3ournal

of

Molecular andCellular Cardiology (1979)

11,803-811

Labetalol Binding to Specific Alpha- and Beta-adrenergic Sites in vitro and its Antagonism of Adrenergic Responses in Y~VO FORREST

C. GREENSLADE, ALFONSO J. TOBIA, SHERRIE M. MADISON, KATHRYN M. KRIDER AND KATHRYN L. NEWQUIST Divisions of Biochemistry qnd Pharmacology, Ortho Pharmaceutical Corporation, Raritan, New Jkrsg 08869, U.S.A. (Received August 1978, accepted 4 January 1979)

F. C. GREENSLADE, A. J. TOBIA, S. M. MADISON, K. M. KRIDER AND K. L. NEWQULST. Labetalol Binding to Specific Alpha- and Beta-adrenergic Sites in vitro and its Antagonism of Adrenergic Responses in vivo. 3oumd of Molecular and Cellular Cardiology (1979) 11, 803-811. Labetalol was found to compete with [sH]dihydroergocryptine for specific alphaadrenergic binding sites in rabbit uterine membrane preparations. Based on IC50 values from binding-competition curves, labetalol had 536 times lower aflinity for alpha-sites than phentolamine. Labetalol competed with [sH]dihydroalprenolol for specific beta-adrenergic binding sites in guinea pig heart and lung membranes. The drug had 53 times lower affinity for the heart beta-sites than propranolol, and was significantly less potent than propranolol in inhibiting stimulation of cardiac adenylate cyclase by 10-s M isoproterenol. Labetalol had 19 times greater binding affinity for beta binding sites in heart membranes than alpha binding sites in uterine membranes. In viva studies in anesthetized dogs indicate that blockade of beta adrenergic responses predominates at lower dose of labetalol and that blockade of alpha responses was observed when the dose was increased by a factor of 10. These data are consistent with the hypothesis that labetalol antagonizes both alpha and beta adrenoceptors, and that labetalol is a more potent antagonist at beta than alphaadrenergic sites. KEY

WORDS:

sites;

in

Labetalol;

Alpha-adrenergic

receptor;

Beta-adrenergic

receptor;

Binding

vitro binding.

1. Introduction Labetalol, Z-hydroxy-5-[-hydroxy-2-[( l-methyl-3-phenylpropyl)amino] ethyl] benzamide hydrochloride, has been reported effective in reducing blood pressure in patients with symptoms of mild to severe hypertension [3, 4, 7, 8, II, 13, 14, 20, 221. Clinical studies have revealed few drug-related untoward effects; the only side-effects reported include postural hypotension, occasional nausea, fatigue, faintness, vivid dreams and retention of urine [7, II, 201. The drug appears effective in reducing blood pressure in patients with phaeochromocytoma and clonidine withdrawal hypertensive crisis [I] and in patients with severe hypertension inadequately controlled by P-adrenoceptor-blocking drugs [19]. 0022~2828/79/08803

+ 09 ,02.00/O

0

1979 Academic

Press Inc.

(London)

Limited

804

F. C. GREENSLADE

ET AL.

Pharmacological evaluation of labetalol in animals and in man has suggested that its mechanism of antihypertensive action involves competitive blockade at both alpha- and beta-adrenergic receptors [S, 9, 15, 221. Recently, radioreceptor techniques have been developed which allow the direct measurement of the interaction of drugs with specific adrenergic binding sites in membrane preparations of catecholamine-sensitive tissues. Radioactively labelled high affinity beta-adrenergic antagonists such as [sH]dihydroalprenolol and [125] hydroxybenzyl-pindolol have been used to define specific P-adrenergic binding proteins in membrane preparations from a variety of tissues 12, 6, 12, 17, 18, 241. Similarly, labelled alpha-blocking agents such as [sH]dihydroergocryptine have been used to label u-adrenergic binding sites [25]. In this report, we compare the interaction of Iabetalol with alpha-adrenergic binding sites in rabbit uterine membranes and with betaadrenergic binding sites in membranes prepared from guinea-pig heart and lung. In addition, to determine an in vivo correlate of alphaand beta-adrenergic responses, we studied the alpha blocking action of labetalol on the tilt reflex response which represents an experimental index of clinical postural hypotension. Antagonism of isoproterenol-induced tachycardia and vasodepression was used to evaluate beta blockade.

