Altered activation by calcitonin and inhibition by somatostatin of human embryonal carcinoma cell adenylate cyclase with retinoic acid-induced differentiation

Human 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 human development, we have characterized the alterations that occur in the hormonal responsiveness of human embryonal carcinoma cell adenylate cyclase with differentiation in response to 10 microM retinoic acid. Two cell lines CL12 and CL13, cloned from Tera 2 cells by Dr. C. F. Graham, have been used in these studies. Adenylate cyclase of CL12 and CL13 cells is stimulated in the presence of 10 microM GTP by epinephrine and calcitonin, with calcitonin being the most potent stimulator of cyclic AMP production. Exposure of these cells to retinoic acid leads to an arrest in growth and within 6 days to a differentiated cell population with a stable nonreversible phenotype. No changes in basal, GTP- and fluoride-stimulated adenylate cyclase activities are observed with retinoic acid treatment, but the cyclase of differentiated cells exhibits a greater stimulation by calcitonin (7.5-fold) and the appearance of a somatostatin inhibitory effect. Somatostatin specifically inhibits, by 25%, the hormonal stimulation of adenylate cyclase of cells treated for 5 days with retinoic acid. The increase in calcitonin stimulation of adenylate cyclase activity of the differentiated cells is related to an increase (congruent to 3-fold) in the number of hormonal receptors and not to a significant change in receptor binding affinity (Kd 4.6 X 10(8) M-1). These alterations in calcitonin and somatostatin responsiveness suggest a possible regulatory role for these hormones during embryonic development. Furthermore, the results indicate that changes in adenylate cyclase hormonal responsiveness might serve as useful markers during early stages of human embryonal carcinoma cell differentiation.     

Calcitonin-responsive adenylate cyclase in cultured F9 embryonal carcinoma cells

Hormonal responsiveness of the adenylate cyclase system of cultured F9 teratocarcinoma cells was investigated. Of numerous hormones tested only calcitonin, (−)isoproterenol, and prostaglandin E₁, stimulate F9 adenylate cyclase activity. Of the active hormones, calcitonin is the most potent stimulator of cAMP formation. Treatment of intact F9 cells with calcitonin results in a time- and hormone concentration-dependent increase in the intracellular concentration of cAMP. cAMP accumulation is enhanced within 5 min after addition of 60 nM synthetic salmon calcitonin to intact F9 cells. These results raise the possibility that calcitonin may play a regulatory role in early embryonic development.     

Production of immunoreactive calcitonin and parathyroid hormone by embryonal carcinoma cells: alteration with retinoic acid-induced differentiation

To determine possible ectopic production of, and altered responsiveness to, specific hormones and growth factors which may be involved in mediating embryonic differentiation and development embryonal carcinoma cells in culture have been employed to serve as an in vitro system of embryogenesis. Exposure of F9 embryonal carcinoma cells to all-trans-retinoic acid previously has been shown to induce differentiation of these undifferentiated stem cells to parietal endoderm and to markedly alter the ability of calcitonin and parathyroid hormone to stimulate adenylate cyclase activity. Evidence is presented that F9 cells secrete immunoreactive calcitonin into the culture medium (200 pg/12 hr/10(7) cells) while parietal yolk sac (PYS) cells secrete immunoreactive parathyroid hormone (800 pg/12 hr/10(7) cells). Retinoic-induced differentiation of F9 cells to endoderm results in a progressive reduction in immunoreactive calcitonin production, while there is an increase in the level of immunoreactive parathyroid hormone found in the conditioned medium. After exposure of F9 cells to retinoic acid for 5 days, little calcitonin is detectable in 12-hr conditioned medium. Changes in the intracellular levels of immunoreactive calcitonin and PTH follow a pattern similar to that noted for changes in the amount of secreted hormones. Thus, immunoreactive calcitonin is produced by undifferentiated F9 cells which possess a calcitonin responsive adenylate cyclase system, while parathyroid hormone is produced by parietal endoderm cells which respond to parathyroid hormone with increased cyclic AMP synthesis. Sephadex G50 gel filtration of F9-conditioned medium shows two peaks of immunoreactive calcitonin with Mr of 3500 and 20,000. Immunoprecipitation of calcitonin from 35S-labeled F9 cells reveals a specific band of 20,000 Mr. Likewise, two peaks of parathyroid hormone immunoreactive material of Mr 8000 and 39,000 are noted after gel filtration of PYS cell-conditioned medium, whereas parathyroid hormone immunoprecipitation from the same cells reveals a specific band of 39,000 Mr. These results raise the possibility that embryo production of these two hormones at specific stages in development may contribute to the regulation of subsequent steps of differentiation.     

