685
Biochimica et Biophysica Acta,444 (1976) 685--693 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 28049
ADENOSINE AND ADENINE NUCLEOTIDES STIMULATION OF SKIN (EPIDERMAL) ADENYLATE CYCLASE
HAJIME IIZUKA, KENIJI ADACHI, KENNETH M. HALPRIN and VICTOR LEVINE
Dermatology Service, Miami Veterans Administration Hospital and Department o f Dermatology, University of Miami School of Medicine, Miami, Fla. 33125 (U.S.A.) (Received April 12th, 1976)
Summary Adenosine, AMP, ADP and ATP activated adenylate cyclase in pig skin (epidermis) slices resulting in the accumulation of cyclic AMP. This effect was highly potentiated by the addition of the cyclic AMP-phosphodiesterase inhibitor, papaverine. But another inhibitor, theophylline, strongly blocked the activation of adenylate cyclase by adenosine and adenine nucleotides. Theophylline apparently competed with adenosine for the cell surface receptor. Like theophylline, the addition o f adenine alone caused no accumulation o f cyclic AMP, but it significantly inhibited the stimulatory effect of adenosine. Guanosine, or guanine, cytidine, uridine, or thymidine nucleotides had no effect on the accumulation of cyclic AMP. Among other adenine nucleotides we tested, adenosine 5'-monophosphoramidate, but n o t adenosine 5'-monosulfate, significantly increased cyclic AMP especially with the addition of papaverine. Neither 2'- nor 3'-adenylic acid were effective. Our data indicate that pig epidermis has four specific and independent adenylate cyclase systems for adenosine (and adenine nucleotides), histamine, epinephrine and prostaglandin E.
Introduction
Since Sattin and Rail [1 ] reported that adenosine and adenine nucleotides caused an increase in the cyclic AMP concentration of guinea-pig cerebral cortex slices, there have been several reports on the effect of these compounds [2--7]. Usually these are stimulatory effects for the accumulation of cyclic AMP, as in brain tissue slices [2], astrocytoma cells [3], neuroblastoma cells [4], isolated bone cells [5] and platelets [6]. On the other hand, it was reported that adenosine inhibits the stimulation of adenylate cyclase activity by norepinephrine in fat cell ghosts [7]. Also there are some tissues Hke thyroid and skeletal muscle [1] which were n o t sensitive to adenosine. Our results reported in this communication indicate that pig skin is another tissue where ex-
686
posure to adenosine and adenine nucleotides results in the accumulation of cyclic AMP. Also, evidence is presented which indicates that this activation by adenosine is independent of the three sites we have reported previously; i.e. adenylate cyclase of pig skin (epidermis) is activated by epinephrine [8], prostaglandin E [9] and histamine [10]. Materials and Methods
The basic experimental procedures are essentially the same as described previously [8]. Skin slices were taken from the backs of domestic pigs by a keratome (Storz Instruments, St. Louis, Mo.) adjusted to a 0.3 m m setting without anesthetics. The skin thus obtained was predominantly epidermis (80--90%) with a little dermal contamination. The slices were cut into 5 × 5 m m squares and preincubated in Hank's medium for 15 rain at 37aC to standardize the initial cyclic AMP level in the epidermal slices [8,11]. After the preincubation, epidermal slices were floated for 5 rain at 37°C in Hank's media containing various compounds to be tested. At least two squares were used for each condition. The cyclic A M P content in the skin squares were measured by Gilman's protein binding method [12] with minor modifications [11,13]. The binding assay was done in duplicate. Protein was measured by the method of Lowry et al. [14]. Chemicals and drugs were all prepared fresh before each experiment and the p H of the incubation media was adjusted to 7. Adenosine, AMP, ADP, A T P and adenine were obtained from Calbiochem (Los Angeles, Calif.),epinephrine was the product of Parke Davis (Detroit, Mich.). Metiamide was a kind gift from Dr. Brimblecombe, Smith Kline abd French Lab., England. All other chemicals were purchased from Sigma Chemical Co. (St. Louis, Mo.). Results
