The 25‐hydroxycholecalciferol‐1‐hydoxylase activity of chick kidney cells: direct effect of parathyroid

la,25(OH)2D,) is the product of the hydroxylation of 25-hydroxycholecalciferol (25(OH)D,) in the kidney [l-4]. Indirect evidence obtained from in vivo studies support the involvement of parathyroid hormone in regulating the activity of the enzyme which catalyzes this hydroxylation, 25-hydroxycholecalciferol-l-hydroxylase (1 -hydroxyl- ase) [1,2,5,6]. An increased production of la,25(OH),D3 was demonstrated in kidney cells in vitro within 15 min, in response to the presence of bovine parathyroid hormone (BPTH) in the incubation medium [7]. An increased production of 10,25(OH),D3 was demon- strated in a kidney cell culture [8], in response only to much higher concentrations of BPTH in the medium and as late as after 6 h incubation. However, in kidney cell culture [9] and with clumps of intact tubular cells in vitro [lo] no stimulation of 1 -hy- droxylase by BPTH could be demonstrated. A marked inhibition by BPTH of 24,25-dihydroxycholecalciferol (24,25(0H)2D3) production was demonstrated in kidney cells in culture, with only a modest stimula- tion of l~r,25(OH)~D~ production [ll]. Here we measured the effect of some sources of parathyroid activity on the production of la,25(OH),D3 by avian kidney cells in vitro. 2. Experimental Kidney cell suspensions were taken from 5-week- old vitamin D-deficient or cholecalciferol-fed (14 days prior to sacrifice) chicks and prepared by a procedure similar to that in [9] but without any trypsin. The cells were resuspended and washed several times before 328 final suspension in Eagle’s (GIBCO) medium without HCOa (containing 20 mM Hepes and 1 .O mM CaClJ and with 0.1% bovine serum albumin (Sigma). The viability of the kidney cells, as estimated by trypan blue exclusion, was usually -90%. The number of cells remained unchanged during 3 h incubation; however, the viability decreased slightly to 85%. To 0.6 ml kidney cell suspension (2 X 10’ cells) was added 0.4 ml either parathyroid gland (PTG) homogenate (20 mg parathyroid glands) from vitamin D-deficient chicks in the same Eagle’s medium, PTG extract (10 min centrifugation, 3500 rev./min), Eagle’s medium, Eagle’s medium with pure BPTH or with ‘trichloroacetic acid (TCA) powder’ of avian PTG [ 121. The relative activity of the PTG material added was estimated on the basis of cyclic AMP response [12] of a similar preparation. 26,27-3H.25(OH)D3 (Radiochemical Centre, Amersham, 9 mCi/mmol), 45 pmol in 10 ~1 ethanol, was added to the cell sus- pension. The vials were then gassed with OL for 1 min and incubated at 37°C for 3 h. Gassing was repeated at 1 h intervals. Kidney homogenates were prepared and incubated as in [3]. Following incubation, either cells or homogenates were extracted and chromatographed on Sephadex LH-20 columns [3] or were subjected to high-pressure liquid chromatography [ 131 for estimation of vitamin D metabolites. Protein in kidney cells or homogenates was determined according to [ 141. The results were subjected to standard statistical procedures [ 151. 3. Results Kidney cells obtained from cholecalciferol deficient chicks, produced in vitro more 1cr,25(OH),D3 and less 24,25(OH)*D3 than cells obtained from cholecalciferol- fed chicks.