European Journal of Pharmacology, 48 (1978) 393--401

393

© Elsevier/North-Holland Biomedical Press

S T E R E O S E L E C T I V E I N T E R A C T I O N O F T E T R A H Y D R O I S O Q U I N O L I N E S IN fi-ADRENOCEPTOR SYSTEMS MICHAEL T. PIASCIK, PETER OSEI-GYIMAH, DUANE D. MILLER and DENNIS R. FELLER * Divisions of Pharmacology and Medicinal Chemistry, College of Pharmacy, The Ohio State University, Columbus, Ohio, U.S.A.

Received 12 July 1977, revised MS received 28 December 1977, accepted 2 January 1978

M.T. PIASCIK, P. OSEI-GYIMAH, D.D. MILLER and D.R. FELLER, Stereoselective interaction of tetrahydroisoquinolines in ~-adrenoceptor systems, European J. Pharmacol. 48 (1978) 393--401. In selected ~1- (heart, lipolysis) and ~2-adrenoceptor (trachea) systems, the interaction of racemic-trimetoquinol (TMQ) and the erythro- and threo-diastereomers of 1-(3',4r,5t-trimethoxy-a-hydroxybenzyl)-6,7-dihydroxy-l,2,3,4-tetrahydroisoquinoline (a-hydroxy TMQ) was investigated. Each tetrahydroisoquinoline possessed agonist activity in these t3-adrenoceptor systems. The rank order of potency observed for these compounds was racemic-TMQ > erythro-a-hydroxy TMQ > threo-a-hydroxy TMQ. Using isolated fat adipocytes, a favorable correlation was observed between the elevation in c-AMP and pharmacological response for the TMQ stereoisomers and diastereomers of a-hydroxy TMQ. The rise in intracellular c-AMP produced by (--)- and (+)-TMQ in fat cells was blocked by the presence of propranolol, and not in the presence of phentolamine. Since considerably higher concentrations (> 10 -~ M) of these compounds were required to produce a significant inhibition of c-AMP phosphodiesterase activity in adipose tissue, it is proposed that the lipolytic response is a result of stereoselective interaction of these tetrahydroisoquinolines at the level of membrane-bound adenylate cyclase. Trimetoquinol

a-Hydroxytrimetoquinol

Stereoisomers

1. I n t r o d u c t i o n T r i m e t o q u i n o l [TMQ; 6 , 7 - d i h y d r o x y - l - f l 3 ' , 4',5'-trimethoxybenzyl)-1,2,3,4-tetrahydroisoq u i n o l i n e ] has been s h o w n t o be a p o t e n t f i - a d r e n o c e p t o r agonist (Iwasawa and Kiyom o t o , 1 9 6 7 ) useful in the t r e a t m e n t o f bronchial a s t h m a and o t h e r r e s p i r a t o r y disorders. T h e i n t e r a c t i o n o f TMQ with 13-adrenoceptor systems is stereoselective (Feller e t al., 1 9 7 5 ; Lee et al., 1 9 7 4 ) , with the S(--)-isomer possessing m u c h o f the p h a r m a c o l o g i c a l activity. A variety o f t e t r a h y d r o i s o q u i n o l i n e s (THI's) s t r u c t u r a l l y related t o TMQ have been prepared and s h o w n to possess activity in several ~-

* Address for correspondence: D.R.F., College of Pharmacy, 500 W. 12th Ave., Columbus, Ohio 43210, U.S.A.

