Photodynamic therapy with 5-aminolaevulinic acid and DNA damage: unravelling roles of p53 and ABCG2.

OBJECTIVES: In spite of high sensitivity of A549 cells (p53(+/+) ) to lethal effects of photodynamic therapy with 5-aminolaevulinic acid (5-ALA/PDT), DNA damage was observed only in H1299 cells (p53(-/-) ), suggesting that p53 may exert a protective effect. Studies on human colon adenocarcinoma cell lines HCT-116, and their cognate knockouts for p53, were not entirely consistent with the assumption above. Exploring alternative explanations for such conflicting behaviour, we observed that expression of the ATP-binding cassette G2 (ABCG2), a regulator of cell component efflux, had important effects on PDT-generated DNA injury in PC3 cells (prostate) which are p53(-/-) and positive for ABCG2. Addition of an ABCG2 inhibitor in ABCG2 positive A549 (p53(+/+) ) and PC3 (p53(-/-) ) cells eliminated resistance to DNA damage. MATERIALS AND METHODS: All cell lines investigated were incubated with 5-ALA and irradiated. Effects of PDT were evaluated assessing residual cell viability, cell-cycle profiles, PpIX localization, comet assay and Western blotting. Identical measurements were made in the presence of ABCG2 inhibitor, in cells expressing the transporter. RESULTS: Our data show that cell aptitude to defend its DNA from PDT-induced injury was mainly ruled by ABCG2 expression. These findings, while providing helpful information in predicting effectiveness of 5-ALA/PDT, may indicate a way to shift PDT from a palliative to a more effective approach in anti-cancer therapy.

5-aminolaevulinic acid/photo-dynamic therapy and gefitinib in non-small cell lung cancer cell lines: a potential strategy to improve gefitinib therapeutic efficacy.

OBJECTIVES: Often, non-small cell lung cancers (NSCLC) respond only poorly to the tyrosine kinase inhibitor (TKI) gefitinib, which targets the epidermal growth factor receptor (EGFR), these poor responders EGFRs lacking activating mutations. In this study, we have attempted to improve TKI response of NSCLC cell lines (A549 and H1299) devoid of EGFR mutations, by combination of gefitinib and 5-ALA/photodynamic therapy (PDT). MATERIALS AND METHODS: Cells of the two lines were incubated with gefitinib (from 0.5 to 50 mm, for 48 h) then irradiated at doses ranging from 4 to 20 J/cm(2) ; 5-ALA concentration and incubation time were kept constant (1 mm for 3 h). We analysed cell viability, colony-forming efficiency, cell cycle parameters, proteasome and NF-κB activity and expression patterns of specific proteins, after individual or combined treatments. RESULTS: Effects (antagonistic, additive or synergistic) of combination treatment were evaluated using a predictive model (combination index) for expected interactive effects and results are consistent with mutual potentiation exceeding simple additivity. Investigation of molecular mechanisms underlying cytotoxic effects indicated that combination treatment impaired proteasome function, inhibited NF-κB transcriptional activity and hampered AKT pro-survival signalling. CONCLUSIONS: The results of this study show that poor response of cells devoid of EGFR activating mutations to TKIs, can be overcome by combining gefitinib with 5-ALA/photodynamic therapy (PDT).

Inhibition of farnesylation blocks growth but not differentiation in FRTL-5 thyroid cells.

The cholesterol lowering drug lovastatin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, blocks DNA synthesis and proliferation of thyrotropin (TSH) primed FRTL-5 rat thyroid cells. The blockade can be completely prevented and/or reversed by mevalonate and largely prevented and/or reversed by farnesol whereas cholesterol and/or other non-sterol mevalonate derivatives such as ubiquinone, dolichol or isopentenyladenosine are ineffective. TSH-dependent augmentation of cyclic-AMP and cAMP dependent differentiated functions, such as iodide uptake, are unaffected by lovastatin. 3H-Thymidine incorporation into DNA is also decreased by alpha-hydroxyfarnesyl-phosphonic acid, an inhibitor of protein farnesylation which mimicks the effect of lovastatin since it also leaves unaffected TSH stimulated iodide uptake. It is suggested that the HMG-CoA reductase inhibitor lovastatin affects cell proliferation mainly through inhibition of protein farnesylation which results in altered function proteins relevant for proliferation control, notably p21ras and/or other small GTPases.

