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Biomedical subjects

V Levine

Publications and source records attributed to V Levine.

At least 37 records · Page 2Linked to original sources
PubMed1983

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.

Adenylyl Cyclases↗
PubMed1983

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.

1-Methyl-3-isobutylxanthine↗
PubMed1983

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.

Animals↗
PubMed1982

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.

Animals↗
PubMed1982

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.

Adenylyl Cyclases↗
PubMed1981

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.

Cyclic GMP↗
PubMed1981

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.

Animals↗
PubMed1980

Inhibitors of epidermal cell DNA synthesis in surviving pig skin in vitro.

Keratome slices of domestic pig skin were used to study the DNA synthesis phase of epidermal cell DNA synthesis. Cyclic AMP and agents which elevate intracellular concentrations of cyclic AMP have no direct effect on the "S" phase of DNA synthesis. Theophylline, isobutylmethylxanthine, and adenosine inhibit DNA synthesis immediately by a mechanism which is reversible and is not dependent on cyclic AMP. This inhibition is not associated with an increase in intracellular thymidine phosphates. Hydroxyurea, however, inhibits DNA synthesis immediately and does produce an elevated pool of thymidine phosphates.

1-Methyl-3-isobutylxanthine↗
PubMed1980

Stimulation of protein phosphorylation by epidermal growth factor in pig skin (epidermis).

Incorporation of 32P-orthophosphoric acid into pig epidermal proteins was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The incorporation was observed in 2 major and 2 minor peaks with approximate molecular weights of 52,000 and 27,000, 17,000, and 12,000 respectively. Treatment with epidermal growth factor (EGF) consistently increased 32P incorporation into all 4 peaks. The stimulation was both time- and dose-dependent, and was not inhibited by the simultaneous addition of cycloheximide. In our previous study (submitted to J Invest Dermatol), we observed that EGF increased the cyclic GMP level in the same pig skin (epidermal) system. The possibility that the increased cyclic GMP leads to the protein phosphorylation observed in the present study was considered but ruled out because (1) the addition of cyclic GMP does not mimic the EGF treatment, (2) simultaneous addition of EGF and cyclic GMP stimulate phosphorylation to the same degree as the addition of EGF alone and (3) EGF stimulates phosphorylation in 20 min whereas the EGF induced increase in the cyclic GMP level takes 60 min.

Animals↗
PubMed1980

Epidermal cyclic AMP is not decreased in psoriasis lesions.

The cyclic AMP level in epidermis of psoriatic patients was reappraised with a highly sensitive radioimmunoassay method in conjunction with an improved skin biopsy technique to avoid any artificial rise of cyclic AMP due to ischemia. Local intradermal injection before biopsy was avoided, since even saline injection caused a clear-cut ischemia effect. The results concur with our previous study: i.e., on a tissue dry weight or protein basis, the cyclic AMP level in the involved epidermis is 20% higher than that in the uninvolved spidermis of psoriatic patients, and on a DNA basis, there was no significant difference. The cyclic AMP level in normal epidermis from non-psoriatic subjects is the same as that in the uninvolved epidermis of psoriatic patients. Such characteristics in psoriatic lesions as the increased mitosis, incomplete, differentiation and increased glycogen content cannot be simply related to a cyclic AMP deficiency.

Cyclic AMP↗
PubMed1979

Cyclic GMP System in epidermis: I. Effect of ischemia.

When keratome-sliced pig epidermis was floated on Hank's balanced salt solution, we observed a rapid decrease in the intracellular level of cyclic GMP. A portion of the lost cyclic GMP was detected in the incubation medium. When the epidermis was kept in air at room temperature, the cyclic GMP level also decreased rapidly but to a lesser degree. Incubating the epidermal slice at 37 degrees C in Hank's balanced salt solution with the addition of 3-isobutyl-1-methyl xanthine (IBMX) prevented the decrease. Also, after the cyclic GMP level had fallen, it could be raised to be the in vitro level by the addition of IBMX. Increased amounts of cyclic GMP were detectable in the medium in this case. These data indicate that the decrease in cyclic GMP in ischemic epidermis is due to sudden activation of epidermal cyclic GMP-phosphodiesterase and also in part due to leakage of cyclic GMP extracellularly. In contrast to the rapid decline in the cyclic GMP level, ischemia caused a rapid and transient increase in epidermal cyclic AMP. This confirms previous data by ourselves and by others (Br J Dermatol 92: 249-254, 1975; J Invest Dermatol 68:125-127, 1977). These "ischemic effects" must be avoided in order to measure the "in vivo level" of cyclic nucleotides in epidermis.

