Hormone-specific responses and biosynthesis of sulfolipids in cell lines derived from mammalian kidney.

The established cell lines isolated from mammalian kidney were characterized by its receptor activities against hormones and the ability to synthesize sulfolipids localized in the renal tubule. The level of 3':5'-cyclic AMP in JTC-12.P3 (monkey kidney) cells increased in 2 min as much as 2.5-5-fold on activation with 1.0 unit/ml of bovine parathyroid hormone or 1.9 units/ml of synthetic parathyroid hormone (1-34) resulting in intracellular cyclic AMP concentration of more than 40 pmol/mg protein. Prostaglandin E1 (14 micronM) and isopropylnorepinephrine (10 micronM) were also found to increase the concentration of cyclic AMP by more than 30- and 9-fold, respectively. Addition in medium of calcitonin, arginine vasopressin, adrenocorticotropic hormone and glucagon caused no significant changes of cyclic AMP level in the cell. In contrast, MDCK, a cell line isolated from canine kidney, reacted to arginine vasopressin, isopropylnorepinephrine and prostaglandin E1 and only slightly to parathyroid hormone. MDBK cell line derived from bovine kidney or fibroblast cell lines from rat lung and guinea pig kidney did not react to any of the hormones specific to kidney, i.e. arginine vasopressin, calcitonin or parathyroid hormone in the presence of theophylline. However, in the presence of 2 mM isobutylmethylxanthine, small but significant elevation of cellular cyclic AMP levels in response to calcitonin, arginine vasopressin, isopropylnorepinephrine and prostaglandin E1 was observed. The cell lines JTC-12, MDCK and MDBK, when incubated with H235SO4, incorporated the isotope into sulfolipids assigned as sulfatides and ceramide dihexoside sulfate or in MDCK also into cholesterol sulfate. The results suggested that JTC-12, MDCK and MDBK cell lines are epithelial origin and also JTC-12 and MDCK originated most probably from renal tubular cells of cortex and medulla, respectively.     

A cell strain cultured from porcine kidney increases cyclic AMP content upon exposure to calcitonin or vasopressin.

Cells originally dispersed from whole juvenile male Hampshire pig kidney and maintained in monolayer culture, increased cyclic AMP content in response to incubation with salmon calcitonin or antidiuretic hormone. Parathyroid hormone and epinephrine did not affect cyclic AMP content. The apparent K_m for arginine vasopressin in the porcine cells was 3.0 nM which is similar to the value obtained in single segments of rabbit kidney tubule. The apparent K_m for salmon calcitonin of 2.7 nM is higher than that reported for the rabbit nephron segments, but comparable to the K_m obtained in rat kidney homogenates. Exposure of the porcine cells to exogenous prostaglandin E₂ did not affect cyclic AMP responses to other hormones. In the cultured porcine kidney cells the pattern of hormone response is similar to that observed in nephron segments prepared from the medullary portion of the thick ascending limb of the loop of Henle, and these findings suggest that the porcine cells may be related to cells present in the medullary region of the kidney tubule.     

Alterations in calcitonin and parathyroid hormone responsiveness of adenylate cyclase in F9 embryonal carcinoma cells treated with retinoic acid and dibutyryl cyclic AMP

Teratocarcinoma cells in culture offer an in vitro system for studying certain aspects of embryonic differentiation. To gain some insight into regulatory systems that might be operative during early development, we have characterized the alterations that occur in the hormonal responsiveness of the F9 embryonal carcinoma cell membrane adenylate cyclase with differentiation. Adenylate cyclase of F9 cells is stimulated in the presence of 10 μM GTP by calcitonin, prostaglandin E₁, (?) isoproterenol, and epinephrine, while parathyroid hormone is only slightly effective. Of these active hormones, calcitonin is the most potent stimulator of cyclic AMP production. Exposure of F9 cells to retinoic acid induces differentiation to parietal endodermal cells. Basal, GTP-, and fluoride-stimulated adenylate cyclase activities show a progressive increase with the retinoic acid-induced change to the endodermal phenotype. Differentiation to the endodermal cell type markedly alters the adenylate cyclase response to calcitonin and parathyroid hormone; the cyclase of endodermal cells exhibits a low response to calcitonin while parathyroid hormone dramatically enhances cyclic AMP formation. Treatment of the retinoic acid-generated endodermal cells with dibutyryl cyclic AMP converts these cells to a type exhibiting neural-like morphology. The adenylate cyclase system of these cells is only stimulated by parathyroid hormone, prostaglandin E₁, isoproterenol, and epinephrine. Calcitonin responsiveness has been lost in these cells. These variations in calcitonin and parathyroid hormone responsiveness suggest a possible regulatory role for these hormones during embryonic development. Further more, the results indicate that changes in adenylate cyclase hormonal responsiveness might serve as useful markers during early stages of differentiation.     

