Biomedical subjects
K M Halprin
Publications and source records attributed to K M Halprin.
UVB is additive when repeated within an 8-hour interval.
Explore the source record for details and available documents.
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.
"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.
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.
Phosphorylation of pig epidermal soluble protein by endogenous cAMP-dependent protein kinase.
The distribution of adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase and its substrate proteins was analyzed using soluble and particulate fractions of pig epidermal homogenates. When histone was used as a substrate for this enzyme reaction, protein kinase activity was distributed almost equally between the soluble and particulate fractions. However, the effect of exogenously added cAMP was confined almost exclusively to the soluble enzyme. Endogenous protein phosphorylation in the absence of exogenous histone was higher in the particulate fraction than in the soluble fraction, but the stimulating effect of cAMP was observed only in the soluble fraction. These results indicate that cAMP-dependent protein kinase is predominantly localized in the soluble fraction and phosphorylates soluble epidermal proteins. The particulate fraction contains protein kinase which is cAMP-independent and phosphorylates particulate-bound proteins as well as histone. Based on these observations, the soluble fraction was incubated with [gamma-32P]-ATP in the presence or absence of cAMP, and phosphorylated protein was analyzed by SDS disc- or slab-gel electrophoresis followed by autoradiography. Among many proteins whose phosphorylation was slightly increased by cAMP, a protein with Mr approximately 45,000 was found which was markedly phosphorylated in the presence of cAMP. Although this protein corresponds to one of the richest proteins in the epidermal soluble fraction, an important physiologic role for this phosphorylation has not been clarified.
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.
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.
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.
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.
Constant low-dose ultraviolet light therapy for psoriasis.
In a bilateral comparison study, symmetric plaques of psoriasis were used to compare the effects of a constant low dose (3 minutes, 30 seconds) of ultraviolet light therapy to the standard increasing ultraviolet light dose. The low constant dose of ultraviolet light was as efficacious in clearing psoriasis lesions as the higher increasing dose.
Cancer in patients with psoriasis.
In a study of 150 non-PUVA-treated patients with psoriasis matched against a control group of patients with diabetes, the incidence of skin cancer in the psoriasis group was 1.96/yr, which was three times that of the diabetes patients. This figure is statistically significant. The prevalence of internal cancer was only half that of the patients with diabetes. This figure, however, is not significant.
Topical nitrogen mustard therapy in multicentric reticulohistiocytosis.
Explore the source record for details and available documents.
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.
Epidermal surface receptors which link pharmacological mediators to the adenylate cyclase system.
The major purpose of our studies has been to investigate various stimulators of the epidermal adenylate cyclase system. We have recognized the occurrence of four distinct adenylate cyclase systems which respond respectively to catecholamine, histamine, prostaglandin and adenosine. The exposure of floating skin slices in vitro to a stimulator causes a rapid intracellular accumulation of cyclic AMP, which is always transient. Further addition of the same stimulator will not stimulate the same receptor system against the state of 'refractoriness'. The addition of any of the other stimulators can increase the cyclic AMP level. Furthermore, the fact that each stimulator can yield an 'additive' stimulatory effects leads to the conclusion that the epidermis has four distinctly specific and independent adenylate cyclase systems. Our recent investigations have been directed to the analyses of subunits of these skin surface receptor-adenylate cyclase systems. We used two experimental systems, i.e. one being a 'leaky' cell system in which its subunits such as receptor, GTP-regulatory protein and the catalytic unit (adenylate cyclase) are still linked together, and the other being independent preparations of the receptor and catalytic units (with GTP-regulatory protein). These systems allowed us to probe the cell membrane from the inside as well as from the outside. Some of the preliminary kinetic data are herein introduced.
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.
Activation of cAMP-dependent protein kinase in epidermis by the compounds which increase epidermal cAMP.
Pig epidermal slices were incubated with various compounds which increased epidermal cAMP (adenosine 3',5'-monophosphate), and the change in cAMP-dependent protein kinase activity ratio was studied by the method of Cherrington et al (J Biol Chem 251:5209-5218, 1976) with modification. Epinephrine (5 x 10(-5) M), histamine (10(-4) M) and adenosine (10(-3) M), potent agonists of epidermal adenyl cyclase, fully activated the protein kinase (PK) during an incubation of 30 to 45 seconds, that was much shorter than that required for maximal cAMP accumulation under the same conditions (5 min). With such a brief stimulus, the epidermal cAMP-PK system did not become refractory and responded to repeated stimuli. Prostaglandin E2 (PGE2) and isobuthylmethylxanthine (IBMX) and ethanol only partially activated the enzyme. Prostaglandin F2 alpha (PGF2 alpha) and theophylline which were much less effective in increasing epidermal cAMP, activated the enzyme to the same extent as PGE2 and IBMX respectively. These results suggest that protein kinase activation takes place in response to a cAMP increase in small locus of the cell. Such an increase in cAMP can be very small or even not measurable when measured as total cAMP in the tissue homogenate. Also, increases above this level may not be physiologic. It is concluded that measurement of cAMP-dependent protein kinase activity ratio is a more direct and more sensitive way to study the effect of compounds which act through cAMP mediated mechanisms.
Epidermal cyclic GMP is increased in psoriasis lesions.
Cyclic GMP levels in epidermis of normal subjects and of psoriatic patients were measured with a highly sensitive radioimmunoassay method. Technical improvements for the assay are 2-fold: (1) skin samples were frozen in vivo before biopsy and local injection of any anesthetic was avoided to overcome ischemia effect which could lower cyclic GMP artificially; (2) epidermis was microdissected to avoid contamination of dermis and keratin layers. The results show that on a per mg tissue dry weight basis the cyclic GMP levels are about 200 fmol in the involved lesional epidermis and 70 fmol in the uninvolved or normal epidermis. Similarly increases in the cyclic GMP levels in the lesional epidermis are observed when the data are expressed either on a DNA or protein basis. The cyclic GMP level in normal epidermis from nonpsoriatic subjects is the same as that in the uninvolved epidermis of psoriasis patients.