Altered hormonal responses: markers for embryonal and embryonic carcinoma stem cells and their differentiated derivatives

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 membrane adenylate cyclase of different embryonal carcinoma cell lines with differentiation. Each undifferentiated embryonal carcinoma stem cell studied (F9, PCC4, PC13, P19) has an adenylate cyclase system predominantly activated by calcitonin. Of great interest is the fact that cAMP production is also enhanced specifically by calcitonin in an embryo-derived stem cell line. Differentiation of the embryonal carcinoma stem cell population toward parietal endoderm results in a decrease in calcitonin activation with a concomitant appearance of sensitivity to parathyroid hormone. Differentiation toward visceral endoderm is characterized by a lack of response of the adenylate cyclase system to both calcitonin and parathyroid hormone. These results indicate that the changes noted in adenylate cyclase hormonal responsiveness might serve as useful markers during early stages of differentiation.     

Effects of choleragen on hormonal responsiveness of adenylate cyclase in human fibroblasts and rat fat cells

Choleragen increases cyclic AMP content of confluent human fibroblasts. Maximally effective concentrations of isoproterenol and prostaglandin E₁ also induce large increases in cyclic AMP content of human fibroblasts and in confluent cultures the effect of prostaglandin E₁ is much greater than that of isoproterenol. After incubation with choleragen, the increment in cyclic AMP produced by 2 μM isoproterenol is increased and approaches that produced by 5.6 μM prostaglandin E₁. Although the concentration of isoproterenol which produces a maximal increase in cyclic AMP is similar in both control and choleragen-treated cells, lower concentrations of isoproterenol are more effective in the choleragen-treated cells. In choleragen-treated cells, although the response to 5.6 μM prostaglandin E₁ is reduced by as much as 50%, the concentration of prostaglandin E₁ required to induce a maximal increase in cyclic AMP is $\text{1}\text{10}$ that required in control cells. Thus the capacities of intact human fibroblasts to respond to isoproterenol and prostaglandin E₁ can be altered independently during incubation of intact cells with choleragen. Differences in responsiveness to the two agonists were not demonstrable in adenylate cyclase preparations from control or choleragen-treated cells.In rat fat cells, the effects of choleragen on cyclic AMP content were much smaller than those in fibroblasts. In contrast to its effect on intact fibroblasts, choleragen treatment of rat fat cells did not alter the accumulation of cyclic AMP in response to a maximally effective concentration of isoproterenol. The responsiveness of adenylate cyclase preparations to isoproterenol was also not altered by exposure of fat cells to choleragen.     

Loss of adenylate cyclase hormonal sensitivity in chemically transformed epithelial cells.

The hormonal sensitivity of adenylate cyclase from a normal rat liver epithelial cell line (K16) and its chemically transformed derivative (W8) were compared. Intact normal rat liver cells had markedly increased cAMP levels after brief exposure to epinephrine, isoproterenol, norepinephrine or prostaglandin E₁. In contrast, the cAMP levels of chemically transformed cells were relatively unaffected by these same compounds even after prolonged incubation. A comparison of broken cell adenylate cyclase activities revealed a decreased basal activity in the chemically transformed cells; the response to NaF was similar in the two cell lines, while the response to catecholamines and prostaglandins paralleled the intact cell studies. These data suggest that one reason for loss of adenylate cyclase hormonal responsiveness in chemically transformed rat liver epithelial cells may be a dysfunction or loss of hormone binding sites.     

Hormone responsiveness of plasma membrane-bound enzymes in normal and regenerating rat liver.