Modulation of cyclic AMP-dependent protein kinase by vasopressin and calcitonin in cultured porcine renal LLC-PK1 cells

We have previously demonstrated that a cultured porcine kidney cell, LLC-PK(1), maintains the characteristics of a polar renal epithelial cell in culture, and responds to salmon calcitonin and [arginine]vasopressin by increasing cyclic AMP content. To demonstrate the usefulness of this cell line as a model for the study of the biochemical events distal to cyclic AMP production, the activation of cyclic AMP-dependent protein kinase was examined. Intact cells in monolayer demonstrated progressive increases in cyclic AMP content and activation of protein kinase in response to [arginine]vasopressin (2-200nm) and salmon calcitonin (0.03-30nm) with both hormones fully activating the enzyme at a cell cyclic AMP content of 35pmol/mg of protein. Of the total cyclic AMP-dependent protein kinase activity, 80% was found in the 27000g supernatant fraction of sonicated cell material, and this soluble protein kinase could be fully activated by hormone. Conversely, the 27000g pellet contained a significant proportion of cyclic AMP-independent protein kinase and only 20% of total cell cyclic AMP-dependent protein kinase; the latter showed little response to hormone. On the basis of DEAE-cellulose chromatography, type II protein kinase was the predominant isoenzyme in both soluble and particulate fractions of the LLC-PK(1) cells and the soluble fractions of rat and guinea-pig renal medulla. Thus, the LLC-PK(1) cell line can serve as a model for hormonal modulation of protein kinase and as a potential source for defining the endogenous substrates for these enzymes.     

The possible mediation by cyclic AMP of the stimulation of thymocyte proliferation by vasopressin and the inhibition of this mitogenic action by thyrocalcitonin

Lysine vasopressin (antidiuretic hormone), like cyclic adenosine 3',5'-monophosphate (cyclic AMP), rapidly (in less than 1 hour) stimulates the initiation of deoxyribonucleic acid synthesis and thereby increases the flow of cells into mitosis in rat thymic lymphocyte populations in vitro. This mitogenic action of vasopressin, again like that of cyclic AMP, is potentiated by caffeine, an inhibitor of the intracellular phosphodiesterase which catalyzes the degradation of cyclic AMP. On the other hand, vasopressin's mitogenic action (also like that of cyclic AMP) is blocked by imidazole, an activator of cyclic nucleotide phosphodiesterase activity. The hormone, thyrocalcitonin (calcitonin) which is known to block the cyclic AMP-mediated mitogenic effect of parathyroid hormone by interfering with cyclic AMP action, also blocks the mitogenic action of vasopressin. The inhibitory effects of imidazole and thyrocalcitonin on vasopressin's mitogenic action are both overcome by the phosphodiesterase inhibitor, caffeine. It is concluded from these observations that the mitogenic action of vasopressin is mediated by cyclic AMP.     

Studies in vivo on the effects of parathyroid hormone upon kidney cyclic adenosine 3',5'-monophosphate content using rapid tissue fixation by microwave irradiation.

Microwave irradiation is shown to be a useful method for simultaneously killing chicks and fixing tissues. Renal adenylate cyclase and phosphodiesterase activities were rapidly abolished by microwaving. The increase in chick kidney cyclic adenosine 3',5'-monophosphate (cyclic AMP) content produced by intravenous bovine parathyroid hormone (PTH) injection was much greater in microwaved birds than in those killed by cervical dislocation with subsequent tissue fixation in liquid nitrogen. After PTH injection there was a prolonged elevation of renal cyclic AMP content. At the time of maximum response (2 minutes), log. dose-response curves were linear in the dose range 0.1-10 U. The responses to three different bovine PTH preparations were indistinguishable. Arginine vasopressin, arginine vasotocin, salmon calcitonin and prostaglandin E1 did not affect kidney cyclic AMP content within 2 minutes. Because of its specificity and precision, the method is of use for the in vivo bioassay of PTH. Injection of CaCl2 (20 mumoles) 1 minute before, or conjointly with, bovine PTH inhibited the subsequent increase in kidney cyclic AMP content. The synthetic bovine PTH peptide fragments BPTH (1-34) and BPTH (2-34) both increased chick kidney cyclic AMP content. The use of such fragments allows investigation of the structural requirements of PTH for interaction with the systems regulating cyclic AMP metabolism in the kidney in vivo.     

