J. CRAIG VENTER, JOHN ROSS, JR., JACK E. DIXON, STEVEN E. MAYER, AND NATHAN 0. KAPLAN. Departments of Chemistry and Medicine, University of ...
Proc. Nat. Acad. Sci. USA Vol. 70, No. 4, pp. 1214-1217, April 1973
Immobilized Catecholamine and Cocaine Effects on Contractility of Cardiac Muscle (glass beads/immobilized drugs/cat papillary muscles/cocaine potentiation)
J. CRAIG VENTER, JOHN ROSS, JR., JACK E. DIXON, STEVEN E. MAYER, AND NATHAN 0. KAPLAN Departments of Chemistry and Medicine, University of California, San Diego, La Jolla, Calif. 92037
Contributed by Nathan 0. Kaplan, February 20, 1973 ABSTRACT Isoproterenol, norepinephrine, and epinephrine covalently bound to glass beads exert a positive inotropic effect on isometrically contracting papillary muscles from cats. Immobilized isoproterenol maintains increases in force and velocity of contraction for more than 5 hr. 1 MM Cocaine potentiates the action of immobilized norepinephrine, isoproterenol, and epinephrine, but not of isoproterenol in solution. The data presented indicate that the effects of immobilized catecholamines are not due to their coming off the glass. The effects observed with cocaine and immobilized catecholamines are not altered by prior treatment of the muscle with reserpine. These results suggest that the major site of catecholamine action is on receptors located on the extended surface of myocardial cells and a post-junctional site for cocaine potentiation.
personal communication), with the following modifications: the glass was heated to 5550 to remove unwanted protein; a Teflon stirring paddle was used to mix the glass with a-aminopropyl triethoxysilane. This procedure minimized reduction of the size of the glass particles. The glass was extensively washed with water, and the alkyl amine derivative gave a positive reaction when tested with picrylsulfonic acid (7, 8). 1 g of alkyl amine glass was then refluxed with 2 g of p-nitrobenzoyl chloride and reduced with dithionite at 25° for 6 hr, thus forming the aryl amine glass. The succinyl glass derivative was prepared from alkyl amine glass and succinic anhydride (2.0 g/1.5 g of glass). This material did not react with picrylsulfonic acid, indicating that all amino groups were blocked. Nitric acid, chloroform, and toluene were all reagent grade. Succinic anhydride (Matheson, Coleman and Bell) had a melting point of 119-120'; p-nitrobenzoyl chloride (Eastman) was recrystallized from carbon tetrachloride. Triethylamine (Mallinckrodt), sodium dithionite (Matheson, Coleman and Bell), a-aminopropyl triethoxysilane (Aldrich), i-epinephrine (Sigma), picrylsulfonic acid, sodium salt dihydrate (Aldrich), Di,-isoproterenol (Sigma), and 1-cyclohexyl 3-(2-morpholinoethyl)-carbodiimide methoxy p-toluenesulfonate were used without further purification. The succinyl glass (0.1 g) was reacted with 20 mng of iepinephrine and 250 mg of the water-soluble carbodiimide at pH 4.5 for 1 hr. The glass was then extensively washed with 0.1 N HCl to remove any unbound catecholamine. The catecholamines, inorepinephrine, iepinephrine, and DL-iSOproterenol, were coupled to the aryl amine glass according to the procedure of Venter et al. (ref. 1, with an additional wash of the glass with several liters of 0.1 N HCl). The isotopic labeled L-epinephrine was that used previously (1). DL-[H]Isoproterenol (New England Nuclear Corp.) had a specific activity of 2.11 Ci/mmol. Isotopic labeled catecholamines were prepared as described (1); however, radioactive catecholamines were diluted 200 to 1 and 20 to 1 with unlabeled amines rather than 400 to 1, thus allowing for a more sensitive assay. The glass gave 50-150 cpm per bead in counting solution (1). Immediately before use, the immobilized catecholamines were extensively washed with 0.8% NaCl to ensure the absence of unbound catecholamines. Papillary muscles were rapidly removed from the right ventricles of male cats (1.0-3.0 kg) anesthetized with intraperitoneally administered sodium pentobarbitol (40 mg/kg). The papillary muscle was placed horizontally in a Leucite