1-Methyl-3-isobutylxanthine↗
PubMed1979

A combined alkali extraction--ethidium bromide technique for the measurement of DNA in small pieces of tissue.

Alkaline solutions (0.1--0.5 N NaOH) at elevated temperatures can be used to extract DNA from small pieces of tissue. RNA is destroyed by the treatment. In tissues which have been previously exposed to tritiated thymidine, aliquots from the extracting solution can be used directly for the determination of DNA by ethidium bromide fluorescence and radioactivity in both DNA and the nucleotide pool.

Animals↗
PubMed1978

Epinephrine activation of pig skin adenylate cyclase in vivo and subsequent refractoriness to activation.

Epinephrine injected intradermally activated pig skin adenylate cyclase and increased the epidermal cyclic AMP level in vivo. This biphasic response reached a maximum in 5 min and gradually decreased thereafter. The simultaneous injection of a cyclic AMP phosphodiesterase inhibitor, isobutyl methyl xanthin (IBMX) potentiated the increase. The simultaneous injection of a specific beta-adrenergic receptor inhibitor, propranolol, inhibited this accumulation of cyclic AMP. After the first activation by epinephrine in vivo, there was a marked refractoriness of the skin (epidermal) adenylate cyclase to subsequent epinephrine stimulation vivo and in vitro. This refractoriness was specific for catecholamine stimulation as responses to histamine were normal. Recovery from refractoriness started at 48 hr and was completed at 1 week after the injection of epinephrine.

Adenylyl Cyclases↗
PubMed1978

Cyclic nucleotide-phosphodiesterase in the uninvolved and involved skin of psoriasis.

In the present study we have compared cyclic nucleotide-phosphodiesterase activities and affinity of phosphodiesterase for substrates (Km) in enzyme preparations obtained from the involved and uninvolved skin of psoriatic patients. With crude skin homogenates we consistently obtained two Km values (high and low) for both the involved and uninvolved, and both Km values were nearly identical between the involved and uninvolved. The same conclusion is also drawn from the Km determinations with partially purified preparations. Cyclic AMP-phosphodiesterase activities with crude homogenates showed no statistically significant differences between the involved and the uninvolved skin. However, when cyclic AMP- and cyclic GMP-phosphodiesterase activities were compared with a highly sensitive assay method in "pure" epidermal samples, which were microdissected free from stratum corneum, dermis and skin appendages, the involved skin contained 40% more activity of the low Km enzyme and 100% more of the high Km enzyme of both cyclic AMP- and cyclic GMP-phosphodiesterase. It is suggested that this may be due to a higher proportion of germinative cells in the lesional epidermis.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1978

Cyclic AMP accumulation in psoriatic skin: differential responses to histamine, AMP, and einephrine by the uninvolved and involved epidermis.

Using the uninvolved and involved skin from psoriatic patients, we investigated the effects of histamine and AMP (or adenosine) in vitro on the intracellular cyclic AMP levels. Both agents activated adenylate cyclase of the uninvolved and involved resulting in the accumulation of cyclic AMP. Without a cyclic nucleotide phosphodiesterase (PDE) inhibitor, these responses were biphasic and the maximal accumulation was observed in 5 min. With the PDE inhibitor both responses were markedly potentiated and high levels of cyclic AMP were observed for more than 20 min. The response to histamine by the involved skin was much greater than that by the uninvolved. The degree of the response to adenosine was approximately equal. In accordance with our previous work, the response to epinephrine by the involved skin was much less than that by the uninvolved. Thus adenylate cyclases of involved skin from psoriatic patients exhibit a markedly diminished response to epinephrine while at the same time exhibiting a markedly enhanced response to histamine. This precludes the possibility that the unresponsiveness to epinephrine can be due to a generalized inability of the epidermal psoriatic plaque cell to make a functioning cell membrane.

Adenosine Monophosphate↗