Primary rabbit kidney proximal tubule cell cultures maintain differentiated functions when cultured in a hormonally defined serum-free medium

Summary A primary rabbit kidney epithelial cell culture system has been developed which retains differentiated functions of the renal proximal tubule. In addition, the cells have a distinctive metabolism and spectrum of hormone responses. The primary cell were observed to retain in vitro a Na⁺-dependent sugar transport system (distinctive of the proximal segment of the nephron) and a Na⁺-dependent phosphate transport system. Both of these transport processes are localized on the apical membrane of proximal tubule cells in vivo. In addition, probenicid-sensitivep-aminohippurate (PAH) uptake was observed in basolateral membranes of the primary tubule cells, and the PAH uptake by these vesicles occurred at a rate that was very similar to that observed with membranes derived from the original tissue. Several other characteristics of the primary cells were examined, including hormone-sensitive cyclic AMP production and phosphoenolpyruvate carboxykinase (PEPCK) activity. Like the cells in vivo, the primary proximal tubule cells were observed to produce significant cyclic AMP in response to parathyroid hormone, but not in response to arginine vasopressin or salmon calcitonin. Significant PEPCK acivity was observed in the particulate fraction derived from a homogenate of primary rabbit kidney proximal tubule cells. This paper was presented at a Symposium on the Physiology and Toxicology of the Kidney In Vitro co-sponsored by The Society of Toxicology (SOT) and the Tissue Culture Association held at the 27th annual meeting of the SOT in Dallas, Texas in 1988. This work was supported by Grant 9 RO1 DK40286-07 from the National Institutes of Health, Bethesda, MD, and NIH Research Career Development Award 1 K04 CA 0088-01 to M.T.     

Localization of cell groups sensitive to parathyroid hormone and calcitonin in rat skeletal tissue.

The level of cyclic AMP in various fractions of rat skeletal tissue was measured after in vitro or in vivo administration of parathyroid hormone and calcitonin. Incubations of bone fractions prepared from young (5 weeks of age thyroparathyroidectomized rats revealed that both parathyroid hormone and calcitonin increased the cyclic AMP level in fractions of epiphysis, metaphysis and marrow cells. Cyclic AMP accumulation in incubated perisoteum and diaphysis were induced solely by parathyroid hormone. In in vivo experiments the cyclic AMP level in the tibia of the thyroparathyroidectomized rat was increased by infusion of either parathyroid hormone or calcitonin, and the simultaneous administration of each maximally effective dose of the two hormones exhibited an additive effect. Within 2 min, parathyroid hormone infusion caused an elevation of cyclic AMP content in periosteum and metaphysis. Rapid increase of cyclic AMP in the metaphysis was also induced by calcitonin, and the effect of the two hormones on cyclic AMP accumulation in this fraction was additive. Small but significant increase of cyclic AMP in the diaphysis was detected at 5 min after the administration of parathyroid hormone. Calcitonin infusion did not show any consistent effects on periosteum and diaphysis.     

Prostaglandin stimulation of adenosine 3′,5′-monophosphate accumulation in cultured chondrocytes in the presence or absence of parathyroid hormone

