AMERICAN

Vol.

JOURNAL

OF

228, No. 6, June

PHYSXOLOGY

1975.

Mechanism

Printed

in U.S.A.

of methylxanthine

of norepinephrine STANLEY

sensitization

responses

KALSNER,

L)ejmrtment of ~‘harmaco~agy, KALSNER, STANLEY, ROBERT r). $hr-rrr . Mechanism of methylxunthine responses in a coronary artery. Am. J

in a coronary

ROI3ERT D. FREW, AND GREGORY Facdty of ~edhw, University of Ottawa,

FREW, AND GREGORY M. sensitization of norepinephrine

Physiol. 228(6) : 17024707. 1975.-Beta-adrenergic receptor-mediated relaxation to norepinephrine was enhanced by caffeine and aminophylline in a coronary artery preparation of the beef in vitro. Augmented responses were not obtainable in the presence of knojvn inhibitors of the extraneuronal uptake and nzetabolisrn of norepinephrine, estradiol-17p, and the haloalkylamine G-D- 13 I, which themselves potentiate responses. In addition, the effect on the norepinephrine dose-response curve of the combination of a methyixanthine and U-0521, the latter a potent inhibitor of catechol O-methyltransferase, the major enzyme of catecholamine inactivation in vascular tissue, did not differ from that of L-0521 alone. Studies of the extraneuronal accumulation of 3H-labeled norepinephrine revealed that caffeine and aminophylline, along with the known inhibitors, materially reduced the accumulation of label in coroIt is concluded that the methylxanthines enhance nary tissue. beta-adrenergic receptor-mediated responses via a blockade of catecholamine uptake, giving rise to an increased concentration of agonist at receptors, and not by an action linked to cyclic AMP accumulation, consequent to receptor activation.

adrenergic extraneuronal

n~echanisms; coronary

artery

responses;

sensitization;

uptake

THE METHYLATED XANTHXNES enhance the alpha-adrenergic receptor-mediated contractile responses of vascular smooth muscle to norepinephrine and epinephrine by inhibiting the extraneuronal inactivation of these amines (ZO), rather than by an action linked to cyclic AMP metabolism as was earlier proposed (3). Much work now suggests that an elevation of cyclic AMP levels is rnore probably associated with beta-adrenergic receptor-mediated events, which are also enhanced by the methylxanthines in a variety of test preparations (10, 29, 30, 38). The methylxanthines inhibit phosphodiesterase and the resultant accumulation of cyclic AMP is generally invoked to explain the sensitization. This interpretation, in turn, has provided major support for the hypothesis linking beta-adrenergic receptors to the adenyl cyclase system (3 1, 37). It was reported recently that the action of norepinephrine on the beta receptors of the coronary arteries is terminated by extraneuronal uptake and metabolism and that inhibitors of uptake sensitize responses (22). In the present investigation it: was found that the methylxanthines enhance responses of coronary artery strips to norepinephrine and the possibility was explored that this is related to an

artery

M. SMITH Ottawa, Canada

increased agonist concentration at receptors, due to inhibition of catecholamine inactivation, rather than to an elevated level of cyclic AMP mediating enhanced betareceptor effects. METHODS

Hearts were removed immediately after slaughter of the beef of either sex and immersed in oxygenated Krebs solution and transported to the laboratory. The total time taken was approximately 20 min. The left circumflex coronary artery was dissected out, cleaned of visible fat and adherent tissue, and cut into spiral strips of about 23 x 2.5 mm. Both left- and right-hand spirals were cut with no detectable difference in any parameter of response performance. The strips were suspended under 2 g tension in 15-ml muscle chambers at 37°C containing KrebsHenseleit solution of the following composition (in mM) : NaCl, 115.3; KCI, 4.6; CaCls, 1.8; MgSOd, 1.1; SaHCOs, 22.1; KHZP04, 1. I ; and glucose, 7.8. To this medium disodium ethylenediaminetetraacetate (EDTA) (0.03 mM) was added to retard heavy-metal-catalyzed oxidation of catecholamines. A 60-min period was allowed for equilibration before eliciting drug responses. Isotonic contractions were recorded by means of frontal writing levers on a slowly moving kymograph drum (about 1.8 mm/min) with a lever magnification of 6.8-fold. All drug concentrations are expressed in terms of molarity. Sodium nitrite, caffeine, and aminophylline (theophylline ethylenediamine) were dissolved to the appropriate concentration in distilled, demineralized water. I-Norepinephrine was diluted to the desired concentration in NaCl containing 0.0 1 N HCl. GD- 13 1 (h’-cyclohexylmethyl-LVethyl-fl-chloroethylamine; Smith Kline 8r. French Laboratories) was dissolved on the day of use in acidified (0.01 N HCl) saline at a concentration of 4.2 mM. Estradiol-17/3 was prepared in propylene glycol to give a stock concentration of 37 n&I. The volume of propylene glycol used (0.015 ml) had no effect on the basal tone of strips or on responses to norepinephrine or potassium (22). Catechol O-methyltransferase was inhibited with U-052 1 (3’,4’-dihydroxy-2methyl propiophenone, 56 PM) (12, 18). Strips were used to obtain only one dose-response curve to a given agonist and then discarded. &7-3H]norepinephrine (6.9 mCi/pmol) (New England Nuclear Corp.) was diluted to a stock concentration of 6.9 uCi/ml (1 PM) in ascorbic acid (50 pg/ml) and stored frozen in 1-1~1 aliquots under nitrogen gas. Aliquots were