Effect of adenosine and adenine nucleotides As shown in Fig. 1, adenosine caused a marked increase in the intracellular content of cyclic AMP. The m a x i m u m increase was attained by 5 rain and then gradually decreased over the next 60 rain. The addition of papaverine greatly potentiated the effect of adenosine, and the cyclic A M P level was stillincreasing after 30 rain. Similar curves were obtained in response to A M P with and without papaverine. (Data not shown.) Table I shows the effect of adenosine and adenine nucleotides at 1 m M concentrations. Adenosine, AMP, A D P and A T P all significantly increased the intracellularconcentration, of cyclic AMP. The addition of these four compounds together had no additive effect on the accumulation of cyclic AMP; the fact suggests these compounds acted on the same site.Adenine had no effect. The effect of different concentrations of A M P is shown in Fig. 2. The increase in the intracellularcyclic A M P level was dose-dependent and was linear up to an A M P concentration of about 200 #M, then the rate of stimulation gradually decreased. The maximal effect was obtained at 1 raM. LineweaverBurk plots (the insert of Fig. 2) show the apparent Ka for 5'-AMP at about 160 #M. Similar results were obtained by the addition of various concentrations of
687 70 .E
•~, 30
-~ 50 E
~ 20,
20
0
~i0 c.I
|,,
0
10
20 .30 minutes
6O
lOxlO 3
01s
mM
I
20xlO3
l:o' "'
5'.0
Fig. I. Time course of the effect of adenosine on the intracellnlar c o n t e n t of cyclic AMP. Details of t he expe~nental a O ~ are l i v e n u n d e r Market/sis and Methods. The e o n e e n t m t i o n of ad4mo~ne ~and p a p a v e d n e ad ded were b o t h I 0 0 pM. o, adenos/ne only; X, c ont rol (no add/t/on); z~ adeno,~Ine + papavedne). Fig. 2. The effect of 5'-AMP coneentrath)n. Tissues were i nc uba t e d w i t h vmdous c m z e e n t ~ t t o n s o f 5'AMP for 5 rain. Nel ther t h e o p h y l l i n e n o r p a p a v e d n e was added. The insert shows L/neweave~Bu~k p l o t s of the c o n c e n t r a t i o n curve.
adenosine and the maximal effect was obtained at about 1 mM. (Data n o t shown).
Effects of papaverine and theophylline Papaverine potentiates the effect of adenosine as has been shown in Fig. 1. Table II shows further that this effect of papaverine is dose
Biochimica et Biophysica Acta,444 (1976) 685--693 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 28049
ADENOSINE AND ADENINE NUCLEOTIDES STIMULATION OF SKIN (EPIDERMAL) ADENYLATE CYCLASE
HAJIME IIZUKA, KENIJI ADACHI, KENNETH M. HALPRIN and VICTOR LEVINE
Dermatology Service, Miami Veterans Administration Hospital and Department o f Dermatology, University of Miami School of Medicine, Miami, Fla. 33125 (U.S.A.) (Received April 12th, 1976)
Summary Adenosine, AMP, ADP and ATP activated adenylate cyclase in pig skin (epidermis) slices resulting in the accumulation of cyclic AMP. This effect was highly potentiated by the addition of the cyclic AMP-phosphodiesterase inhibitor, papaverine. But another inhibitor, theophylline, strongly blocked the activation of adenylate cyclase by adenosine and adenine nucleotides. Theophylline apparently competed with adenosine for the cell surface receptor. Like theophylline, the addition o f adenine alone caused no accumulation o f cyclic AMP, but it significantly inhibited the stimulatory effect of adenosine. Guanosine, or guanine, cytidine, uridine, or thymidine nucleotides had no effect on the accumulation of cyclic AMP. Among other adenine nucleotides we tested, adenosine 5'-monophosphoramidate, but n o t adenosine 5'-monosulfate, significantly increased cyclic AMP especially with the addition of papaverine. Neither 2'- nor 3'-adenylic acid were effective. Our data indicate that pig epidermis has four specific and independent adenylate cyclase systems for adenosine (and adenine nucleotides), histamine, epinephrine and prostaglandin E.