Diastereomers

J3-Adrenoceptors

cAMP

systems (Miller et al., 1975a, 1 9 7 5 b ) . T h e data o b t a i n e d f r o m these s t r u c t u r e activity studies reveal t h a t t h e p a r e n t c o m p o u n d , S(--)-TMQ, is the m o s t p o t e n t agent w i t h i n the THI-seties. Osei-Gyimah et al. {1976) have synthesized the e r y t h r o - and t h r e o - a - h y d r o x y isomers [ 1-(3',4',5'-trimethoxy-a-hydroxybenzyl)6,7-dihydroxy-1,2,3,4-THI; a-hydroxy TMQ] o f TMQ with the p r o s p e c t t h a t generating a s e c o n d a s y m m e t r i c c e n t e r o f TMQ w o u l d p r o d u c e a m o r e p o t e n t or selective f l - a d r e n o c e p t o r agent (see fig. 1). T h e m e c h a n i s m o f f l - a d r e n o c e p t o r activity within the T H I class is still equivocal. S(--)TMQ is k n o w n t o p r o d u c e a rise in c-AMP levels in tracheal muscle (Inamasu et al., 1 9 7 4 ) . Since T H I ' s also bear a s t r u c t u r a l r e s e m b l a n c e t o papaverine, a k n o w n i n h i b i t o r f o r cyclic n u c l e o t i d e p h o s p h o d i e s t e r a s e (PDE), the elev a t i o n in c-AMP m a y be m e d i a t e d in p a r t b y

394

M.T. PIASCIK ET AL.

CH2 CH 3 0 " ~ O C H3 OCH3 (A)

CH2 [ ~ O C H3 OCH3 (B)

chambers contained Krebs-Henseleit buffer maintained at 37°C and aerated with 95% O: and 5% CO2. Tissues were allowed to equilibrate for 1--1.5 h. Drug-induced effects were recorded on a Grass polygraph (Model 7C) via a force displacement transducer, and cumulative dose--response curves were obtained with each drug. Drug responses are expressed in terms of percent of the maximal effect obtained in the presence of 10 -s M (--)-isoproterenol. 2.2. Isolation o f fat cells

HCOH

HOCH

OCH3 (C)

OCH3 (D)

Fig. 1. Structures of trimetoquinol (A), papaverine (B) and the erythro- (C) and threo- (D)-isomers of ~-hydroxytrimetoquinol. Asterisks indicate centers of asymmetry.

an inhibition in the activity of cyclic nucleotide PDE. The present study was initiated to clarify the mechanism of fl-adrenoceptor action possessed by the TMQ stereoisomers. Furthermore, the comparative pharmacological potency of erythro- and threo-diastereomers of a-hydroxy TMQ in selected fl-adrenoceptor preparations was assessed along with their ability to (a) produce a change in intracellular c-AMP levels and (b) interact with a partially purified c-AMP phosphodiesterase preparation in rat adipose tissue.

2. Materials and methods 2.1, Isolated trachea and atria Guinea pigs of either sex (300--500 g) were employed. The isolation and mounting of the tissues were carried out as previously described (Buckner and Patil, 1971; Krell and Patil, 1971). The 12 ml jacketed muscle

Male Sprague--Dawley rats weighing 1 6 0 220 g were employed in all experiments. Animals (10--15/experiment) were stunned and killed by cervical dislocation. The lower peritoneal cavity was exposed and epididymal fat pads removed and placed in a Krebs--Ringer bicarbonate buffer of the following composition (raM): NaC1 118; KC1 4.7; CaCl: 1.2; KH:PO4 1 . 1 : M g S O 4 1.1; NaHCO3 24.0. The buffer was aerated for 10 min with a 95% 0 : - - 5 % CO: mixture and adjusted to pH 7.4 prior to the addition of 2% albumin (Bovine fraction V, Sigma). Fat cells were isolated by the method o f Rodbell (1964) employing crude bacterial collagenase (Worthington Biochemical). Following several washings with the albumin buffer (37°C), a fat cell suspension was obtained by centrifugation at 750 X g for 15 sec. Cells were distributed by a siliconized 1 ml glass tuberculin syringe. 2.3. Lipolysis To analyze the ability of selected THI's to p r o m o t e glycerol release, techniques previously described in our laboratory were employed (De Santis et al., 1974). Incubation mixtures contained 0.2 ml of the fat cell suspension, and drugs in a volume of 0.05 ml. Drug concentations ranged from 10 -9 to 3 × 10-4M. The final reaction mixture was brought to volume (2.5 ml) by the addition of Krebs 2% albumin buffer. Reaction flasks