Catecholamines covalently bound to glass beads are biologically active and can enhance the frequency of contraction of cardiac muscle (1). Isoproterenol, epinephrine, and norepinephrine also effect the inotropic state of the heart (2), as well as producing increases in contractile force and rate of rise of tension in isometrically contracting, isolated papillary muscles (3). Accordingly, since catecholamine effects can best be studied when they are uncomplicated by secondary actions, we have undertaken a study of the response of isolated cat papillary muscle to immobilized catecholamines. The effects of cocaine on adrenergic effector systems have been known since 1910 (4), when cocaine potentiation of catecholamine action was first observed. Potentiation of norepinephrine effects by cocaine is commonly attributed to inhibition by cocaine of the neuronal uptake of this amine, thereby resulting in a translocation of norepinephrine from the blocked nerve ending to the cell receptors that induces an increase in response of the affected organ, and gives a state of supersensitivity (5, 6). The availability of immobilized catecholamines has provided an opportunity to examine this hypothesis in isolated cardiac muscle. The present experiments on isolated cardiac muscle provide evidence that: (i) immobilized catecholamines exert their action on cardiac tissue while covalently bound to glass beads; (ii) immobilized catecholamines exert a positive inotropic effect in isolated cardiac muscle; and (iii) cocaine potentiates the action of immobilized catecholamines. METHODS
Porous glass was obtained from Electronucleonics of the Corning Glass Works. This glass was modified according to methods outlined by the Corning Glass Works (H. H. Weetall, 1214
Proc. Nat. Acad. Sci. USA 7o
Immobilized Catecholamines
U97s)
bath and arranged to contract isometrically. One end of the muscle was held by a Leucite clip attached to a force transducer (Statham), and the tendonous end was tied by a 4-0 silk thread to a micrometer, thus allowing the muscle length to be altered. The muscle was stimulated to contract at 12 times per min by means of a Grass stimulator, and two platinum electrodes placed parallel to the muscle provided transverse field stimulation. Peak isometric force and its first derivative (df/dt) were recorded simultaneously, along with the stimulator artifact and a time mark on a forced ink oscillographic recorder (Clevite 200 Brush Instruments). The muscle bath contained a Kreb's solution (3) that was maintained at 21-23° (pH 7.4) and was bubbled continuously with a mixture of 95% 02-5% CO2. The horizontal position of the papillary muscle made it possible to retain the glass beads on the contracting muscles. Beads were removed from the muscles by rinsing the muscle with an aliquot of the bath solution. Beads removed by this method remained in the bath unless the bath solution was completely changed. Cocaine HCl (1 MM) (Mallinckrodt) was stored frozen in 1-ml aliquots until used. Reserpine (Ciba) was injected subcutaneously (1 mg/kg) 24 hr before heart dissection. Isoproterenol and norepinephrine were made up in solution at the indicated concentrations immediately before use. For scanning electron microscopy (Cambridge S4), papillary muscles and beads were fixed in 0.1% gluteraldehyde solution for 1 hr, followed by fixation in a 1% solution for 1 hr. Muscle preparations were critical-point dried according to the procedure of Cohan et al. (9). Samples were coated with 60% gold and 40% palladium in a vacuum evaporator to a thickness of 100-200 A. The data presented represent the analyses of 46 technically satisfactory preparations out of 58 attempted. RESULTS Inotropic effects
Application of the immobilized catecholamines to the papillary muscles resulted in immediate increases in the force and velocity of contraction, the maximum increases being evident within 1-5 min of bead application. A return to the original force and velocity of contraction was seen only on removal of the immobilized catecholamines from the muscle. The effect of the addition of immobilized isoproterenol to a papillary muscle is compared to that of addition of a 1 AM isoproterenol solution to the bath of a separate muscle in Fig. 1. The posi-
1215
FIG. 2. A scanning electron micrograph (magnification X 70) of a cat papillary muscle of 0.5 mm diameter, with immobilized catecholamine glass beads on the surface.