2. Materials Membrane

and

Methods

freparations

Guinea pigs were killed either by decapitation or cervical dislocation, and hearts were immediately placed in ice-cold physiological saline. Lungs were excised from guinea pigs sacrificed by cervical dislocation and either stored frozen at - lOO”C, or placed in ice-cold physiological saline and used immediately. The bronchial trees were exposed by stripping them of alveolar tissue by “combing” through the lungs several times with a steel animal hair brush. Stripping was completed by scraping free any remaining adherent tissue with a scalpel. This procedure, performed while the lungs were immersed in ice-cold physiological saline, produced bronchial tree preparations which were essentially free of extraneous tissue. Frozen type II mature rabbit uteri were purchased from Pel-Freeze Biologicals. After thawing, fat was removed and endometrial cells were scraped free with a scalpel. The heart, bronchial tree, or uterine tissue was sliced and minced with scissors in a solution containing 0.25 M sucrose, 5 mM Tris HCl (pH = 7.4), and 1 mM MgCla. The minced tissue was homogenized 4 times for 5 s intervals using a Tekmar Model SDT tissuemizer at maximum setting. After filtration through cheese cloth, the homogenate was centrifuged at 400 x g for 10 min, and the supernatant fraction collected with a Pasteur pipette. The supernatant liquid was then centrifuged for 10 min at 29 000 x g. The resulting pellet was homogenized

LABETALOL

BINDING

TO ADRENERGIC

SITES

805

using a teflon-glass tissue homogenizer in 50 mM tris HCI (pH 7.4) and 10 mM MgCla. The resuspended pellet was centrifuged for 10 min at 20 000 x g. The heart membrane preparation was finally resuspended in 0.1 M sucrose for storage. The bronchial tree membrane preparation was stored in 74 mM Tris HCl, 75 mM MgClz pH 8.1 buffer. Heart and lung membranes were stored under liquid nitrogen. Uterine membranes were prepared fresh for each binding assay. Purified guinea pig sarcolemma membranes used as the enzyme source for the adenylate cyclase assay were prepared as described by Greenslade and Newquist

CWComFetitive binding

assays

The beta-adrenergic radioreceptor assays were conducted according to the method of Mukherjee et al. [18]. Dihydroalprenolol, levo-[propyl-2,3-sH] specific activity= 32.6 Ci mmol) was purchased from New England Nuclear. Test compounds were dissolved in either dimethylsulfoxide (DMSO) or 75 mM Tris HCl, 25 mM MgCIa (pH 8.1) immediately prior to incubation. The frozen heart or bronchial tree membrane preparation was thawed and maintained on ice. The incubation mixture contained 25 ~1 of the test drug, 50 ~1 of [aH]dihydroanprenolol (40 000 ct/min; 10 nM), and 100 ~1 of the membrane preparation. A 15 min incubation at 37°C was initiated by addition of the membrane followed by five 1 s “bursts” using a Vortex mixer at the lowest setting. The incubation was terminated by adding 5 ml of ice-cold 75 mM Tris HCl and 25 mM MgCls (pH 8.1), and immediately collecting the membranes by fiber filtration on Whatman GF/C discs using a Millipore filtering manifold. The membranes were then washed 5 times with 5 ml of the same ice-cold buffer. The filtration and washing process required less than 20 s per sample. The filter discs were then dried in an oven and counted in Aquasol. Specific binding of [sH]dihydroalprenolol to P-adrenergic receptors was determined by subtracting the radioactivity bound to membranes in the presence of 1.5 x 10-J non-labeled d,Z-propranolol from each value. In the alpha-adrenergic binding assay [2.5], the radioligand used was dihydroergocryptine, 9, lo-[9, 1OsH (N)], purchased from New England Nuclear with a specific activity of 24 Ci/mmol. Non-labeled drugs were dissolved or suspended in Tris buffer/50 mM, ph 7.5 containing 10 mu MgC12. The incubation consisted of 25 pl of non-labeled competitor, 50 ~1 of [aH]dihydroergryptine (40 000 ct/min; 8 nM), and 100 ~1 of the uterine membrane. The incubation was conducted for 15 min at 25°C in 12 x 75 mm plastic test tubes. The binding reaction was terminated by the addition of 5 ml of room temperature buffer, 50 mM Tris HCl (pH=7.5) and 10 mM MgCla. The membranes were collected by fiber filtration on Whatman GF/C glass discs. The membranes were then washed 5 times with five additional 5 ml aliquots of the same buffer. Along with each test drug, incubations were conducted with phentolamine at 5.1 x 10-4 M. Binding of [aH]dihydroergo-

806

F. C. GREENSLADE

ET AL.

cryptine in the presence of this high concentration of phentolamine was considered to represent non-specific binding, and was subtracted from each value to calculate specific binding to alpha-adrenergic receptor sites. The details of the adenylate cyclase assay using [ssP]aATP have been outlined previously [lo]. in vivo studies Adult mongrel dogs were anesthetized with pentobarbital (35 mg/kg, i.v.) and secured to a tilt table. A femoral artery was cannulated to monitor arterial blood pressure and drugs were administered via one cannulated femoral vein. Arterial pressure was detected with a Statham pressure transducer and heart rate was monitored with a cardiotachometer. All recordings were made on a Beckman Dynograph. The tilt reflex response is determined by tilting the dog (head-up, about 65”) for 2 min and measuring the percent recovery of blood pressure after 15 s of tilt. Amount ( Initial

of pressure recovered (mmHg) decrease in pressure (mmHg)

x loo ) ’