1702

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METHYLXANTHINES

AND

BETA-14DRENERGIC

1703

RESPONSES

thawed only once, immediately prior to use. Strips of circumflex coronary artery were prepared as described above and preincubated individually at 37°C for 30 min in test tubes containing 10 ml Krebs solution with disodium EDTA (0.03 mM) added to retard oxidation. Cocaine (29 PM) was routinely included in the Krebs solution to block neuronal uptake in experiments with “H-labeled norepinephrine. Aeration with 95 YC 02-5 ?C CO2 was maintained throughout. The strips were then incubated with [YH]norepinephrine at a final concentration of 15 nM for 10 min. Known inhibitors of extraneuronal uptake or the methylxanthi nes, when used, were added 15 min prior to incubation with the tritiated norepincphrine except in the case of GD- 13 1, where exposure was for 15 ruin, beginning 30 min prior to addition of the tritium, followed by a I5-min period with frequent washes. After incubation with [3H]norepinephrinc for 10 min, the strips were blotted gently, weighed, chopped, and placed in scintillation vials containing 2 ml of solubilizcr (Protosol, N ew England Nuclear) and 0.2 ml of distilled water to promote dissolution of the tissue. The vials were then maintained overnight in a water bath at 50°C to speed solubilization. Fifteen milliliters of scintillation solution (4 g of PPO and 50 mg of POPOP per liter of toluene) were added to each vial and the samples were counted to a 2 % error in a Beckman LS-150 system with autolnatic external standardization to determine efficiency. Uptake of radioactivity is expressed as disintegrations per minute per gram of tissue after correction for extracellular space, assumed to be 0.35 ml/g tissue (36). The concentration of norepinephrine in the bathing medium was confirmed for eachA experiment by counting the radioactivity in 0. l-ml aliquots. The tissue-to-medium ratio was calculated as disintegrations per minute per gram of tissue (without extracellular space correction) divided by the disintegrations per minute per milliliter of bathing medium. ;h\/Tean values of all data are shown with their standard errors. DiiYerences with 1’ values of 0.05 or less wcrc considered significant. Changes in response sensitivity at the mean effective dose (EDso) are expressed as the ratio of geometric mean values (11). Each EDso was converted to its log and the mean for each group was recorded. The antilog of each mean log is presented as the geometric mean. The 95 % confidence limits were calculated for the mean logs and also converted to antilogs.

to obtaining inhibitory responses to agonists in coronary artery strips has been previously described for the dog mean cumulative doseby Baron et al. (2). Th e control response curve to norepinephrine, obtained in the beef, is presented in Fig. 1. These responses are blocked completely in strips pretreated with propranolol (l-10 PM), conirming mediation by beta-adrenergic receptors. To examine the eflect of the methylxanthines on the curve, strips taken from the norepinephrine dose-response same coronary vessels as the controls were cant ratted bY potassium and then exposed to aminophylline or caffeine. washout of the muscle Beginning 15 min later, without chambers, the responses to cumulative additions of norepinephrine (0.6 nM to 59 PM; 1 X lO-xo to 1 X lOA g/ml) were determined. The concentrations of caffeine (0.5 mM; 1 x lo-4 g/ml) and aminophyllinc (79 PM; 3 X lo-” g/ml) ch osen for these experiments were those materially inhibiting the pothat could be used without tassium response ar llplitude. However, in some strips, a direct depressant effect on potassium-induced tone was observed, but this only occasionally exceeded 5 % of the total contraction amplitude, and a plateau level of response was always reestablished prior to norepinephrine administration. The methylxanthines lowered the threshold concentration for response and shifted the dose-response curve for norepinephrinc significantly to the left (Fig. 1). Aminophylline and ca.Reine produced approximately equivalent sensitizations at the concentrations employed. The ratio of geometric mean EDso values (control/treated) was 2.7 and 2.2, respectively (Table I)* The maximal inhibitory response to norepinephrine, which was 38.7 =t 2.1 mm