Introduction
Since Sattin and Rail [1 ] reported that adenosine and adenine nucleotides caused an increase in the cyclic AMP concentration of guinea-pig cerebral cortex slices, there have been several reports on the effect of these compounds [2--7]. Usually these are stimulatory effects for the accumulation of cyclic AMP, as in brain tissue slices [2], astrocytoma cells [3], neuroblastoma cells [4], isolated bone cells [5] and platelets [6]. On the other hand, it was reported that adenosine inhibits the stimulation of adenylate cyclase activity by norepinephrine in fat cell ghosts [7]. Also there are some tissues Hke thyroid and skeletal muscle [1] which were n o t sensitive to adenosine. Our results reported in this communication indicate that pig skin is another tissue where ex-
686
posure to adenosine and adenine nucleotides results in the accumulation of cyclic AMP. Also, evidence is presented which indicates that this activation by adenosine is independent of the three sites we have reported previously; i.e. adenylate cyclase of pig skin (epidermis) is activated by epinephrine [8], prostaglandin E [9] and histamine [10]. Materials and Methods
The basic experimental procedures are essentially the same as described previously [8]. Skin slices were taken from the backs of domestic pigs by a keratome (Storz Instruments, St. Louis, Mo.) adjusted to a 0.3 m m setting without anesthetics. The skin thus obtained was predominantly epidermis (80--90%) with a little dermal contamination. The slices were cut into 5 × 5 m m squares and preincubated in Hank's medium for 15 rain at 37aC to standardize the initial cyclic AMP level in the epidermal slices [8,11]. After the preincubation, epidermal slices were floated for 5 rain at 37°C in Hank's media containing various compounds to be tested. At least two squares were used for each condition. The cyclic A M P content in the skin squares were measured by Gilman's protein binding method [12] with minor modifications [11,13]. The binding assay was done in duplicate. Protein was measured by the method of Lowry et al. [14]. Chemicals and drugs were all prepared fresh before each experiment and the p H of the incubation media was adjusted to 7. Adenosine, AMP, ADP, A T P and adenine were obtained from Calbiochem (Los Angeles, Calif.),epinephrine was the product of Parke Davis (Detroit, Mich.). Metiamide was a kind gift from Dr. Brimblecombe, Smith Kline abd French Lab., England. All other chemicals were purchased from Sigma Chemical Co. (St. Louis, Mo.). Results
Effect of adenosine and adenine nucleotides As shown in Fig. 1, adenosine caused a marked increase in the intracellular content of cyclic AMP. The m a x i m u m increase was attained by 5 rain and then gradually decreased over the next 60 rain. The addition of papaverine greatly potentiated the effect of adenosine, and the cyclic A M P level was stillincreasing after 30 rain. Similar curves were obtained in response to A M P with and without papaverine. (Data not shown.) Table I shows the effect of adenosine and adenine nucleotides at 1 m M concentrations. Adenosine, AMP, A D P and A T P all significantly increased the intracellularconcentration, of cyclic AMP. The addition of these four compounds together had no additive effect on the accumulation of cyclic AMP; the fact suggests these compounds acted on the same site.Adenine had no effect. The effect of different concentrations of A M P is shown in Fig. 2. The increase in the intracellularcyclic A M P level was dose-dependent and was linear up to an A M P concentration of about 200 #M, then the rate of stimulation gradually decreased. The maximal effect was obtained at 1 raM. LineweaverBurk plots (the insert of Fig. 2) show the apparent Ka for 5'-AMP at about 160 #M. Similar results were obtained by the addition of various concentrations of
687 70 .E
•~, 30
-~ 50 E
~ 20,
20
0
~i0 c.I
|,,
0
10
20 .30 minutes
6O
lOxlO 3
01s
mM
I
20xlO3
l:o' "'
5'.0
Fig. I. Time course of the effect of adenosine on the intracellnlar c o n t e n t of cyclic AMP. Details of t he expe~nental a O ~ are l i v e n u n d e r Market/sis and Methods. The e o n e e n t m t i o n of ad4mo~ne ~and p a p a v e d n e ad ded were b o t h I 0 0 pM. o, adenos/ne only; X, c ont rol (no add/t/on); z~ adeno,~Ine + papavedne). Fig. 2. The effect of 5'-AMP coneentrath)n. Tissues were i nc uba t e d w i t h vmdous c m z e e n t ~ t t o n s o f 5'AMP for 5 rain. Nel ther t h e o p h y l l i n e n o r p a p a v e d n e was added. The insert shows L/neweave~Bu~k p l o t s of the c o n c e n t r a t i o n curve.
adenosine and the maximal effect was obtained at about 1 mM. (Data n o t shown).
Effects of papaverine and theophylline Papaverine potentiates the effect of adenosine as has been shown in Fig. 1. Table II shows further that this effect of papaverine is dose
