Agents that activate cyclic AMP-dependent protein kinase inhibit explant culture growth and mitotic activity.

Epidermal cells contain 4 separate surface receptors which are linked to adenylate cyclase. Activation of any one of these receptors leads to the accumulation of cAMP within the cell which in turn leads to the activation of cAMP-dependent protein kinase. The levels of cAMP accumulation within the cell caused by the 4 activators are not the same. Epinephrine, histamine, adenosine, and prostaglandins of the "E" series cause easily measurable concentrations of cAMP within 5 min of exposure. Prostaglandin F2 alpha causes only a small nonsignificant increase. Similarly, 2 phosphodiesterase inhibitors, which inhibit the breakdown of cAMP formed within the cell, differ in their ability to accumulate cAMP when cells are exposed to these agents alone. Isobutylmethylxanthine causes a measurable increase in cAMP, while theophylline, a weak inhibitor of phosphodiesterase, gives a nonsignificant increase in cAMP. Recently, experiments have shown that agents that give only slight increases in cAMP by biochemical measurements, that is, prostaglandins F2 alpha and theophylline, are equally able to activate protein kinase within the cell. Since activation of protein kinase is the only mechanism for an increase in cAMP to have a physiologic effect, all of these agents that do activate protein kinase should cause physiologic effects. Using an explant culture system, we show in this paper that this supposition is correct and that all agents that activate protein kinase do result in inhibition of mitotic activity regardless of whether or not they are able to raise cAMP to a level that can be biochemically measured as being significantly different from the baseline value.

"Leaky" epidermal cells contain a complete receptor-mediated adenylate cyclase system with an accessible GTP regulatory protein.

Hormone sensitivity to epinephrine or histamine of the adenylate cyclase system in pig skin is very labile to homogenization. We have developed a new adenylate cyclase receptor-mediated assay system with trypsinized epidermal cells which are treated with hypotonic shock. This new assay system maintained the hormonal sensitivity, as both epinephrine and histamine clearly stimulated cyclic AMP production. Moreover, the Ka for each hormone on this system was similar to that obtained from the floating pig skin slice system. Receptor-adenylate cyclase unit (coupling) in this assay system is therefore preserved as it occurs in intact tissue or cells. Because of the "leaky" nature of our preparation, phosphorylated compounds such as GTP and its analogue and NaF can penetrate the cell membrane and stimulate cyclic AMP production. In this system refractoriness is still maintained to subsequent stimulation by a receptor activator, and cholera toxin can be shown to dramatically increase the activity of GTP on the GTP binding protein, presumably by preventing GTP hydrolysis.

Forskolin activates adenylate cyclase activity and inhibits mitosis in in vitro in pig epidermis.

The novel adenylate cyclase activator forskolin caused rapid and high intracellular accumulation of cyclic AMP in a floating skin (epidermal) slice system. Increased cAMP levels were also detected in the media. Addition of a phosphodiesterase inhibitor to forskolin-containing medium caused only a slight increase in the intracellular cAMP level and forskolin itself did not inhibit phosphodiesterase activity. Ka of forskolin for epidermal adenylate cyclase was about 2-3 X 10(-5) M. This forskolin activation was rapidly reversed after washing. The forskolin stimulation (Ka 5 X 10(-5) M) was also found when tested with an epidermal membrane preparation which contained the catalytic unit of adenylate cyclase but lacked either the GTP or receptor stimulation. With the epidermal slice system, the combination of forskolin and epinephrine (or histamine) stimulated adenylate cyclase synergistically. The data suggest that forskolin activates not only the catalytic unit but also the nucleotide regulatory protein or the receptor-regulatory protein complex of the adenylate cyclase system. The cAMP accumulation caused by forskolin produced a dose-dependent mitotic inhibition of epidermal cells in an in vitro outgrowth system. This inhibitory effect was reversible 48 h after washing out the forskolin.

Adenylate cyclase activation by cholera toxin in pig epidermis: an obligatory role of the GTP-regulatory protein.