$ F 8C m I Z-

.r’

6C

RESULTS

EJects of methylxanthines on mponses to nost;tu+m~J~rine. The circumflex coronary artery of the beef has a negligible population of alpha-adrenergic receptors and responds to norepinephrine with beta-receptor-mediated relaxation (22). This is in agreement with the results of Bohr (7) with coronary artery preparations from several other species. To obtain inhibitory dose-response curves to norepinephrine a predictable and reproducible degree of tone must first be induced in the preparation. This was done in the present experiments by constricting the artery with potassium (usually 50 MM); after a steady plateau level of response was achieved, the preparation was exposed to progressively increasing concentrations of norepinephrine, without washout of the muscle chamber. This approach

20

0 I

ho

NOREPINEhiRINE

I 5

(M)

I

4

PIG. 1. Effects of methylxanthines, estradiol-17@, and their combillation on the c~m~~rlative dose-response cl. rt-e to norepinephrine. con trol (m), aminophyllinc (79 PM) (A), caffeine (0.5 mAI) (o), estradiol-17p (:* To provide further information on a possible association between the sensitization produced by the methylxanthines and a blockade of norepinephrine metabolism, experiments were done with GD-13 1, a haloalkylamine compound that blocks extraneuronal uptake persistently (24)-a property now known to be shared by a number of haloalkylamines (17). Strips were pretreated with the haloalkylamine (13 or 42 PM; 3 X 10mF or 1 X low5 g/ml) for 15 min, followed by an equivalent period with frequent washes of the muscle chambers, and then were contracted by potassium and exposed to norepinephrine. Some of these strips were additionally treated with caffeine (0.5 mM) for 15 min prior to norepinephrine administration. In agreement with a previous report GD- 13 1 sensitized responses to the catecholamine (ZZ), a shift of 5.3-fold at the EDso level, but the further addition of caffeine did not materially increase the magnitude of the sensitization (Fig. 2). There were no TABLE 1. EJects of methylxanthinas and extraneuronnl uptake inhibitors on norepinephrine responses -----_-No. of

Treatment

Strips

-----

-

Geometric

Mean

KDKo”

(95yo Confidence Intervals) X 10-T 31

Rstio of ED 6&t

----

Control Aminophyfline Caffeine

37 29 18

10.6 3.9 4.8

(9.8-12.7) (3.0-5.0) (3.4-6.9)

2.7 2.2

< .OOl < .OOl

U-052 Uiyiel

19 11

4.1 3.1

(3.3-5.1) (2.1-4.4)

2.6 1.3t

< -001 > .4$

17 14

2.3 1.9

(1.8-2.8) (1.2-3.0)

4.6 1.2$

< ,001 >0.4$

I .7 (1+2-2.4)

I.41

>0.4$

2.0 1.4

5.3 1.41

< ,001 >0.2$

I + aminophyl-

Estradiol-17p Estradiol-170 aminophylline Estradiol-17P feine 1

+ -j- caf-

8

.

GD-133 GD-131

+

caffeine

17 17

(1.3-3.0) (0.8-2.3)

* Mean of concentrations producing 50% of maximal response to norepinephrine for each strip. t Comparison made with untreated control group. $ Comparison made with corresponding group without methylxanthine treatment.

