Glycogen Synthetase [Udp Glucose: α-1, 4-Glucan α-4-Glucosyl Transferase (Ec 2.4.1.11)] of Human Epidermis

Normal human epidermis contains glycogen synthetase in activities which are approximately 10% of that found in brain and muscle. The enzyme is activated by glucose-6-phosphate (Glc-6-P) and seems to have characteristics which are similar to those reported for the same enzyme in other tissues. About 50% of the enzyme is normally in the I form which presumably correlates with the very low levels of glycogen (0.35 µg/mg) normally present in human epidermis

Cyclic Amp Accumulation in Psoriatic Skin: Differential Responses to Histamine, Amp, and Epinephrine 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 5min. With the PDE inhibitor both responses were markedly potentiated and high levels of cyclic AMP were observed for more than 20mm. 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

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 × 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 F2α. (PGF2α) 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 mechanism

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 [γ-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 ∼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, winch 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 11. 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 dissociation 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