STEREOSELECTIVE ACTIONS OF TETRAHYDROISOQUINOLINES were incubated at 37°C in air for 1 h (100 oscillations/rain). Reactions were terminated by addition of an equal volume of trichloroacetic acid (TCA, 10% w/v). The a m o u n t of glycerol release was measured by procedures described previously (Lambert and Neish, 1951; Nash, 1953). Within each experiment, a maximal rate of glycerol release was obtained in the presence of 10-SM isoproterenol and this maximal response was employed to calculate the percent response of adipose tissue to the tetrahydroisoquinolines. Rates of druginduced glycerol release were corrected for basal activity. Maximal glycerol release in the presence of 10 -s M (--)-isoproterenol was calculated to be 650 + 49 nmoles/0.2 ml cells/60 min (mean ± S.E.M. of n = 15).

2.4. Estimation of c-AMP elevation in isolated fat cells Reaction mixtures contained 0.3 ml of the fat cell suspension and 0.1 ml theophylline (10 -6 M). In certain experiments, the adrenoceptor blockers phentolamine (5 X 10 -~ M) or propranolol (5X 10-TM) were added in a replacement volume of 0.05 ml. Reaction flasks were brought to a volume of 1.95 ml by the addition of Krebs 2% albumin buffer. These mixtures were preincubated for 15 min (37°C; 100 oscillations/min) and reactions were initiated by addition of the various compounds in a volume of 0.05 ml. All flasks were then incubated for 7 or 10 min. In preliminary experiments, c-AMP formation was found to be linear over a 10 min period. A 2 ml volume of trichloroacetic acid (TCA, 10% w/v) was added to terminate the reaction and mixtures were centrifugated for 20 min (1000 X g). 1 ml aliquots of the TCA supernatant were acidified with 0.1 ml of 1 N HC1 and extracted with 3 volumes of water-saturated ether, and 50 pl of the remaining aqueous phase were evaporated to dryness. c-AMP levels in these dried extracts were quantitated by the competitive binding assay of Gilman (1970). Final reaction mixtures contained 6 ng of 3H-c-AMP, 0.12 ml of ace-

395

tate buffer (0.05 M, pH = 4), 0.05 ml (15 pg) of c-AMP protein kinase and 0.03 ml (30 pg) of protein kinase inhibitor. Samples were incubated at 0°C for 65 min (100 oscillations/ min). The assay mixture was then filtered under vacuum through Millipore filters (HAWP02400), followed by two 4 ml washings with ice-cold potassium phosphate buffer (0.5 mM, pH = 6). Filters were dissolved in 2 - e t h o x y e t h a n o l prior to the addition of a toluene--cellulose (3 : 1) cocktail with fluors. Efficiency of 3Hdetection was 30--35%. The a m o u n t of c-AMP generated for each sample was determined from a standard curve. In each experim e n t a maximal c-AMP accumulation was obtained with 10 -6 M (--)-isoproterenol and this value was used to calculate the percent of the maximal c-AMP response for each of the test drugs. The maximal c-AMP formation in the presence of 10 -6 M (--)-isoproterenol was calculated to be 950 + 49 pmoles c-AMP/0.3 ml cells/10 min (mean +- S.E.M. of n = 27). Theophylline-induced c-AMP formation rates were subtracted prior to construction of dose--response curves for each drug. Theophylline (10 -6 M) exhibited an increase in c-AMP levels t h a t was 10--15% above the basal levels. Basal c-AMP formation rates were calculated to be 142-+ 65 pmoles c-AMP/0.3 ml cells/10 min (mean + S.E.M. of n = 32).

2.5. Estimation of phosphodiesterase inhibitory activity Cyclic nucleotide PDE was isolated from rat epididymal fat tissue by the m e t h o d of Thompson and Appleman (1971a). The ability of the compounds within the THI series to inhibit PDE was assessed by the m e t h o d of Thompson and Appleman (1971b). Reaction mixtures contained 0.1 ml of TRIS-HC1 buffer (0.16 M, pH = 8), 0.025 ml of 0.2 M MgCI2 and 0.025 ml of 0.2 M mercaptoethanol. Enzyme concentration was varied according to the desired K m form. Preliminary kinetic analysis revealed t h a t 0.1 mg protein/ assay for the low Km form and 0.4 mg pro-