Cholera toxin (CT) stimulates the epidermal adenylate cyclase system in vitro. This stimulation was demonstrated in the skin (slice) floating system and the homogenate (membrane) assay system. With the floating system, the addition of CT to the incubation medium caused a marked accumulation of cAMP intracellularly, which was both dose- and time-dependent. A 1-h lag time was present before activation started. Pretreatment of the skin with CT changed the nature of the stimulatory effect caused by epinephrine and histamine, i.e., the transient accumulation of cAMP (a peak at 5 min and subsequent decrease) was no longer observed but the stimulation became persistent. With the membrane assay system in which the receptor components had been uncoupled, adenylate cyclase activities were markedly stimulated by CT (with guanosine-5'-triphosphate, GTP), guanylyl-beta,gamma-imidodiphosphate (GTP-analog, Gpp[NH]p), or sodium fluoride. The stimulation was both dose- and time-dependent without an initial time lag. Either CT or Gpp[NH]p could fully activate adenylate cyclase, and the simultaneous addition of both did not cause further additive stimulation. These data are consistent with the view that the GTP-regulatory protein plays a key role in the activation of adenylate cyclase, and that CT both activates the catalytic unit and modifies the response to receptor hormones through its action on this protein.

Reversal of beta-agonist-induced refractoriness in skin by tetracaine and mepacrine.

We previously reported that in skin slices stimulated by a beta-adrenergic agonist, the intracellular cyclic AMP level increased transiently. The level returned to a low steady state in 20-30 min and further stimulation by the agonist did not increase the cyclic AMP level. This state of "refractoriness" was found to be specific to the initial stimulator, i.e., histamine but not epinephrine could restimulate the cyclic AMP system after an initial exposure to epinephrine (Biochim Biophys Acta 497:428-436, 1977). We now report that incubation of skin with mepacrine or tetracaine after beta-adrenergic stimulation caused partial recovery from the refractoriness. Neither the simultaneous incubation of skin with epinephrine plus mepacrine (or tetracaine) nor preincubation of skin with mepacrine (or tetracaine) before the beta-adrenergic stimulation prevented the development of the refractoriness. Mepacrine inhibited the skin adenylate cyclase catalytic (or the complex of GTP-regulatory protein and catalytic) unit. The available data suggest that mepacrine and tetracaine interacted with the agonist-receptor complex at the cell membrane.

Cyclic AMP-dependent protein kinase isozymes of pig skin and human skin from normal and psoriatic subjects.

Cyclic AMP-dependent protein kinase isozymes of pig and human skin (epidermis) were separated by DEAE-cellulose column chromatography after micromodification for small biopsy samples. Clear-cut separations of type I and type II isozymes, which were of about equal amounts, could be obtained only when the ischemia effect was avoided by in vivo freezing of skin and homogenization for less than 10 s. Intradermal injections of epinephrine caused dose-dependent activation of type I isozyme, but not of type II. Injections of other skin adenylate cyclase stimulators such as histamine, adenosine, and prostaglandin E2 elevated the local cyclic AMP levels to not more than 5 pmol/mg protein and also stimulated only the type I isozyme. Incubation of keratome-sliced pig skin under various conditions caused both activation by dissociation and inactivation by reassociation of the subunits, which appeared to be dependent on the cyclic AMP content. Epinephrine added to the incubation medium led to complete activation of both type I and type II isozymes (the intraepidermal cyclic AMP contents ranged from 20-50 pmol/mg protein). The isozymes of normal skin and involved skin of psoriatics showed identical peaks of type I and type II isozymes of equal amounts. The data indicate that protein kinase in the involved skin is not in an activated (by cyclic AMP) state.

Effects of methotrexate in vitro on epidermal cell proliferation.