The effect of labetalol on beta-adrenergic responses was studied by tachycardia and vasodepressor responses of isoproterenol (0.5 pg/kg) after pretreatment with labetalol (0.63 or 6.3 mg/kg). For comparative phenoxybenzamine (5 mg/kg) and propranolol (1 or 10 mg/kg) were these test systems. Data were statistically analysed by using limits of least difference which were calculated from control dogs receiving vehicle drug [18].

comparing before and purposes studied in significant in place of

3. Results The ability of labetalol to compete with [sH]dihydroergocryptine for alphaadrenergic binding sites on rabbit uterine membranes, and with [sH]dihydroalprenolol for beta-adrenergic binding sites on guinea pig heart and lung membrane is shown in Figure 1. Labetalol exhibited greater affinity for P-adrenergic sites on heart membranes than for the lung P-adrenergic sites (IC50 = 0.8 and 4.0 PM respectively). Labetalol had slightly less affinity for a-adrenergic binding sites (IC50 = 15 FM). The ratio of labetalol’s IC50 for alpha binding sites to its IC50 for beta binding sites on heart membranes was 19. Labetalol is compared to standard a- and P-blocking agents with respect to affinity for these ccand p binding sites in Table 1. The rank affinity order of these standards for the heart and lung binding sites labeled by [sH]dihydroalprenolol is consistent with the literature which describes them as p-adrenergic receptors. The relative binding affinities of the cardioselective P-blocking agents, tolamolol, metoprolol, practolol and atenolol suggest the binding sites on heart and lung

LABETALOL

BINDING

TO ADRBNERGIC

807

SITES

loo

E 60e25 _ z 60s -?G P 5 40t p

mI 0Go1

0

o-01

0.1

Labetolol

concentration

I

IO

too

(PM)

FIGURE 1. Competition of iabetalol with [sH]dihydroalprenolol for specific beta-adrenargic binding sites in guinea pig heart (betal) and lung (betas) membranes; and with [sH]dihydroergocryptive for specific alpha-adrenergic binding sites in rabbit uterine membranes. (A---A) betal; (m-.-m) betaz; (0 - - 0) alpha.

membranes

to represent @I- and Pa-adrenergic receptors respectively. Labetalol’s IC504abetalol relative affinity for @-receptors was 53, and for pz-receptors ICSO-propranolol was 67. The compound would thus not be considered as cardioselective. The rank order of affinities of the standards for the uterine binding sites titrated by [sH]dihydroergocryptine is similarly consistent with the literature which describes them as a-adrenergic receptors. The relative affinity of labetalol IC504abetalol was 536. IC50-phentolamine TABLE

1. Competition P-adrenergic ergocryptine

(IC50 in PM) of compounds for [sH]dihydroalprenolol sites in guinea pig heart and lung membranes and binding to rabbit uterine membranes

Heart (PI) Labetalol* Phentolamine* Ergocryptine Phenoxybenzamine* Prazosin* Tolazoline* Propranolol* Tolamolol Metoprolol Practolol Atenolol Butoxamine* * Hvdrochloride.

> > > > >

0.8 150.0 150.0 150.0 150.0 150.0 0.015 0.08 3.0 8.0 10.0 70.0

Lung w 4.0 150.0 150.0 150.0 150.0 150.0 0.06 1.0 60.0 > 150.0 100.0 100.0

Uterus

> > > > >

> > > >

binding for [aH]dihydro-

(fx)

15.0 0.028 0.06 0.15 1.0 4.0 25.0 60.0 150.0 150.0 150.0 150.0

to

808

TABLE

F. C. GREENSLADE

ET AL.

2. Activity (FM) of compounds in inhibiting adenylate cyclase from guinea-pig myocardium Compound

10-s M isoproterenol-stimulated

o/o Inhibition

Propranolol* Labetalol*

10-5 M

10-4

71f3

9263 7417

38+1

M

* Hydrochloride. TABLE

3. Effect of labetalol

on tilt reflex response in the anesthetized

dog

Tilt reflex response Dose (mglk) Labetalol*

~2: Percent change from control

0.63 6.3

Propranolol*

2 2 2 2 2

1 10

Phenoxybenzamine*

5

$5 -854

+28§ -14 -85s

* Hydrochloride. t All drugs were infused intravenously over 10 min and tilt responses were determined before drug infusion and 10 min post-infusion of labetalol or propranolol and 30 min post-phenoxybenzamine. $ n, number Q Statistically

TABLE

of dogs. different

from

control

4. Effect of labetalol responses

Dwz

(P < 0.01).

on isoproterenol-induced

Dose (w/k)

Labetalol*

0.63 6.3

Propranolol*

1 10

+ Hydrochloride. t All values are statistically

different

from

n

and vasodepressor

Antagonism of isoproterenol Change from control7 Tachyeardia Vasodepression beatslmin (mmHd

2 2 2 2

control

tachycardia

-79 -98 -87 -92

(P