396 tein/assay for the high Km form were linear with respect to enzyme concentration (Piascik et al., 1976). Drug concentrations, delivered in volumes of 0.05 ml were varied from 10 -7 to 10 -3 M. In all experiments papaverine was employed as a standard inhibitor of the PDE preparation. Reaction mixtures were diluted to a volume of 0.3 ml by the addition of Tris buffer (0.16 M, pH = 8) and preincubated for 5 m i n at 30°C (60 oscillations/min). Enzymatic hydrolysis was initiated by the addition of 0.1 ml 3H-c-AMP (0.1 pCi). The concentrations of c-AMP employed to study the high and low Km forms of PDE were 10 -4 and 10 -6 M, respectively. Reactions were allowed to proceed for 10 min after which time they were terminated by boiling for 5 min. 5'-AMP formed by PDE hydrolysis was converted to adenosine by the addition of 0.1 ml (1 mg/ ml) of snake venom (ophiophagus hannah), and after incubation for 10 min at 37°C the reaction was stopped by the addition of a 1 : 3 slurry of anion-exchange resin (Bio Rad AG1-X2, 200--400 mesh). After centrifugation (1000 X g, 10 min) the resin-free supernatant was assayed for 3H-adenosine by liquid scintillation spectrometry. Detection efficiency was 30--33%.

2.6. Drugs and biochemicals Drug solutions were freshly prepared in demineralized double distilled water containing 0.9% NaC1 and 0.1% sodium metabisulfite. Trimetoquinol stereoisomers were generously provided by Tanabe Seiyaku Co. (Japan). Erythro- and threo-~-hydroxy TMQ were synthesized in our laboratory by methods previously described (Osei-Gyimah et al., 1976). Racemic-TMQ was also synthesized in our laborator~ by the m e t h o d of Miller et al. (1975a). Other drugs employed in the study were (--)-isoproterenol (Aldrich), papaverine (Sigma), phentolamine (Ciba), propranolol (Ayerst), and theophylline (Matheson, Coleman, and Bell). Biochemicals for the c-AMP estimation experiments were purchased commercially (Sigma) and made as 1 mg/ml stock

M.T. PIASCIK ET AL. solutions, c-AMP dependent protein kinase was prepared in 0.5 mM citrate buffer (pH = 6), while the c-AMP protein kinase inhibitor was prepared in demineralized double distilled water. 8-3H-c-AMP (28 Ci/mmole; New England Nuclear) was assayed for radiochemical purity (>98%) by thin layer chromatography in an ethanol--ammonium acetate (5 : 2) system on Silica Gel G. Radiochromatographic analysis yielded a single radioactive peak.

2.7. Statistical analyses Differences between means were analyzed by the Student's t-test using a significance level of 5%.

3. Results

3.1. Isolated trachea and atria The a - h y d r o x y TMQ diastereomers were found to be agonists in both tracheal and atrial preparations (see fig. 2). In each case, e r y t h r o ~ - h y d r o x y TMQ (EDs0 ~-- 3 X 10 -7 M) was more potent than threo-a-hydroxy TMQ (EDs0 ~ 5 X 10 -6 M). Closer examination of this figure shows that the compounds are nearly equipotent in these fil (heart) and fi2 (trachea) adrenoceptor systems. Erythro-ah y d r o x y TMQ was f o u n d to be 10,fold._aad 8-fold more active than threo-~-hydroxy TMQ in guinea pig atria and trachea, respectively. As expected, racemic-TMQ was observed to be a potent agonist (EDs0 ~ 10 -9 M) in both fl-adrenoceptor systems.

