View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector THE JOURNAL OP INVESTIGATIVE DERMATOLOGY Vol. 50, No. 6 Copyright 1968 by The Williams & Wilkins Co. Printed in U.S.A. GLYCOLYTIC ENZYME PROFILES IN THE ARRECTOR PILl MUSCLE AND THE SUBCUTANEOUS MUSCLE COMPARISON BETWEEN SMOOTH AND SKELETAL MUSCLE* MICHAEL J. C. TM, M.S., hiD. AND KENJI ADACHI, MD., Pn.D. Although modern biochemical techniquesfor possible dermal contamination. The purity of have done much to elucidate various metabolicthese samples with respect to muscle structures is more than 95%. The dissected arrector pili and processes in epidermis, data on the metabolismsubcutaneous muscles, averaging 0.5 to 1.0 g, of skin appendages are scanty and limited. Inwere weighed on a "fish pole balance" (1) and particular, muscle structures in skin have neversubsequently used for assay. The enzyme activities been subjected to metabolic study. This pau-assayed in these muscle structures are phos- city of information is largely due to the diffi-phorylase (microadaptation of Ref. 3), hexokinase (4), phosphofructokinase (5), aldolase (6), glycer- culty of obtaining enough material for standardaldehyde-3-phosphate dehydrogenase (7), enolase biochemical analysis. Dissection of the arrector(8), pyruvate kinase (9), lactate dehydrogenase pili muscle, in particular, from unstained fresh(10), glucose-6-phosphate and 6-phosphogluconate dehydrogenases (11), isocitrate dehydrogenase (12), skin is nearly impossible and the prolongedfumarase (12), and adenosine triphosphatase (the dissection procedure lowers the metabolic ca-reagent mixture was the same as described in Ref. pacity of the tissue. In this study, the tech-13, but the product adenosine diphosphate was nical problem was solved by using an adapta-measured fluorometrically as described in Ref. 14). tion of Lowry's method (1, 2), which permits enzyme assay of minute samples (microgram RESULTS quantities) that can be accurately dissected Table I summarizes the levels of glycolytic from frozen-dried sections of the original tissueenzyme activities in the arrector pili and sub- without losing glycolytic enzyme activities. cutaneous muscles as compared with those The present short communication dealsin the epidermis. Little difference in enzyme with profiles of enzyme activities related toactivities, in general, was observed between energy metabolism in both the arrector pilinrrector pili muscle and epidermiS. On the and the subcutaneous muscles. It is hoped thatother hand, there were marked differences these results will substantiate, at least par-between the enzyme activities of the arrector tially, the comparison between smooth andpili and those of subcutaneous muscle. The skeletal muscle metabolism. phosphorylase activity in aubcutaneous muscle was about 25 times higher than in the arrector MATERIALS AND METHODS pili muscle, whereas in the subcutaneous muscle Fresh biopsy specimens were obtained withouthexokinase activity was lower than in the ar- anesthesia from the middle back region of 1½ year old rhesus monkeys, and the frozen-driedrector muscle. Glycolytic enzymes are gen- sections were prepared as described previously (1). erally 1.2 to 2.7 times more active in the From these sections accurate tissue sampling wassubcutaneous muscle than in the arrector pili easily done by free-hand dissection under themuscle. One of the exceptions was lactate de- microscope. All arrector pili muscle samples werehydrogenase activities which were similar in obtained from those attached to terminal anagen V or VI hair follicles (Figs. 1 & 2). Several dis-the two muscle structures and the epidermis. sected tissue samples were stained and examinedNo attempt was made to examine the possible alteration in the isozyme patterns. Publication No. 308 from the Oregon Regional Primate Research Center. Characteristic differences between smooth Supported in part by Grant FR 00163 of theand skeletal muscles were also encountered in National Institutes of Health and by funds from U.S. Pubhc Health Service Grant Nos. AM 08445the levels of the two dehydrogenases partici- and AM 5512. pating in the pentose phosphate cycle. Both Accepted for publication November 30, 1967. glucose-6-phosphate and 6-phosphogluconate de- * From the Department of Cutaneous Biology, Oregon Regional Primate Research Center, Bea-hydrogenase activities were 4—6 times higher verton, Oregon 97005. in the arrector pili muscle than in the sub- 429 430 THEJOURNAL OF INVESTIGATIVE DERMATOLOGY 4., t r - 0/ -4 FIG. 1. Frozen-dried preparation of skin sample. The arrector muscle is easily identified (see arrow). FIG. 2. The dissected arrectores pilorum muscles ENZYMES IN AREECTOR PILl MUSCLE 431 cutaneous muscle. These activities were still TABLE II higher in the epidermis. AdenosineTriphosphatase (A TPase) A cli vilzes* in Table II summarizes adenosine triphospha- Arrector Pill Muscle and Subcutaneous Muscle tase (ATPase) activities in the arrector pili Arcector pili Subcutaneous and the subcutaneous muscles. The ATPase Activator or inhibitor muscle muscle