The hormonal responsiveness of plasma membrane-bound enzymes (Na-+-K-+)-ATPase and adenylate cyclase has been investigated in normal and regenerating rat liver. (Na-+-K-+)-ATPase basal activity is not affected by surgery and only slightly affected by partial hepatectomy; its response to epinephrine and cyclic AMP is decreased only 15 h after hepatectomy. Adenylate cyclase activity of plasma membranes from untreated animals is stimulated by parathyroid hormone and thyroxine; partial hepatectomy increased basal activity as well as the stimulation exerted by the aforementioned hormones, when glucagon and epinephrine sensitivity is essentially unaltered.     

Adenylate cyclase coupled beta adrenergic receptors of Rauscher erythroleukemia cells

Rauscher murine erythroleukemia cells undergo erythroid differentiation in response to the hormonal inducer erythropoietin and to synthetic inducers. Incubation of these cells with (?) isoproterenol or (?) epinephrine results in a 2–3-fold increase in cyclic AMP levels. This effect is completely blocked by propranolol, identifying the response as due to a β-adrenergic receptor·adenylate cyclase system. Experiments with [¹²⁵I]iodohydroxybenzylpindolol demonstrate 1700 β-adrenergic receptors/cell with a K_D of 320 pM. These results form the basis for further studies of the β-adrenergic receptor·adenylate cyclase complex during erythropoiesis.     

Effects of choleragen on hormonal responsiveness of adenylate cyclase in human fibroblasts and rat fat cells.

Choleragen increases cyclic AMP content of confluent human fibroblasts. Maximally effective concentrations of isoproterenol and prostaglandin E1 also induce large increases in cyclic AMP content of human fibroblasts and in confluent cultures the effect of prostaglandin E1 is much greater than that of isoproterenol. After incubation with choleragen, the increment in cyclic AMP produced by 2 muM isoproterenol is increased and approaches that produced by5.6 muM prostaglandin E1. Although the concentration of isoproterenol which produces a maximal increase in cyclic AMP is similar in both control and choleragen-treated cells. In choleragen-treated cells, although the response to 5.6 muM prostaglandin E1 is reduced by as much as 50%, the concentration of prostaglandin E1 required to induce a maximal increase in cyclic AMP is 1/10 that required in control cells. Thus the capacities of intact human fibroblasts to respond to isoproterenol and prostaglandin E1 can be altered independently during incubation of intact cells with choleragen. Differences in responsiveness to the two agonists were not demonstrable in adenylate cyclase preparation from control or choleragen-treated cells. In rat fat cells, the effects of choleragen on cyclic AMP content were much smaller than those in fibroblasts. In contrast to its effect on intact fibroblast choleragen treatment of rat fat cells did not alter the accumulation of cyclic AMP in response to a maximally effective concentration of isoproterenol. The responsiveness of adenylate cyclase preparations to isoproterenol was also not altered by exposure of fat cells to choleragen.     

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.     

Effect of 1,25-dihydroxyvitamin D3 on cyclic AMP responses to hormones in clonal osteogenic sarcoma cells.

The effect of 1,25-dihydroxyvitamin D3 on adenylate cyclase responsiveness was studied in the clonal osteogenic sarcoma cell line, UMR 106-06, which responds to several bone active hormones. 1,25-dihydroxyvitamin D3 treatment had no consistent effect on basal formation of cyclic AMP in intact cells, but the responses to parathyroid hormone, isoproterenol, prostaglandin E2, salmon calcitonin and the plant diterpene, forskolin, were all attenuated, by up to 90%. The effect of 1,25-dihydroxyvitamin D3 was dose-dependent, with half-maximal effectiveness at 0.1 nM, and required 48 h treatment of cells before it became apparent. The relative potencies of other vitamin D3 compounds correlated closely with their relative affinities for the 1,25-dihydroxyvitamin D3 receptor and their biological activities in other systems. 1,25-dihydroxyvitamin D3 treatment had no effect on the kinetics of labelled calcitonin binding to UMR 106-06 cells. Furthermore, the fact that such a range of hormones was affected made a receptor mediated mechanism unlikely. Nucleotide stimulatory (Ns) unit activity was assayed after 1,25-dihydroxyvitamin D3 treatment and found to be unchanged. Islet activating protein, an inhibitor of nucleotide inhibitory unit (Ni) activity, failed to modify the 1,25-dihydroxyvitamin D3 effect. Thus the effect of 1,25-dihydroxyvitamin D3 appeared to be exerted beyond hormone receptor and nucleotide regulatory components of the adenylate cyclase complex. It is concluded that 1,25-dihydroxyvitamin D3 attenuates adenylate cyclase response to hormones by a direct or indirect action on the catalytic component of adenylate cyclase.     