3.2. Lipolysis in fat adipocytes Similarly, the results with isolated rat adipocytes are in good agreement with the data obtained in: guinea pig atria and trachea (see fig. 2). E r y t h r o ~ - h y d r o x y TMQ (EDs0 ~ 10 -6 M) was more active than the corresponding threo-diastereomer at promoting glycerol release. T h r e o ~ - h y d r o x y TMQ was n o t capable of eliciting a maximal response at the

S T E R E O S E L E C T I V E A C T I O N S OF T E T R A H Y D R O I S O Q U I N O L I N E S

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highest concentration used; only a 40% lipolytic response was attained at 10 -s M {fig. 2). Racemic-TMQ was more active than either erythro- or threo-a-hydroxy TMQ. The stereoselective interaction of the TMQ isomers on lipolysis is illustrated in fig. 3. As can be seen, S(--)-TMQ is considerably more active than the corresponding R(+)-isomer. Further, the R(+)-enantiomer was unable to elicit a maximal lipolytic response in this system.

3.3. Elevation o f c-AMP in fat adipocytes Racemic-TMQ, erythro- and threo-a-hydroxy TMQ were capable of producing dosedependent rises in c-AMP (see fig. 4A). The

Fig. 2. Illustrated log dose--response curves obtained for (+_)-trimetoquinol (o o), erythro-~-hydroxyt r i m e t o q u i n o l (~ n) and t h r e o - a - h y d r o x y t r i m e t o q u i n o l (u R) in guinea pig trachea (A, n = 6--8), guinea pig atria (B, n = 5) and rat adipocytes (C, n = 6--9). Each point represents the mean ± S.E.M. Ordinate (A): % relaxation; (B and C): % response. Abscissae: drug c o n c e n t r a t i o n (M).

erythro-isomer (EDs0 = 2 X 10 -7 M) was again more potent than threo-a-hydroxy TMQ (EDs0 = 8 × 10 -7 M). Significant differences (P < 0.05) in c-AMP accumulation were observed between the ~-hydroxy TMQ isomers at 10 -8 and 10 -6 M. Both of these compounds were capable of promoting near-maximal rises in c-AMP, but at much higher concentrations than observed with racemic-TMQ (EDs0 ~ 3 X 10 -s M). Comparison of these data with fig. 2 reveals that these tetrahydroisoquinoline compounds are capable of producing a pharmacological response at concentrations which produce elevations in c-AMP within fat adipocytes. The stereoisomers of TMQ were also observed to produce dose-dependent increases in

398 IO0

M.T. PIASCIK ET AL.

T/~*'-'"~~.

=-

- LIPOLYSIS

nearly equipotent with (--)-isoproterenol (EDs0 = 10 -9 M, u n p u b l i s h e d results). As can be seen in table 1, rises in c-AMP g e n e r a t e d b y R(+)- and S(--)-TMQ were b l o c k e d b y prop r a n o l o l (5 X 10 -7 M) b u t n o t in the p r e s e n c e o f p h e n t o l a m i n e (5 X 10 .7 M). P r o p r a n o t o l (5 X 10 -7 M) also b l o c k e d ( - - ) - i s o p r o t e r e n o l i n d u c e d c-AMP f o r m a t i o n in rat a d i p o c y t e s (see t a b l e 1).

80 60 40 20 O

t 10 -7

] 0 -6

3.4. Inhibition o f cyclic nucleotide PDE activity from adipose tissue

I l 0 -5

10 -4

i0 -3

Fig. 3. Illustrated log dose--response curves obtained

for the stereoisomers of trimetoquinol to promote glycerol release in rat adipocytes. Key: S(--)-trimetoquinol (A --~) and R(+)-trimetoquinol (A A). Each point represents the mean + S.E.M. of n --- 4--8. Ordinate: % response; abscissa: drug concentration (M). c-AMP (see fig. 4B), and these d a t a also d e m o n s t r a t e a stereoselective effect. S ( - - ) - T M Q e x h i b i t e d an activity w h i c h was g r e a t e r t h a n t h e c o r r e s p o n d i n g R(+)-isomer. When c o m p a r e d t o S(--)-TMQ t h e R ( + ) - i s o m e r was observed to be a partial agonist w h i c h possessed a l o w e r e d a f f i n i t y and intrinsic activity f o r c-AMP a c c u m u l a t i o n in r a t a d i p o c y t e s . S(--)T M Q (EDs0 = 2 × 1 0 - 9 M ) was f o u n d t o be

lo0

(A)