Epidermal cell migration activity and epidermal cell proliferation are clearly dissociable in explant culture. Epidermal cell migration requires a non-dialysable, 65,000 mol. wt factor which is destroyed at 100 degrees C but is stable at 80 degrees C for at least 30 min. In the presence of dialysed serum or heated (80 degrees C for 30 min) or DNA synthesis inhibitors (methotrexate or hydroxyurea), cells will migrate from the explant but will not proliferate. At least two factors are required for normal proliferation under these restricted conditions--an adequate supply of DNA precursers, i.e. nucleosides, and a heat liable (80 degrees C) non-dialysable serum component. Methotrexate in concentrations of 1.0 microgram/ml or greater added to cultures in normal fetal calf serum significantly inhibited mitoses; however, when added to serum dialysed to remove thymidine, mitotic inhibition occurred at a concentration of 0.1 microgram/ml of methotrexate and when added to dialysed serum and kept in dialysed serum, inhibition occurred with 0.01 microgram/ml of methotrexate. Methotrexate did not inhibit outgrowth. Hydroxyurea also inhibited mitoses but did not effect outgrowth.

Measurement of adenosine 3',5'-monophosphate-dependent protein kinase and phosphorylase activities in in vivo conditions.

Microassay procedures for cAMP-dependent protein kinase and phosphorylase were developed which detected these activities in less than 25 micrograms of frozen-dried epidermis from a punch biopsy of skin without homogenization. Using these procedures, the activation of cAMP-dependent protein kinase and phosphorylase by beta-adrenergic stimulation in mouse skin was studied in vivo. Cyclic AMP-dependent protein kinase was stimulated by isoproterenol and inhibited by propranolol. Isoproterenol stimulation also activated phosphorylase a in mouse skin. In normal epidermis and uninvolved and involved epidermis from psoriatic patients no significant differences were found in the activities of cAMP-dependent kinase and phosphorylase a. In all experiments we observed that the unstimulated activity ratios of phosphorylase a/total phosphorylase were around 20-30%; these values were much lower than those hitherto reported and show a preponderance of phosphorylase b rather than a. We suggest that in previous reports where phosphorylase a domination was found, phosphorylase b to a activation occurred during homogenization. The data also suggest that in the steady state no obvious defect in basic activities of cAMP-dependent protein kinase and phosphorylase is observed in psoriatic skin.

Failure of periodic ultraviolet radiation treatments to prevent sensitization to nitrogen mustard: a case report.

We have reported that prior ultraviolet radiation (UVR) exposure significantly delayed development of contact sensitivity to nitrogen mustard (Halprin et al., 1981). We felt that this effect was due to disruption of functional Langerhans cells in skin by UVR and suggested that periodic UVR treatments might prevent sensitization to the mustard. We now report on a patient with mycosis fungoides whose epidermal Langerhans cell count was monitored with the ATP-ase stain in order to determine when such 'booster" UVR therapy was to be given. Our attempts to interfere with Langerhans cell function in this manner failed to prevent delayed contact sensitivity to nitrogen mustard and may have been partly responsible for the development of contact urticaria to nitrogen mustard after 28 days of use. Whether the reaction was a delayed, cell-mediated reaction, or an antibody mediated reaction is not clear, but the use of UVR did fail to prevent contact sensitivity to the nitrogen mustard in our patient.

The effect of histamine on epidermal outgrowth: its possible dual role as an inhibitor and stimulator.

We have investigated the effect of histamine on pig epidermal cell outgrowths in vitro. Histamine inhibited the epidermal cell outgrowths (and also mitosis). This inhibition was partially counteracted by a specific H2 antagonist, cimetidine. Inhibition was maximal at a histamine concentration of 10(-4) M and was less at 10(-3) M. These histamine concentrations respectively coincide with the optimal concentrations for accumulating intracellular cyclic AMP (via H2 receptors) and cyclic GMP (via H1 receptors) in the same pig epidermal slice system. 4-Methyl-histamine, a pure H2 agonist, which only increased the intracellular cyclic AMP level but not the cyclic GMP level, caused a maximal outgrowth inhibition at 10(-3) M. Attempts to counteract the histamine effects due to cyclic GMP accumulation by various H1 antagonists (so that 10(-3) M histamine would have caused maximal outgrowth inhibition) were unsuccessful, since the addition of each H1 antagonist alone strongly inhibited the outgrowth. These data strongly suggest a dual role of histamine through the cyclic nucleotide system; i.e., histamine inhibits epidermal cell growth by elevating the intracellular cyclic AMP level via an H2 receptor, while histamine at high concentrations (10(-3) M) partially counteracts the inhibition by increasing cyclic GMP via an H1 receptor.