The effect of prostaglandin analogues on the cycle AMP level in cultured chondrocytes were examined. Prostaglandin E₁ at 0.4 to 30 μM, increased the intracellular concentration of cyclic AMP in chondrocytes. Its effect was rapid, being evident within 1 min and reaching a maximum in 10 to 20 min. The maximum level was sustained until 30 min after its addition and then decreased gradually. Prostaglandin D₂ and E₂ also increased the cyclic AMP level in chondrocytes, but they had less effect than prostaglandin E₁. Prostaglandin A₁ had no effect on the nucleotide level in chondrocytes, although they markedly increased the level in fibroblasts. The time course of stimulation of cyclic AMP accumulation in chondrocytes by prostaglandin E₁, D₂ or E₂ was quite different from that by parathyroid hormone (PTH): the effect of prostaglandin was slower and more sustained than that of PTH. PTH potentiated the effect of prostaglandin E₁, E₂, or D₂ on the cyclic AMP level in chondrocytes and that the combined effects of prostaglandin, PTH or both produced a synergistic effect on the accumulation of cyclic AMP in the chondrocytes. These findings suggest that prostaglandin E₁, E₂, and D₂ increase the synthesis of cyclic AMP and that the combined effect of the prostaglandins and PTH on the cyclic AMP level in chondrocytes is partly attributed to the synergistic synthesis of cyclic AMP in the cells.     

Loss of the PGE1 requirement for MDCK cell growth associated with a defect in cyclic AMP phosphodiesterase

Prostaglandin E₁(PGE₁), one of the components in the hormone-supplemented, serum-free medium for Madin Darby Canine Kidney (MDCK) cells (Medium K-1), is required for both long-term growth and for dome formation. Variant cells have been isolated from MDCK populations, which lack the PGE₁, requirement for long-term growth in Medium K-1. These variants will be useful in identifying the molecular events initiated by PGE₁ which are necessary for the growth response to be observed. The growth and functional properties of five independently isolated PGE₁ independent clones have been examined. Normal MDCK cells grew at an equivalent rate in Medium K-1 and in serum-supplemented medium; the growth rate was lower in Medium K-1 lacking PGE₁. In contrast, PGE₁ independent clone 1 grew at an equivalent rate in Medium K-1 minus PGE₁, and in serum-supplemented medium. When PGE₁ was added to K-1 minus PGE₁, less growth of PGE₁ independent clone 1 was observed. A similar observation was made with one other PGE₁ independent clone which was studied. A hormone deletion study indicated that PGE₁ independent clone 1 still retained growth responses to the other four supplements in Medium K-1 (insulin, transferrin, T₃, and hydrocortisone). The molecular alterations associated with loss of the PGE₁ requirement for long-term growth were examined. At confluency, all of the PGE₁ independent clones studied had higher intracellular cyclic AMP levels following PGE₁ treatment, as compared with normal MDCK cells. The increased cyclic AMP levels in the variant cells could result from a number of different types of defects, including reduced cyclic adenylic acid (cyclic AMP) efflux, an increased affinity of PGE₂ for the PGE₁ receptor, or a defect in cyclic AMP metabolism. However, in all of the variant clones studied a decreased rate of cyclic AMP degradation by cyclic AMP phosphodiesterase was observed. Thus, the increased cyclic AMP levels in the PGE₁ independent variants may result from alterations which affect cyclic AMP metabolism. The effect of PGE₁ on dome formation by the variant cells was also examined. The frequency of dome formation by PGE₁ independent clone 1 was enhanced in a dosage-dependent manner, like normal MDCK cells. This observation suggests that PGE₁ affects MDCK cell growth and dome formation by different mechanisms.     

Regulation of the receptor-mediated cyclic AMP response of kidney to parathyroid hormone in the vitamin D-deficient rat

Rats fed a diet deficient in vitamin D were found to exhibit a refractory cyclic AMP response of kidney slices to parathyroid hormone and a marked decrease in membrane parathyroid hormone-dependent adenylate cyclase activity. Both the characteristic calcium deficiency (hypocalcemia) and secondary elevation of circulating parathyroid hormone appeared before the first noticeable decrease in hormone-dependent enzyme activity. After repletion of D-deficient rats with vitamin D₂, we found that serum calcium and parathyroid hormone were both restored to normal levels before the depressed enzyme response to the hormone was reversed. Moreover, infusion of parathyroid hormone into vitamin D-replete rats led to a marked reduction in parathyroid hormone-dependent adenylate cyclase activity, which was partly restored to control level 3 hours after discontinuing the hormone infusion. Taken as a whole, this study suggests that the elevated endogenous parathyroid hormone in the vitamin D-deficient rat is involved in the “down-regulation” of renal cyclic AMP responsiveness to the hormone. However, these experiments do not rule out the possibility that calcium deficiency and/or vitamin D per se participate in the regulation of the renal cyclic AMP response to parathyroid hormone.     