T h e results o f these studies are p r e s e n t e d in fig. 5. As can b e seen, n o n e o f the T H I derivatives t e s t e d were effective inhibitors o f PDE until relatively high c o n c e n t r a t i o n s w e r e emp l o y e d ( > 1 0 -4 M). F r o m these d a t a , it is evid e n t t h a t the T H I ' s are n o t c a p a b l e o f inhibiting e i t h e r the high or the l o w Km f o r m o f P D E b e y o n d 30%. Papaverine, on the o t h e r h a n d s h o w e d a d i f f e r e n t i a l i n h i b i t i o n o f the l o w and high f o r m s . In c o n t r a s t to c-AMP elev a t i o n , t h e i n h i b i t i o n ( w h e n o b s e r v e d ) o f PDE was n o t stereoselective f o r the T M Q isomers. With regard to the ~ - h y d r o x y T M Q diastereom e r s , a stereoselective i n t e r a c t i o n was n o t observed until v e r y high c o n c e n t r a t i o n s (10 -3 M) w e r e used.

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Fig. 4. Illustrated log dose--response curves for the ability of various trimetoquinol analogs to bring about rises in intracellular c-AMP from rat adipocytes. Agonists employed are (+-)-trimetoquinol (o o), erythro-~-hydroxytrimetoquinol (• D), threo-~-hydroxytrimetoquinol ( I =), S(--)-trimetoquinol (~ ~) and R(+)-trimetoquinol ( & - - A ) . Each point represents the mean _+S.E.M. of n = 3--12. Time of incubation was 10 rain. Ordinate: % response ; abscissa: drug concentration (M).

STEREOSELECTIVE ACTIONS OF TETRAHYDROISOQUINOLINES

399

TABLE 1 Effects of adrenoceptor blockers (5 × 10 -7 M) on the c-AIVIP rise induced by the stereoisomers of trimetoquinol in fat adipocytes 1. Time of incubation was 10 min. Treatment Control

Propranolol

Phentolamine

Experiment I R(+)-Trimetoquinol (10 -7 M) (--)-Isoproterenol (10 -6 M)

251 +_ 12 676 +_ 20

19 _+11 2 325 _+50 2

252 _+ 7 _

Experiment II S(--)-Trimetoquinol (10 -8 M) (--)-Isoproterenol (10 -6 M)

553 _+32 950 ± 75

238 _+50 2 650 _+ 19 2

491 ± 59 _

1 Data are expressed as net pmoles c-AMP/0.3 ml cells/10 min (mean ± S.E.M. of n = 4). All values obtained in each agonist were corrected by subtraction of the theophylline-induced c-AMP formation rate. 2 Values differ significantly (P < 0.05) from the control.

4. D i s c u s s i o n Within the tetrahydroisoquinoline series of compounds, TMQ and tetrahydropapaveroline have been shown to exhibit a stereoselective interaction with ~-adrenoceptor systems ( B u c k n e r a n d A b e l , 1 9 7 4 ; S h o n k e t al., 1 9 7 1 ; L e e e t al., 1 9 7 4 ; F e l l e r e t al., 1 9 7 5 ) . I n t h e present study, a dependency upon stereochemistry was also noted with erythro- and

100

Low KmFORM PDE: cAMP = 166 / " ~..~100

80

t h r e o - a - h y d r o x y T M Q ( s e e fig. 2). M o r e o v e r , these data indicate that the addition of an a-hydroxyl group to the TMQ moiety does sot yield a more selective or potent fi-adrenoceptor agent. Although the interaction of T M Q w i t h f i - a d r e n o c e p t o r s y s t e m s is i n f l u enced by the presence of a second asymmetric center on the methylene carbon of the 1-benzyl substituent, the postulated role of the a - h y d r o x y l g r o u p in t h e i n t e r a c t i o n o f a - h y -

High KmFORM POE: cAMP = 164 M --

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Fig. 5. Log dose--response curves for the inhibition of low and high K m forms of c-AMP PDE in rat epididymal adipose tissue. Inhibitors employed were papaverine (o o), erythro-a-hydroxytrimetoquinol (D ~), threo-a-hydroxytrimetoquinol (I --), S(--)-trimetoquinol (A ~), and R(+)-trimetoquinol (A A). Each point represents the mean + S.E.M. of n = 3--12. Ordinate: % inhibition;abscissa: drug concentration (M).