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.     

β-2-adrenergic stimulation of ornithine decarboxylase activity in porcine granulosa cells in vitro

Epinephrine, norepinephrine, and isoproterenol produced dose-dependent stimulation of ornithine decarboxylase (EC 4.1.1.7) activity in isolated porcine granulosa cells maintained under defined conditions in vitro. β- but not α-receptor-blocking agents prevented enzyme stimulation by catecholamines. Application of preferential β-1 and β-2-receptor antagonists and agonists localized the epinephrine effect to β-2-adrenergic mediation. Epinephrine action was enhanced by the phosphodiesterase inhibitor, 1-methyl-3-isobutyl-xanthine, but not by saturating concentrations of the cyclic AMP analogue, 8-bromocyclic AMP, of follicle-stimulating hormone, or of prostaglandin E₂. However, stimulation by epinephrine was additive to that of luteinizing hormone. Follicular fluid obtained from immature Graafian follicles contined concentrations of norepinephrine and epinephrine active in vitro.Thus, catecholamines may participate in the regulation of ornithine decarboxylase activity in the ovary. Catecholamine effects may be mediated by β-2-receptors linked to the adenylate cyclase system.     

Enhanced GTP-dependent activities of the adenylate cyclase system: basis for increased hormonal responsiveness.

Normal rat kidney (NRK) cells growth arrested by picolinic acid and isoleucine deprivation exhibit an increased response to certain agents (i.e., prostaglandin E₁, (?)-isoproterenol, and cholera toxin) which elevate intracellular cyclic AMP levels. The enhanced hormonal response is apparently due, at least in part, to increased adenylate cyclase activity. Adenylate cyclase activities measured in the presence of GTP, GTP plus prostaglandin E₁, and GTP plus (?)-isoproterenol are increased two- to threefold in membranes prepared from treated cells. In contrast, basal activity is potentiated only 20 to 50% and activity determined in the presence of fluoride is only marginally altered. Also of interest is the increase in cholera toxin activation of cyclase activity in the treated cells. Lower concentrations of cholera toxin (5 ng/ml) are required to achieve maximal stimulation of cyclase activity from picolinic acid-treated and isoleucine-deprived cells; maximal stimulation of control cell adenylate cyclase is attained with 25 to 50 ng/ml cholera toxin. Picolinic acid treatment and isoleucine deficiency both have been shown to arrest NRK cell growth in the G₁ phase of the cell cycle. However, results with cells arrested in G₁ by serum starvation and by growth to high cell population density indicate that G₁ specific growth arrest does not appear to account for the increase in hormonal responsiveness. Chelation of inhibitory metals and proteolytic activation also do not appear to be involved in the mechanism by which picolinic acid enhances cyclic AMP formation. Rather, the results suggest that the treated cells have an increased amount of an active GTP-dependent function required for hormone and cholera toxin stimulation of adenylate cyclase. Thus, picolinic acid treatment and isoleucine deprivation may provide a useful means of modulating the GTP-dependent step required to potentiate hormonal responsiveness.     

Effects of parathyroid hormone and calcitonin on adenylate cyclase in murine mononuclear phagocytes

Peritoneal mononuclear phagocytes elicited by thioglycollate demonstrate responsiveness to parathyroid hormone (PTH) and calcitonin (CT) which differs from that seen in the normal resident population. PTH causes a twofold stimulation of adenylate cyclase activity in elicited cells but inhibits this activity in resident cells. CT causes a greater stimulation of adenylate cyclase in elicited than in resident cells. Both CT and PTH cause an increase in cyclic AMP accumulation in cultures of elicited mononuclear phagocytes. These results indicate that cells of the mononuclear phagocyte lineage have functional receptors for both PTH and CT. This is the first biochemical evidence to support the hypothesis that mononuclear phagocytes are precursors of the bone resorbing osteoclast.     

