization, or by changes in intravesicular volume. Glucose uptake was not affected. The acute addition of ethanol (up to 1 M) in vitro to the control BBMVs increased both membrane fluidity and calcium accumulation. No difference in the fluidizing effect of ethanol in BBMVs between ethanol-ingesting chicks and control chicks could be demonstrated, although the acute effect of ethanol on calcium accumulation was blunted in the BBMVs from chicks ingesting ethanol. Increasing the temperature of the incubation medium also increased membrane fluidity and calcium accumulation in BBMVs from control and ethanol-ingesting chicks, with a greater increase in calcium uptake by the control BBMVs despite comparable increases in fluidity in BBMVs from the control and ethanol-ingesting chicks. We conclude that the chronic ingestion of ethanol by chicks, although markedly altering growth rates, has minimal impact on the intestinal absorption of calcium when assessed in vivo. However, chronic ethanol ingestion does appear to alter the intestinal brush-border membrane to make it less permeable to calcium and less susceptible to the stimulation by ethanol of calcium flux across this membrane; this adaptation may prevent increased flux of calcium across the brush-border membrane into the cell in the presence of ethanol.