400 droxyl TMQ with ~-adrenoceptor systems cannot be accurately assessed until the two diastereomers are resolved and their pharmacological activities are characterized. Stereoselective differences between compounds within a series of tetrahydroisoquinolines provide an opportunity to explore mechanisms of adrenoceptor activation. Lipolysis is recognized to be a /31-adrenoceptor system, the activity of which can be regulated by intracellular c-AMP. The tissue concentration of c-AMP may be elevated by a stimulation of adenylate cyclase and/or an inhibition of c-AMP phosphodiesterase activities (Fain, 1973). In this regard, none of the THI compounds used in this study were capable of blocking the c-AMP phosphodiesterase systems (high and low Km forms) until relatively high concentrations are used (fig. 5). Furthermore, the absence of a significant stereoselective effect for these THI c o m p o u n d s on c-AMP phosphodiesterase activity in adipose tissue suggests that their lipolytic action in fat adipocytes is likely mediated via stimulation of membrane-bound adenylate cyclase. In accord with this view, the dose-dependent ability of the TMQ isomers and a-hydroxy TMQ diastereomers to increase c-AMP accumulation was similar to their observed quantitative differences in pharmacological response (figs. 2--4). For both of these parameters, S(--)-TMQ and erythro-a-hydroxy-TMQ were more effective agonists than their corresponding isomers. The retention of stereoselective interaction for these THI analogs can be taken as evidence to support their involvement with adenylate cyclase and c-AMP as a mediator of the pharmacological response in adipose tissue. Such a proposal has been suggested previously by DeSantis et al. (1974) to explain the stereoselective action of the norepinephrine isomers on lipolysis. Little evidence has accumulated which has demonstrated an interaction of tetrahydroisoquinolines with adenylate cyclase in fl-adrenoceptor systems. Grunfeld et al. (1974) showed TMQ to be a stimulant of frog erythrocyte

M.T. PIASCIK ET AL. adenylate cyclase; TMQ produced an inhibition of isoproterenol-induced c-AMP elevations in this fi2-adrenoceptor preparation. Inamasu et al. {1974), using (--)-TMQ in guinea pig trachea (a fi2-adrenoceptor system) found that the drug-induced c-AMP elevation in this tissue was blocked by propranolot and was not associated with an inhibition of c-AMP phosphodiesterase activity in tracheal muscle homogenate. Our study confirms the latter findings in rat adipose tissue, a fll-adrenoceptor system. In addition, the TMQ-induced c-AMP elevation in fat adipocytes by both stereoisomers was blocked by propranolol and unaffected by a low concentration of phentolamine (table 1). This observation agrees favorably with our earlier report on the competitive and noncompetitive inhibition of racemic-TMQ-induced lipolysis by propranolol and phentolamine, respectively (Shonk et al. (1971). If the mechanism of lipolytic action for the tetrahydroisoquinolines used in this study involves c-AMP and a subsequent protein kinase activation as proposed for catecholamines (Fain, 1973), then a sequential stimulation of adenylate cyclase, c-AMP accumulation, protein kinase activation, triglyceride lipase activation and glycerol release would be expected. While it is possible to speculate that the p-adrenoceptor activation by TMQ and related THI's is mediated by this sequence of reactions, this hypothesis must await final confirmation. Studies are currently in progress to examine the nature of the interaction of these compounds with adenylate cyclase in fat cell ghost preparations.

Acknowledgements

This s t u d y was s u p p o r t e d in part by a grant from the N a t i o n a l Institutes o f Health, U S P H S ( N S 1 0 8 9 6 ) . One o f us (M.T.P.) is a F e l l o w o f the American F o u n d a t i o n for Pharmaceutical E d u c a t i o n . We also thank Dr. Popat Patil for his constructive c o m m e n t s in the design o f these studies, and Mrs. Raji Raman for her technical assistance.

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