Potentiation by GTP of hormone-responsive adenylate cyclase in renal cortex plasma membrane preparations

GTP potentiated the stimulation by parathyroid hormone and prostaglandin E₁ of adenylate cyclase in a renal cortex preparation enriched in proximal tubule basal-lateral plasma membranes. Adenylate cyclase in these membranes did not respond to epinephrine nor glucagon, in the absence or presence of GTP. Activation of basal activity by GMP-PNP was strongly inhibited by GTP. GTP also increased the sensitivity of renal adenylate cyclase to parathyroid hormone and prostaglandin E₁. The synergistic effect of GTP was not inhibited by chelating nor thiol-reducing reagents.     

Adenylate cyclase activity in cultured epithelial cells.

The cyclic AMP metabolism of cultured epithelial cells was investigated. Epinephrine or 1-methyl,3-isobutylxanthine (MIX) alone had no effect on cyclic AMP levels in intact cells, whereas the combination of the two agents yielded a 6- to 10-fold increase in cyclic AMP levels. Both basal and stimulated cyclic AMP levels decreased with increasing cell density. Cell-free adenylate cyclase preparations were stimulated markedly by epinephrine or isoproterenol in the absence of MIX. Since the epithelial cells were found to have a relatively small amount of cyclic nucleotide phosphodiesterase (PDE) activity, the requirement for MIX to visualize intact cell responsiveness to epinephrine could be explained only partially by its PDE inhibitory properties.     

Endothelial cell adenylate cyclase: activation by catecholamines and prostaglandin I2+

Following incubation of intact vascular endothelial cells with 1 mM 3-isobutyl-1-methylxanthine, and isoproterenol or PGI₂, cyclic AMP levels increased 4- and 3-fold, respectively. Isoproterenol-stimulated adenylate cyclase activity of cell lysates was selectively inhibited by the β-adrenergic blocking agent propranolol. Catecholamines stimulated adenylate cyclase with the potency series: isoproterenol > epinephrine > norepinephrine. Prostaglandin did not stimulate adenylate cyclase activity in cell lysates, even in the presence of guanine nucleotides or following preincubation of the intact cells with prostaglandins prior to freeze-thaw lysis.     

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 μM) and isopropylnorepinephrine (10 μM) 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.     

Activation of renal adenylate cyclase by forskolin: assessment of enzymatic activity in animal models of the secondary hyperparathyroid state

The effects of forskolin on kidney slice cyclic AMP content and membrane adenylate cyclase activity were studied in order to determine whether or not activation of the enzyme by forskolin was affected in experimental animal models of the secondary hyperparathyroid state. Forskolin was found to be a potent activator of renal adenylate cyclase in rats and chicks, and the diterpene produced a marked potentiation of the cyclic AMP response to parathyroid hormone (PTH). The diterpene had no effect on the binding of PTH to renal receptors. Activity of adenylate cyclase in the presence of forskolin was similar in renal membranes from either vitamin D-deficient rats or chicks compared to control. Forskolin did not restore full responsiveness to PTH in renal slices from chicks raised on diets that were deficient in either vitamin D or calcium although the diterpene was capable of potentiating the cyclic AMP response to PTH in these tissues. Forskolin also augmented the activation of membrane adenylate cyclase by PTH although this effect of the diterpene was much less prominent in membrane preparations than that observed in renal slices. This study provided additional evidence that the downregulation of renal PTH-dependent adenylate cyclase in experimental models of secondary hyperparathyroidism is due to a specific reduction in receptor-mediated regulation of cyclic AMP formation. Adenylate cyclase activity as assessed by forskolin-stimulated enzyme activity was fully maintained in kidney membranes from these animal models. Thus, forskolin appears to be a useful drug for measuring total enzymatic activity in situations where altered responsiveness of adenylate cyclase to hormones has been demonstrated to be mediated by changes in hormone receptors.