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase gene expression in FRTL-5 cells. I. Identification and characterization of a cyclic AMP-responsive element in the rat reductase promoter.

Thyrotropin (TSH) increases 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene transcription in FRTL-5 rat thyroid cells, and the effect of TSH can be mimicked by cAMP. Sequence analysis of the rat reductase promoter has revealed a hitherto unnoticed cAMP-responsive element (CRE)-like octamer. This octamer is located between 53 and 60 nucleotides downstream of the sterol regulatory element 1; its first 6 nucleotides are identical to the consensus somatostatin CRE, and the entire octamer is identical to the fos CRE. A synthetic oligonucleotide containing the HMG-CoA reductase CRE-like octamer (RED CRE) formed protein-DNA complexes with nuclear extracts from FRTL-5 cells, which could be prevented by unlabeled CRE-containing oligonucleotides whose flanking sequences were otherwise nonidentical. The complexes were specifically supershifted by anti-CREB antibodies. FRTL-5 cells transfected with a fusion plasmid carrying the bacterial chloramphenicol acetyl transferase (CAT) under the control of the HMG-CoA reductase promoter displayed CAT activity, which was specifically stimulated by TSH. In contrast, CAT activity in FRTL-5 cells transfected with similar constructs carrying mutations in the reductase CRE was significantly lower and did not increase after TSH challenge. We suggest that the HMG-CoA reductase gene contains a functional CRE, important for TSH regulation of transcription. The data presented provide the molecular basis for a novel regulatory mechanism for HMG-CoA reductase gene expression in rat thyroid cells, which involves the direct effect of cAMP.

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase gene expression in FRTL-5 cells. II. Down-regulation by v-K-ras oncogene.

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity and mRNA levels were significantly reduced in FRTL-5 cells transformed with the Kirsten-Moloney sarcoma virus (KiMol); these cells have lost thyrotropin dependence and express high levels of p21ras. FRTL-5 cells, transformed with a temperature-sensitive mutant of the v-K-ras oncogene (Ats cells: 33 degrees C, permissive; 39 degrees C, nonpermissive), showed significant reduction of HMG-CoA reductase expression when exposed to 33 degrees C. In KiMol cells, as well as in Ats cells at 33 degrees C, the transcription driven by cAMP-responsive element was probed by measuring chloramphenicol acetyl transferase (CAT) levels after transfection with a chimeric plasmid containing the reporter gene linked to the rat reductase promoter. Basal CAT activity in KiMol cells transfected with wild-type promoter was lower than in FRTL-5 cells but was increased by forskolin to the levels attained in thyrotropin-stimulated FRTL-5 cells. Forskolin failed to increase CAT activity in KiMol cells transfected with the plasmid harboring a reductase promoter in which the cAMP-responsive element octamer was mutated to a nonpalindromic sequence. The effect of v-K-ras could be mimicked in FRTL-5 cells by tetradecanoyl phorbol acetate and reverted in KiMol and Ats cells, expressing active Ras protein, by increasing intracellular cAMP and/or by protein kinase C inhibition. The data are consistent with the contention that v-K-ras, through protein kinase C and depletion of intracellular cAMP, is inhibitory for the protein kinase A pathway. This is the first demonstration that active v-K-ras down-regulates HMG-CoA reductase expression.

Congenital hypothyroidism: etiology and pathogenesis.

Congenital hypothyroidism is a frequently occurring condition with possibly severe and irreversible consequences. Most of the cases are due to thyroid ectopia, aplasia or hypoplasia and are sporadic in occurrence. Inherited defects of thyroid hormone biosynthesis, secretion and utilization represent a minor, although not insignificant, fraction of the cases of congenital hypothyroidism. In a number of cases, transient congenital hypothyroidism can be due to such causes as maternal exposure to antithyroid drugs or excess iodine, transplacental transfer of blocking antibodies or endemic iodine deficiency. The latter is still a matter of concern in selected geographical areas. Both sporadic and familial cases of hypothalamic-pituitary hypothyroidism are quite rare. Early diagnosis of congenital hypothyroidism by mass screening programs is of the foremost importance for the prevention of long-term sequelae. The molecular defect has been elucidated in a number of inherited defects of thyroid hormone biosynthesis, secretion and utilization. These include impaired thyroidal response to thyroid-stimulating hormone (TSH) due to an altered TSH receptor, defective synthesis of thyroglobulin, defective synthesis of thyroid peroxidase, generalized resistance to thyroid hormone, and familial isolated TSH deficiency. It is anticipated that, as more mutations become available for detailed molecular analysis, further advances in our knowledge of the molecular aspects of thyroid function will ensue in the near future.