Cortical cell populations from rabbit kidney isolated by free-flow electrophoresis: characterization by measurement of hormone-sensitive adenylate cyclase

Free-flow electrophoresis allows the separation of different cell populations from a cell suspension isolated from rabbit kidney cortex after perfusion of the kidneys with a calcium-binder, followed by gentle mechanical treatment. After electrophoretic separation, analysis of the adenylate cyclase activities after stimulation by various hormones allows the precise determination of the origin of the cell populations with different electrophoretic mobilities. Adenylate cyclase from the slow-moving main cell population was only sensitive to parathyroid hormone. These cells had also high alkaline phosphatase content, further demonstrating their proximal origin. The various fast- moving cell populations had adenylate cyclase sensitive to isoproterenol and arginine vasopressin but were less sensitive to parathyroid hormone than the slow-moving cells. Their alkaline phosphatase content was also much lower. This indicates that these fast- moving cell populations originate from both the granulous segment of the distal tubule and from the collecting ducts. The adenylate cyclase activity and the cyclic AMP contents of isolated proximal cells maintained in culture medium were also investigated.     

Calcium-mediated effects of calcitonin on cyclic AMP formation and lymphoblast proliferation in thymocyte populations exposed to prostaglandin E 1

The calcitonin (SCT) from salmon ultimobranchial bodies which (like mammalian calcitonins) lowers the plasma calcium concentration in mammals can also affect cyclic AMP (cyclic adenosine 3′,5′-monophosphate) metabolism and proliferation of lymphoblasts in normal and prostaglandin E₁ (PGE₁)-treated rat thymocyte populations in three different ways. In the first case, low concentrations (0.5–5.0 ng per milliliter) of SCT lower (by a calcium-mediated process) the ability of PGE₁ to transiently increase cyclic AMP synthesis, but the reduced surge of cyclic AMP production is still ample to stimulate lymphoblasts in the cell population to initiate deoxyribonucleic acid (DNA) synthesis. Secondly, these low SCT concentrations affect the eventual progression of the PGE₁-stimulated, DNA-synthesizing lymphoblasts into mitosis by a calcium-mediated process. Depending on the extracellular calcium concentration and the magnitude of the initial increment in the intracellular cyclic AMP content, SCT can either promote or inhibit the progression of the stimulated cells into mitosis. SCT's third action is a rapid (within 5 minutes), calcium-independent elevation of the cellular cyclic AMP content in otherwise untreated thymic lymphocyte populations exposed to a very high concentration (100 ng per milliliter) of the hormone. This early, transient rise in the cyclic AMP level is followed by a calcium-dependent increase in lymphoblast proliferation. An attempt is made to interrelate and explain the different actions of SCT on cyclic AMP metabolism and mitogenesis.     

Comparison of the effects of prostaglandin I2 and prostaglandin E2 stimulation of the rat kidney adenylate cyclase-cyclic AMP systems

Prostacyclin (Prostaglandin I₂) effects on the rat kidney adenylate cyclase-cyclic AMP system were examined. Prostaglandin I₂ and prostaglandin E₂, from 8 · 10^?4 to 8 · ^?7 M stimulated adenylate cyclase to a similar extent in cortex and outer medulla. In inner medulla, prostaglandin I₂ was more effective than prostaglandin E₂ at all concentrations tested. Both prostaglandin I₂ and prostaglandin E₂ were additive with antidiuretic hormone in outer and inner medulla. Prostaglandin I₂ and prostaglandin E₂ were not additive in any area of the kidney, indicating both were working by similar mechanisms. Prostaglandin I₂ stimulation of adenylate cyclase correlated with its ability to increase renal slice cyclic AMP content. Prostaglandin I₂ and prostaglandin E₂ (1.5 · 10^?4 M) elevated cyclic AMP content in cortex and outer medulla slices. In inner medulla, with Santoquin® (0.1 mM) present to suppress endogenous prostaglandin synthesis, prostaglandin I₂ and prostaglandin E₂ increased cyclic AMP content. 6-Ketoprostaglandin F_1α, the stable metabolite of prostaglandin I₂, did not increase adenylate cyclase activity or tissue cyclic AMP content. Thus, prostaglandin I₂ activates renal adenylate cyclase. This suggests that the physiological actions of prostaglandin I₂ may be mediated through the adenylate cyclase-cyclic AMP system.     