5

FREW,

AND

SMITH

80

I

8

I

6

7 - LOG/()

NOl3EPINEPHRINE

(Ml

5

I

4

FIG. 2. Effects of GD-13 1 and of GD-13 1 plus caffeine on doseresponse curve to norepinephrine. Control (m), Go-131 (13 or 42 PM) (o), and GD-131 plus caffeine (0.5 mM) (a). Number of values are 37, 17, and 17, respectively. Population of controls same as in Fig. 1. Means + SE are shown wherever they do not reduce clarity.

statistically significant differences between the d ose-response curves of strips pretreated with GD- 13 1 alone or GD- 13 1 plus caffeine except at one concentration (18 nM). In addition, the geometric mean ED& of these two groups did not differ significantly (Table 1). The major enzymatic pathway for norepinephrine, once it is taken up extraneuronally in vascular tissue, is O-methylation (18, 23, 24), and experiments were done with a known inhibitor of catechol O-methyltransferase to substantiate further the data appearing to relate the sensitizing effects of the methylxanthines to inhibition of norepinephrine metabolism. The inhibitory dose-response curve to norepinephrine was shifted significantly to the left (2.6-fold at EDjo) by treatment with U-0521 (56 PM; 1 X 10V5 g/ml), administered after contractions to potassium had plateaued, but the responses were not magnified significantly at any point along the curve by additional treatment of strips with aminophylline (79 PM) (Fig. 3). Also, the geometric mean ED 50 values of these groups - did not differ significantly (Table 1). The speciiicity of the enhancing effects of the methylxanthines on adrenergic responses was confirmed on strips contracted by potassium and exposed to cumulatively increasing concentrations of sodium nitrite. As shown in Fig* 4, neither caffeine (0.5 mM), nor aminophylline (79 PM) altered significantly the dose-response curve to the nitrite. It was reported previously that GD-131 and estradioLl7p do not modify detectably the inhibitory responses to sodium nitrite (22). Efects of methylxanthines and known inhibitors on exfraneuronal uptake. Direct evidence for the extraneuronal uptake of norepinephrine and i ts inhibition by methylxanthines,

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METHYLXANTHINES

AND

BETA-ADRENERGIC

1705

RESPONSES

150 E 01 l-. m 0 P x E a -a

100

5 * m

50

0 3.7 Con tro I

I 7

8

- LOG ,.

I 5

f 6 NOREPINEPHRINE

FIG. 3. Effects of U-0521 and of dose-response curve to norepinephrine. PM) (A), and U-0521 plus aminophylline values are 37, 19, and 11 1 respectively. as in Fig. 1. Means =tr SE are shown clarity.

I 4

(Ml

U-0521

plus aminophylline on Control (m), U-0521 (56 (79 PM) (A). Number of Population of controls same wherever they do not reduce

IOO-

17P-Estrad

4.2 iol

GD-131

7.9

26

79

Aminophylline

51 150

X10-5M

Caffeine

FIG. 5. Effects of methylxanthines, estradiol-17p, and GD-131 on extraneuronal uptake of [3H]norepinephrine in coronary artery strips. Means + SE are presented. P values (two-tailed) are presented above bars and number of values within bars. Strips were exposed to pH]norepinephrine (15 nM), in presence and absence of inhibitors, and accumulation of radioactivity after a lo-min incubation period is expressed after correction for extracellular space. Mean tissueto-medium ratio in 21 control strips was 1.06. Details in text.

5; GD-13 1 and estradiol-l7p significantly inhibited the uptake of norepinephrine, by 42.1 % and 30.0 %, respectively, and it was also reduced by aminophylline and caffeine over a range of concentrations tested. DISCUSSION

2 2 X

60-

0

/

/fl’ L, cl-E--p I 6 5

1

4

-LOGlO

NaN02

I

1

3

2

(Ml

Effects of caffeine and aminophylline on cumulative dosecurves to sodium nitrite. Control (m), caffeine (0.5 mM) (0), and aminophylline (79 PM) (n). Number of values are 9, 4, and 7, respectively. Means =t SE are shown wherever they do not reduce clarity. FIG.

4.

response

in the coronary vessels, was obtained by incubating strips with [3H]norepinephrine (15 nM) in the presence of cocaine (29 PM; 1 X 10m5 g/ml) to block neuronal uptake. The effects on extraneuronal uptake of known inhibitors and of the methylxanthines are presented in Fig.

It has been proposed by several groups of workers that the beta-adrenergic receptor-induced relaxation of smooth muscle is mediated by activation of adenyl cyclase with a resultant accumulation of cyclic AMP (1, 8, 10, 27, 37). This nucleotide is presumed to act as a second messenger linking catecholamine-receptor interactions to a subsequent step in the contractile process. Major evidence in support of the hypothesis that, ‘