Purinergic (P2) receptor-operated calcium entry into rat thyroid cells.

In the epithelial cell line FRT, derived from rat thyroid, extracellular ATP, at a concentration as low as 1 x 10(-7) M, specifically increases cytosolic Ca++ two fold over the basal level of 255 +/- 45 nM. A maximum increase of 5 fold over basal is seen at 1 x 10(-5) M ATP. The effect occurs in the absence of any measurable phosphatidyl inositol metabolism and requires the presence of extracellular Ca++, but is independent of extracellular Na+; it is duplicated by ATP gamma S but not by adenosine, AMP, ADP, AMP-PNP, AMP-CPP, or AMP-PCP. In the presence of the P2-receptor antagonist suramin, the ATP induced Ca++ influx is completely inhibited, whereas Mg++, La , and verapamil are ineffective. It appears that the most likely (and unique) mechanism of ATP induced increase of cytosolic Ca++ in FRT cells in an increased influx through the activation of a P2 receptor operated Ca++ channel.

Mevalonate controls cytoskeleton organization and cell morphology in thyroid epithelial cells.

Blockade of mevalonate synthesis by the 3-hydroxy-3-methylglutaryl Coenzyme A reductase inhibitor mevinolin (lovastatin) causes FRTL-5 thyroid cells to undergo significant morphological changes; these include a transition from a flat, polygonal to a round shape, the development of cytoplasmic arborizations, and the loss of contact between neighboring cells. Immunofluorescence studies of cytoskeletal structures show that, at early times after administering the drug, and before the round phenotype develops, stress fibers disassemble while the peripheral actin filaments, which are adjacent to the cytoplasmic face of the plasma membrane, appear largely unaffected. Subsequently, when this cortical actin network becomes fragmented, cells start to round up and become separated from neighbors. Microtubules become disconnected from the plasma membrane and retract toward the cell center, although they do not appear depolymerized; indeed, at this stage, cytoplasmic elongations contain mostly intact microtubules. After exposure to mevinolin FRTL-5 cells also lose vinculin-related substrate contacts. Treatment of cells with either cycloheximide or colchicine abolishes morphological changes induced by mevinolin, suggesting that ongoing protein synthesis and microtubule integrity are prerequisites for the drug to be effective. Both cytoskeletal and morphological perturbations can be reversed by mevalonate, but not by cholesterol or the non-sterol derivatives of mevalonate such as dolichol, ubiquinone, and isopentenyladenine, individually or in combination. It is suggested that mevalonate deficiency may impair formation of isoprenylated proteins important for cytoskeletal organization and stability.

Increased levels of DHEAS in serum of patients with X-linked ichthyosis.

The metabolic basis of X-linked ichthyosis is a deficiency of steroid sulphatase, a microsomal enzyme which removes sulphate groups from sulphated steroids. We report on a carefully controlled group of 15 patients with recessive X-linked ichthyosis, selected in a narrow age range (22-33 years), in whom, through the use of gas chromatographic analysis and conventional radioimmunoassay, we have measured not only elevated serum cholesterol sulphate levels but also significantly elevated serum dehydroepiandrosterone sulphate levels. The latter finding has been controversial in previous reports. We believe that the radioimmunoassay procedure generally used should be held responsible for such controversy since it often gives rise to false positive and/or false negative values. Gas chromatography, although more exacting, appears to be far more reliable for the assessment of elevated serum dehydroepiandrosterone.

Thyrotropin modulates low density lipoprotein binding activity in FRTL-5 thyroid cells.