Effect of parathyroid hormone and cyclic AMP on protein phosphorylation in rabbit kidney cortex

Suspensions of renal cortical tubules were incubated with ³³P_i and exposed to parathyroid hormone (40 μg/ml) or 1 mM dibutyryl cyclic AMP. In other experiments homogenates of renal cortex were assayed for protein kinase and phosphoprotein phosphatase activity using [γ-³²P]ATP with or without 5 mM cyclic AMP. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and phosphorylation of proteins measured by liquid scintillation counting of gel slices. The pattern of protein phosphorylation was similar in control tissue from both tubule suspensions and homogenates. In intact tubules, parathyroid hormone stimulated the phosphorylation of four proteins with molecular weights of approx. 1500 000, 125 000, 100 000 and 50 000 by 28%, 24%, 13%, and 20%, respectively. Results with dibutyryl cyclic AMP were comparable but more variable. Stimulation of phosphorylation by cyclic AMP in homogenates was more generalized with the major effect on a 50 000 dalton protein (50% stimulation). No effect of cyclic AMP on dephosphorylation of proteins was observed. The results are interpreted as indicating that increased phosphorylation of cell proteins is part of the cyclic AMP-mediated response of the renal cortex to parathyroid hormone.     

Characterization of primar cell cultures derived from rat renal proximal tubules

Summary Proximal tubules were prepared from rat kidney cortex by collagenase digestion and purified by percoll gradient centrifugation. Their enrichment was estimated by comparing the specific activities of various cell-specific enzymes in homogenates of renal cortex and of the isolated tubules. The tubules were cultured in a 50:50 mixture of Dulbecco’s modified Eagle’s and Ham’s F12 media supplemented with insulin, transferrin, epidermal growth factor, hydrocortisone, and prostaglandin E₁. After 2 to 3 d an extensive outgrowth of epithelial cells developed from the attached tubules. After 5 to 7 d near confluent monolayers were obtained. Hormonal responsiveness, marker enzyme activities, and transport properties were determined to further characterize the primary cultures. The cultured cells exhibited increased cyclic AMP production in response to parathyroid hormone but not calcitonin or vasopressin, consistent with the absence of cells derived from distal and collecting tubules. The cells also retained significant levels of 25-hydroxyvitamin D₃-lα-hydroxylase, alkaline phosphatase, and ψ-glytamyltranspeptidase, three enzymes that are primarily associated with the proximal tubule. The cultured epithelial cells also exhibit a Na⁺-dependent phosphate and glucose transport systems. Therefore, the cells retain many functional properties that are characteristic of proximal tubules. Thus, the primary cultures should be suitable for the study of processes that occur specifically within this segment of the rat nephron. This work was supported in part by the Veterans Administration (JBP), Washington, DC, by grant DK-37124 (NPC) from the National Institutes of Health, Bethesda, MD, and by grant BNS-86-17004 (CFL) from the National Science Foundation, Washington, DC.     

Regulation of 24,25-dihydroxyvitamin D-3 production by 1,25-dihydroxyvitamin D-3 and synthetic human parathyroid hormone fragment 1–34 in a cloned monkey kidney cell line (JTC-12)

