DNA synthesis and mitosis have been induced in vitro in fully differentiated neurons from the central nervous system by depolarization with a variety of agents that produce a sustained rise in the intracellular sodium ion concentration and a decrease in the potassium ion concentration. Depolarization was followed in less than 1 hour by an increase in RNA synthesis and in 3 hours by initiation of DNA synthesis. Apparently normal nuclear mitosis ensued, but cytokinesis was not completed in most cells; this resulted in the formation of binucleate neurons. The daughter nuclei each contained the same amount of DNA as the diploid preinduction parental neurons; this implies that true mitogenic replication was induced.
A new case of vitamin D dependent rickets (Type II) with alopecia in a 5 yr old child is reported. Skin fibroblasts were propagated in culture and analyzed for cytoplasmic 1,25(OH)2D3 receptors. The rachitic cells failed to exhibit specific, high-affinity binding sites by either Scatchard analysis or sucrose density gradient. Furthermore, a rise in 24-hydroxylase activity could not be elicited following incubation of the rachitig cells with 1,25(OH)2D3. Fibroblasts from a non-rachitic child examined in parallel experiments demonstrated high affinity binding sites (Kd = 0.1 nM, Nmax = 33 fmol/100 micrograms DNA) and the induction of 24-hydroxylase activity. The molecular basis of the unresponsiveness of the cells from the rachitic child appears to be due to defective or absent 1,25(OH)2D3 receptors.
Skeletal unloading in the growing rat induces a temporary inhibition of bone formation and thereby a deficit in bone calcium compared with age-matched, normally loaded animals. To determine whether this deficit can be restored by skeletal reloading we measured bone formation rate at the tibiofibular junction and total bone calcium in the tibia and lumbar vertebra in rats whose hindlimbs were unloaded for 2 wk and then reloaded by return to normal weight bearing. Continuously loaded or unloaded animals were also studied. Skeletal unloading reduced bone formation by 34% and tibial and vertebral calcium by 12 and 22%, respectively. Reloading significantly increased the rates of bone formation and calcium accretion 30-34% above normally loaded animals, and by 2 wk had decreased the deficit in tibial and vertebral calcium by 36 and 23%, respectively. These data indicate that the deficit in bone calcium induced by skeletal unloading in the growing rat can be restored in part by return to normal weight bearing. However, the time required to restore bone calcium exceeds the time required to produce the original calcium deficit.
In this study we report the demonstration of receptors for 1,25-(OH)2 vitamin D3 in fresh and cultured human skin. Cultured fibroblasts grown from infant foreskin exhibit a binding site which by Scatchard analysis had a Kd for [3H]1,25-(OH)2D3 of 0.2 nM and an Nmax of approximately 40 fmol/mg cytosol protein. On sucrose density gradients the receptor sediments at 3.2S. Receptors could also be identified in skin biopsies from adult patients when assayed either in fresh epidermis or cultured keratinocytes and dermal fibroblasts. The human receptors are similar to rodent receptors assessed in classical target organs such as intestine, bone and kidney. The findings that receptors can be measured in cultured human skin after several arterial passages indicates that skin biopsy may provide a means of assessing the 1,25-(OH)2D3 receptor status of patients.
To investigate further the cellular defects of vitamin D-dependent rickets type II with alopecia, we studied 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptors and the response to 1,25-(OH)2D3 in cultured skin fibroblasts from rachitic patients. Our studies included cells from four affected patients from three kindreds and their parents and cells from five normal subjects. We measured total 1,25-(OH)2D3 receptor binding in cell extracts and the capacity of 1,25-(OH)2D3 to induce the enzyme 25-hydroxyvitamin D3-24-hydroxylase (24-hydroxylase) as a marker of functional response. In normal fibroblasts, the 1,25-(OH)2D3 maximal binding capacity was 52 +/- 5 fmol/100 micrograms DNA (mean +/- SE), and the apparent dissociation constant (Kd) was 0.05 +/- 0.01 nM. The maximal induced 24-hydroxylase activity after 1,25-(OH)2D3 treatment was 11.5 +/- 1 fmol/10(6) cells X 30 min, and the dose of 1,25-(OH)2D3 that achieved half-maximal induction was 2.3 +/- 0.3 nM. Fibroblasts from all four rachitic patients had the same defect: no measurable 1,25-(OH)2D3 receptor binding and no detectable response above basal activity even after high doses of 1,25-(OH)2D3. Cells from all parents except one had normal 1,25-(OH)2D3 binding characteristics and normal 24-hydroxylase bioresponse to 1,25-(OH)2D3. One parent despite a normal phenotype had only half the normal level of binding sites and only half the normal bioresponse. In summary, the cultured fibroblasts from four affected children representing three different kindreds with 1,25-(OH)2D3 resistance failed to exhibit detectable 1,25-(OH)2D3 receptors. We postulate that this biochemical defect produced both the inability to respond to 1,25-(OH)2D3 in vitro and the 1,25-(OH)2D3 resistance in vivo. The obligate heterozygotic parents were normal, except for one who had both half the normal number of receptors and half the normal response to 1,25-(OH)2D3. The data confirm the critical role of the receptor in 1,25-(OH)2D3 action and the close coupling of receptor content and functional responsiveness.
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