FRTL-5 cells possess high affinity low density lipoprotein (LDL) receptors which bind, internalize, and degrade LDL. When FRTL-5 cells are deprived of thyrotropin (TSH) the binding of LDL increases more than 2-fold. Upon addition of TSH, at a concentration of 1 x 10(-10) M or greater, LDL binding decreases rapidly and within 24 h reaches the level which is typical of FRTL-5 cells chronically stimulated by TSH. The data available suggest that TSH-dependent down-regulation of LDL receptor activity is exerted through a reduction of the number of active LDL receptors, with no change in affinity. It is unlikely that the synthesis of LDL receptors is impaired, since LDL receptor messenger RNA is not decreased by TSH. The effect of the hormone on LDL receptor activity can be mimicked by 8-Br-cAMP and is completely abolished by the protein synthesis inhibitor cycloheximide but not by actinomycin D. TSH regulation of LDL receptor activity is lost in v-ras Ki-transformed FRTL-5 cells (Ki Mol) which also have lost TSH dependence for adenylate cyclase activation and growth. However, 8-Br-cAMP decreases LDL binding in Ki Mol FRTL-5 cells. The reduced availability of LDL receptor in TSH-stimulated FRTL-5 cells may be related to the increased membrane fluidity (Beguinot, F., Beguinot, L., Tramontano, D., Duilio, C., Formisano, S., Bifulco, M., Ambesi-Impiombato, F. S., and Aloj, S. M. (1987) J. Biol. Chem. 262, 1575-1582) or may reflect increased degradation of LDL receptors. We propose that a lower cholesterol uptake is needed in an actively proliferating cell population, to increase the production of isoprenoids whether it be for cholesterol biosynthesis or for the synthesis of other compounds requiring isoprenoid precursors.

Cell cycle progression and 3-hydroxy-3-methylglutaryl coenzyme A reductase are regulated by thyrotropin in FRTL-5 rat thyroid cells.

The incorporation of [14C]acetate into cholesterol shows that FRTL-5 cells possess an active cholesterol biosynthetic pathway. When these cells were made quiescent, and synchronized by thyrotropin (TSH) starvation, in the presence of low serum (0.2%), addition of this hormone increased acetate conversion into cholesterol up to a maximum of 8-fold. Feedback inhibition of sterol synthesis by exogenous cholesterol occurs in FRTL-5 cells since, in the presence of higher serum concentration (5%), acetate conversion into cholesterol was significantly depressed. Even in high serum TSH increased sterol synthesis, albeit to a lesser extent. The time course of the TSH effect on cholesterol synthesis, strongly suggests that this process is necessary for quiescent FRTL-5 cells to enter the cell cycle. Thus, the rate of cholesterol synthesis was maximal 12-16 h after TSH challenge and declined thereafter, returning to levels slightly above the basal at 48 h. Thymidine incorporation into DNA, measured under identical conditions of TSH starvation/challenge, increased after 20 h, was maximal at 36 h, and returned to pre-TSH level at 70 h. The effect of TSH on cholesterol synthesis is not a general feature of lipid synthesis in FRTL-5 since [14C]acetate incorporation into triglycerides after TSH treatment has a different magnitude and time course. TSH increases cholesterol synthesis through the induction of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase. This is due to an increase in the level of 3-hydroxy-3-methylglutaryl-CoA reductase messenger RNA up to 8-fold caused by a proportional increase in the rate of gene transcription, as assessed by nuclear "run on" experiments. The effect of TSH on cholesterol synthesis and reductase gene expression is likely to be mediated by cAMP since 8-bromo-cAMP mimicked the effect of the hormone. The data presented suggest that an active cholesterol biosynthetic pathway is required for DNA synthesis to occur.

Thyrotropin regulation of malic enzyme in FRTL-5 rat thyroid cells.