Regulation of 25-hydroxyvitamin D-3 24-hydroxylase by 1,25-dihydroxyvitamin D-3 and synthetic human parathyroid hormone fragment 1–34 (PTH_1–34) was investigated using a cloned monkey kidney cell line, JTC-12. Treatment of the cells with 1,25-dihydroxyvitamin D-3 markedly enhanced the conversion of [³H]-25-hydroxyvitamin D-3 into a more polar metabolite. The metabolite was identified as 24,25-dihydroxyvitamin D-3 by normal phase and reverse phase high-performance liquid chromatography and periodate oxidation. The 24-hydroxylae activity appeared to follow Michaelis-Menten kintics, and 1,25-dihydroxyvitamin D-3 treatment increased the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of 24-hydroxylase from 33 to 95 pmol/h per 10⁶ cells without affecting the apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value of the enzyme (220 nM in control vs. 205 nM in 1,25-dihydroxyvitamin D-3 treated cells). The enzyme activity reached a maximum between 4 and 8 h of treatment with 1,25-dihydroxyvitamin D-3. The dose of 1,25-dihydroxyvitamin D-3 required to cause a half-maximal stimulation was about 3 · 10^?10 M. The 1,25-dihydroxyvitamin D-3-induced increase in 24-hydroxylase was almost completely inhibited by the presence of 1 μM cycloheximide. Treatment of the cells with PTH_1–34 caused a dose-dependent increase in cyclic AMP production. Half-maximal stimulation of cyclic AMP production was obtained at about 5 · 10^?9 M PTH_1–34. When 2.4 · 10^?9 M PTH_1–34 was added after 1,25-dihydroxyvitamin D-3 treatment, the 1,25-dihydroxyvitamin D-3-stimulated 24-hydroxylase was inhibited to $\text{70.7 ± 2.9\%}$ of control. Higher concentrations of PTH_1–34 caused less inhibition of the enzyme activity. When cyclic AMP was added instead of PTH_1–34, the enzyme activity was also suppressed significantly. These results indicate that, in JTC-12 cells, 1,25-dihydroxyvitamin D-3 stimulates 24-hydroxylase in a dose- and time-dependent manner by increasing the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of the enzyme through a mechanism dependent upon new protein synthesis, and suggest that PTH_1–34 inhibits the 1,25-dihydroxyvitamin D-3-induced stimulation of 24-hydroxylase through its effect on cyclic AMP production.     

Actions of calcitonin, parathyroid hormone, and prostaglandin E2 on cyclic AMP formation in chicken and rat osteoclasts

The effects of calcitonin, parathyroid hormone, and prostaglandin E2 on cyclic AMP production were studied in osteoclast-rich cultures derived from medullary bone of laying hens and from the long bones of newborn rats. Cyclic AMP was assayed biochemically in replicate cultures, and furthermore, changes in cytoplasmic fluorescence were sought by indirect immunofluorescence with rabbit anti-cyclic AMP and FITC-labelled goat anti-rabbit IgG. Treatment of rat osteoclasts with calcitonin increased cyclic AMP formation as measured biochemically, and this was confirmed by the immunofluorescence method. No such increase took place in chick osteoclasts. Prostaglandin E2 increased cyclic AMP production in both rat and chick osteoclasts as determined by both methods. Since the immunofluorescence method failed to detect a response to parathyroid hormone either in chick or rat osteoclasts, its variable biochemical effects were concluded to be due to actions on contaminating osteoblasts in the cultures. Thus it has been possible with a combined biochemical and immunocytochemical approach to define the cyclic AMP responses to the calcium-regulating hormones in rat and chick osteoclasts. The failure of calcitonin to increase cyclic AMP in chick osteoclasts identifies a need to investigate the nature of calcitonin action on avian osteoclasts, which may contribute to understanding of its actions on mammalian cells.     

Long-term stimulation of cAMP production in LLC-PK1 pig kidney epithelial cells by salmon calcitonin or a photoactivatable analogue of vasopressin

A photoreactive analogue of vasopressin, [1-(3-mercapto)propionic acid, 8-(N⁶-4-azidophenylamidino)lysine]-vasopressin, was compared to salmon calcitonin and [8-arginine]-vasopressin with respect to stimulation of cAMP synthesis in the LLC-PK₁ pig kidney epithelial cell line. Without photoactivation, the vasopressin analogue-elicited responses were identical to those induced by vasopressin, in that cAMP synthesis returned to the basal, unstimulated level about 4 h after hormonal treatment. In contrast, the levels of activation of cAMP-dependent protein kinase induced by salmon calcitonin returned to basal approx. 12 h after hormone addition. When activated by ultraviolet irradiation, the vasopressin analogue induced ‘permanent’ stimulation of adenylate cyclase, whereby cAMP production could be detected even 12.5 h after treatment. Both salmon calcitonin and the photoactivated vasopressin analogue inhibited growth of LLC-PK₁ cells, in contrast to vasopressin or the nonactivated analogue. Growth inhibition appeared to be a consequence of the prolonged stimulation of adenylate cyclase. This conclusion was supported by the fact that a LLC-PK₁ cell mutant in cAMP-dependent protein kinase was resistant to growth inhibition by salmon calcitonin and activated vasopressin analogue. The results imply that the cAMP-dependent protein kinase is the mediator of the hormone-stimulated growth inhibition.