activities in both muscles arc activated by Mg and inhibited by the simultaneous addi-Mg (5mM) 1.0 .482.8 1.3 tion of Na and Kt It must be stated thatNoMg .55 .061.6 .30 under our assay condition, both muscle tissueMg (5 mM) 1 Na(80mM)plus .64 .02 1.0 .51 may contain an endogenous amount of these K (40 mM)) ions. Therefore the actual rates of inhibition or activation may be greater than the values * Allvalues are expressed as moles of ATP presented in this Table. converted to ADP per kg dry wt tissue per hour standard deviation. DISCUSSION Perusal of the available literature indicatesskin may be a suitable organ since it contains that a comparative study of the energy me-skeletal muscle (panniculus camnosus) imme- tabolism between the smooth and skeletal mus-diately below the panniculus layer and smooth cle is scanty. In spite of abundant data on themuscle (arrcctores pilorum) in the corium. skeletal muscle, metabolic studies on smooth Data on the enzyme activities in these two muscle have been confined to the carbohydratemuscle structures provoke some speculation on metabolism of the uterus (15, 16). For com-the different metabolic capacities of smooth parison of smooth and skeletal muscle metab-and skeletal muscles. One such difference is the olism, the muscle samples should be obtainedniarkedly different levels of both glucose-6- from the same organ. Accordingly, monkeyphosphate and 6-phosphogluconate dehydro- TABLE I Enzyme Aclivities* in Arrector Pill Muscle, Subcutaneous Muscle and Epidermis of Rhesus Monkey Skin Enzymes" Arrector pili muscle Subcutaneous muscle Epidermist Phosphorylase .03 .001 .73 .16 .03 .01 Hexokinase .42 .18 .17 .07 .52 .05 Phosphofructokinase 3.1 .44 7.8 .78 4.2 .48 Aldolase 1.1 .14 7.8 2.3 .42 .17 G-3-PDH 0.2 1.6 19.6 3.8 7.0 .88 Enolase 9.2 .48 25.0 6.5 13.2 1.1 Pyruvate kinase 13.4 3.0 22.2 2.5 14.3 1.7 LDH 15.6 2.3 17.4 2.3 14.5 8.8 G-6-PDH .26 .07 .04 .01 .61 .25 6-PGDH .15 .03 .04 .01 .38 .18 ICDH .61 .09 2.5 .54 .93 .23 Fumarase 2.8 .54 2.8 .57 3.1 .91 * Activitiesare expressed as moles/kg dry wt. tissue/hrstandard deviation. Each figure represents an average enzyme activity from 3 separate animals. Quintuplicate tubes were run for each enzyme assay. **Theabbreviations used are G-3-PDH =glyceraldehyde-3-phosphatedehydrogenase; LDII = lactatedehydrogenase; G-6-PDH =glucose-6-phosphatedehydrogenase; 6-PGDH =6-phosphoglu- conate dehydrogenase; ICDH =isocitratedehydrogenase. tDataon the enzyme activities in epidermis are taken from previous reports (see References 4—11; data on phosphorylase, isocitrate dehydrogenase and fumarase activities are unpublished ones) and are based on the activities in scalp epidermis. 432 THEJOURNAL OF TNVESTIGATIVE DERMATOLOGY genases in the two different muscle structures.adenosine triphoapbatase activities were assayed In spite of certain limitations and assumptionsin both arrector pili and subcutaneous mus- in the use of C-i and C-6 labeled glucose forcles in young rhesus monkey skin. While the the evaluation of the pentose cycle contribu-profiles of the enzyme activities in the arrector tion (17, 18), the literature affirms that normalpili muscle differ considerably from those in the skeletal muscle (in a physiological condition)subcutaneous muscle, both muscles possess a has an insignificant pentose cycle (19), whereascommon enzymatic feature for their physiolog- the smooth muscle (uterus) shows an appre-ical role—"contraction"——i.e. high adenosine ciable one (15). Therefore, our data suggesttriphosphatase activities which are activated by the active participation of this alternative path-Mgtt and inhibited by the simultaneous addi- way of glucose catabolism in the arrector mus-tion of Nat and Kt cle and practically no participation of it in subcutaneous muscle. REFERENCES No further speculation on the different levels 1. Lowry, 0. II.: Quantitative histochemistry of of other carbohydrate enzymes will be dis- brain. Histological sampling. J. Histochem. Cytochem., 1:420,1953. cussed here since there are many alternatives. 2. Lowry, 0. H., Roberts, N. R., Wu, M. L., An over-interpretation of data can be erro- Hixon, W. S. and Crawford, E. J.: The neous, and the steady state measurements and quantitative histochemistry of brain. II. Enzyme measurements. J. Biol. Chem., 207: considerations of allosteric relationships are re- 19,1954. quired to yield the most probable speculation. 3. Halprin, K. M. and Ohkawara, A.: Glucose Present methods are, unfortunately, not sen- and glycogen metabolism in the human epidermis. J. Invest. Derm., 46: 43, 1966. sitive enough to measure the intermediate 4. Adachi, K. and Yamasawa, S.: Quantitative substrate levels. Further studies on the over-all histochemistry of the primate skin. I. Hexo- kinase. J. Invest. Derm., 46: 473, 1966. metabolic pathways of these muscles are also 5. Adachi, K. and Yamasawa, S.: Quantitative necessary to draw a final conclusion although bistochemistry of the primate skin. X. Phos- in order to carry them out a number of tech- phoglucoisomerase, phospbofructokinase and triose isomerase.