TSH-induced increases in malic enzyme mRNA levels in FRTL-5 rat thyroid cells are paralleled by increases in malic enzyme activity and are mimicked by 8-bromo-cAMP. Apparent approximately 4 h after TSH challenge and maximal after 16 h, they decline by 24 h and are at basal levels by 48 h. The increase occurs in the absence of a measurable effect of TSH on DNA synthesis related to cell growth, since [3H] thymidine incorporation into DNA is still at basal levels 24 h after TSH challenge and is maximal only at 48 h. A protein(s) whose formation is inhibited by cycloheximide appears to be critical to the ability of TSH to increase malic enzyme mRNA levels. Thus, cycloheximide given 30 min before TSH prevents the hormone-induced increase in malic enzyme mRNA; also, when given 24 h after TSH, cycloheximide accelerates the loss of the TSH-induced increase in malic enzyme mRNA. In neither case does cycloheximide affect beta-actin mRNA levels. A second factor(s) whose formation is prevented by actinomycin-D appears to be important for the decrease in malic enzyme mRNA levels seen 24 and 48 h after TSH challenge. Thus, in experiments in which it is given 24 h after TSH, actinomycin-D preserves the hormone-induced increase in malic enzyme mRNA levels rather than accelerating the decrease, as does cycloheximide. In the same experiment, beta-actin mRNA levels decrease to less than 10-20% of control values over the same period; this factor also, therefore, appears to exhibit some degree of specificity.(ABSTRACT TRUNCATED AT 250 WORDS)

Thyrotropin increases malic enzyme messenger ribonucleic acid levels in rat FRTL-5 thyroid cells.

The addition of TSH to FRTL-5 thyroid cells induces a 7- to 8-fold increase in the steady state level of malic enzyme [L-malate-NADP+ oxidoreductase (decarboxylating); EC 1.1.1.40] mRNA, but does not alter beta-actin mRNA levels. Insulin alone or together with TSH has no effect on malic enzyme mRNA. The effect of TSH is not the result of thyroid hormone formation, since the addition of T3 in the presence or in the absence of TSH and the addition of 5% serum (which includes T3 and T4) have no effect. Forskolin (10(-6) M) reproduces the TSH effect, suggesting that cAMP is involved.

The arachidonic acid signal system in the thyroid: regulation by thyrotropin and insulin/IGF-I.

The present study and the previous report (6) show that the cyclooxygenase path is a primary route of metabolism of arachidonic acid in FRTL-5 rat thyroid cells. The production of PGD2 and PGE2 is an active process in intact cells treated with complete medium including TSH, insulin and 5% calf serum. In contrast, PGF2 alpha and HHT are probably nonenzymatic degradation products of an unstable intermediate, PGH2, since the two compounds are produced and occupy a significant proportion of the cyclooxygenase metabolites only in the homogenate system; this is true in other cells. Although the production of prostaglandins involves three steps, i.e. the release of free arachidonic acid, the production of PGH2 by PGH synthase (cyclooxygenase) and the conversion of PGH2 to various prostaglandins by specific isomerases or synthetases, the first step, the release of free arachidonic acid, has been, until recently, believed to be the sole step important for the regulation of prostaglandin synthesis. This presumption rested on the following observations. Only the free form of arachidonic acid is converted to prostaglandins and the intracellular free arachidonic acid pool is very small compared to the esterified form in phospholipids. The size of the free arachidonic acid pool is regulated by the balance between release from phospholipids by phospholipases and reacylation into phospholipids. When resting cells are stimulated, the release of arachidonic acid and the production of prostaglandins increase concomitantly. The present study shows, however, that all three steps of prostaglandin synthesis are under regulatory control in FRTL-5 rat thyroid cells and that the control is a complex process involving TSH, insulin/IGF-I, and serum. The first step is primarily under the control of TSH. TSH increases the synthesis of arachidonic acid and also, like norepinephrine (5, 6) induces the release of arachidonic acid from the cell by a mechanism involving a pertussis toxin-sensitive G protein. Regulation of the second step can be estimated by measuring cyclooxygenase activity. The present report shows that TSH increases cyclooxygenase activity, presumably by increasing gene expression, but that the TSH effect on cyclooxygenase activity requires insulin/IGF-I or serum. This result is similar to studies showing the effect of TSH and insulin/IGF-I on glycosaminoglycan synthesis, thyroglobulin synthesis, and growth in FRTL-5 thyroid cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Cholinergic control of cyclic nucleotide metabolism in human thyroid cells.

In the presence of Ro 20-1724, a selective inhibitor of cyclic nucleotide phosphodiesterase, carbamylcholine increases cAMP and cGMP levels in human thyroid cells in primary culture. The increase of cAMP exhibited at concentrations of carbamylcholine between 10 fM and 10 pM, is dose- and time-dependent, it is maximum after 30 min and is abolished after 60 min. At higher carbamylcholine concentration (10 microM), cAMP increases rapidly, becoming maximum after 15 min, but returns to unstimulated values after 30 min. The increase of cGMP is also dose-dependent (0.1 nM-10 microM); it reaches the maximum after 30 min and returns to unstimulated values after 120 min. A significant increase of phosphodiesterase activity is observed at 10 microM carbamylcholine. Atropine, a muscarinic receptor antagonist, blocks carbamylcholine effects on both cAMP and cGMP production without affecting the thyrotropin-induced cAMP accumulation. Hexamethonium, a nicotinic receptor antagonist does not affect the cholinergic effects. In the presence of Ro 20-1724, 10 microM carbamylcholine significantly inhibits the effect of thyrotropin on cAMP production, while the combined addition of low doses of carbamylcholine and thyrotropin (0.1 nM and 10 pM, respectively) results in an additive effect on cAMP levels. Inhibition of thyrotropin activity on cAMP production, similar to that exerted by 10 microM carbamylcholine is produced by increasing free intracellular calcium; this inhibition is relieved by using a calmodulin-sensitive phosphodiesterase inhibitor, M and B 22948 at 50 microM dose. High concentrations (10 microM) of carbamylcholine increase the adenylate cyclase activity, without any significant effect on the thyrotropin-induced activation of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Thyrotropin regulation of membrane lipid fluidity in the FRTL-5 thyroid cell line. Its relationship to cell growth and functional activity.

The mitogenic effect of thyrotropin on functional rat thyroid cells of the line FRTL-5 is correlated with membrane lipid fluidity as evaluated by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. Continued exposure of FRTL-5 cells to a medium lacking thyrotropin causes cessation of cell proliferation and a decrease in membrane lipid fluidity which reaches its minimum in approximately 8 days. The change in lipid fluidity is due to an absolute increase (greater than 2-fold) of membrane cholesterol, with an increased cholesterol/phospholipid ratio and an increased ratio of saturated to unsaturated fatty acids of the membrane phospholipids, contributed primarily by a nearly 4-fold increase in the ratio of saturated to unsaturated C16 fatty acids. It is also associated with a variation of the relative proportions of the major membrane phospholipids; thus, phosphatidylinositol and phosphatidylethanolamine decrease while phosphatidylcholine increases. Both membrane fluidity and lipid composition can be restored by thyrotropin to their original levels, i.e. levels measured under continuous exposure to the hormone. Complete reversal requires at least 48 h, i.e. approximately the same time required for resumption of growth when FRTL-5 cells, starved in thyrotropin, are re-exposed to the hormone. Changes in lipid composition and fluidity can be prevented or can be reversed if FRTL-5 cells are exposed to dibutyryl cAMP while being deprived of thyrotropin. Dibutyryl cAMP has only a modest direct effect on growth; however, this pretreatment eliminates the 48-h lag phase with respect to thyrotropin stimulation. It is proposed that the effects of thyrotropin on growth of FRTL-5 cells requires a modification of the molecular structure and the physical state of cell membranes, which can be mediated by cAMP, although cAMP is not sufficient by itself to promote growth.

Loss of adrenergic regulation of cAMP production in the FRTL-5 cell line.

Rat thyroid cells in primary culture augment cAMP production when challenged with beta-adrenergic agonists; at 10(-5) M the potency is isoproterenol greater than norepinephrine greater than epinephrine. In analogy with human thyroid cells, rat thyroid primary cultures display alpha-adrenergic-stimulated cGMP production which inhibits TSH and norepinephrine stimulation of cAMP. Adrenergic regulation of cyclic nucelotide production is lost in the cloned thyroid cell line of rat origin known as FRTL-5. Also the potentiating effect of phentolamine on TSH stimulation of cAMP production in thyroid primary culture becomes an inhibitory one in the FRTL-5 cells. Neither 'soluble factors' nor contamination of other cell populations could account for the different behaviour of the primary culture and the cell line toward adrenergic regulation. The reported activation by norepinephrine of iodide efflux in FRTL-5 cells rules out the loss of specific adrenergic receptors in the FRTL-5 cells. It is proposed that the cloning of FRTL-5 cells from primary cultures causes an 'alteration' in the coupling of adrenergic receptors to the adenylate cyclase system. This alteration does no affect those mechanism of message transduction that do not involve cAMP as the signal.