tive inotropic effect of the immobilized isoproterenol was relatively well sustained and completely reversible by removal of the beads after more than 5 hr. In contrast, the effect of the soluble -catecholamine was complete in about 30 min. The reversible effects on removal of the immobilized catecholamines were demonstrated in every muscle tested. A scanning electronmicrograph (magnification X70) of a 0.5-mm diameter papillary muscle showing the position of the glass beads is reproduced in Fig. 2. The random size, shape, and surface area at bead contact is clearly visible. The glass beads varied from 300-500 ,m in diameter. The average percent changes in force and velocity of contractions due to immobilized catecholamines are listed in Table 1. Immobilized isoproterenol exhibited a greater potency than immobilized epinephrine or norepinephrine. The difference between immobilized isoproterenol and the other immobil zed amines was statistically significant (P < 0.05; Student's t-test). In many muscles, the addition of a single glass bead with bound isoproterenol was sufficient to cause as much as a 30% increase in the force of contraction. Cocaine effects
The effects of immobilized norepinephrine beads on a papillary muscle when 1 pM cocaine * HCO was added to the bath 14 min later are shown in Fig. 3. The immobilized norepinephrine produced a positive inotropic response. Within 1 min after the addition of cocaine, there was an additional increase in the force and velocity of contraction in the eight muscles tested. TABLE 1. Positive inotropic effects produced by
30
immobilized catecholamines
I.
0
30
60
90
120
150
180
.
210
240 270 300 330 360
MINUTES
FIG. 1. The effect of the addition of immobilized isoproterenol (A) to a cat papillary muscle is compared with that of addition of 1 MM isoproterenol solution (C) to the bath of a separate muscle. (B) Denotes the time when the isoproterenol beads were removed from the muscle.
Immobilized isoproterenol (n Immobilized norepinephrine (n = 8)*
=
Average % changes Force Velocity 11) 43. 8 i 6. 4 104. 8 4- 28. 2 15.7 i 1.3 36.0 ±t 3.0
* The difference in the means is statistically significant (P < 0. 05) by a t-test for nonpaired data.
Medical Sciences: Venter et al.
1216
4
0
8
12
16
20
24
28
Proc. Nat. Acad. Sci. USA 70
32
36
MINUTES
FIG. 3.
An example of the effects of immobilized norepineph-
rine added at (A ), and the additional effects produced by
1,uAM
(1973)
increase representing the amount of catecholamine bound to the inner surfaces of the porous glass beads. A similar group of 10 ['H]isoproterenol-labeled beads was placed on a papillary muscle, producing the usual positive inotropic effect. The beads were placed on and removed from the muscles with forceps. Table 2 shows the cpm found on the beads and on the muscle after 30 min of incubation in two of the experiments. After they were ground, the incubated beads gave more than 9000 cpm. The muscles did not yield counts above background, and a 100-jul sample of the Krebs solution also showed no significant radioactivity. Experiments with ['H]isoproterenol in solution (0.01 jAM) showed that counts greatly in excess of background could be detected on treated papillary muscle.
cocaine added at (B).
DISCUSSION Similar responses to cocaine were shown with norepinephrine in solution. In the absence of catecholamines, cocaine usually had a slight depressant effect on the force and velocity of
contraction. Fig. 4 shows the effects of adding immobilized isoproterenol beads to a papillary muscle when 1 27
mmn later.
,u
cocaine
* HCl
was added
The cocaine clearly caused an additional increase
in the force and velocity of contraction over that produced by immobilized isoproterenol alone. However, when cocaine was added to the bath of muscles activated by isoproterenol (1
,uM)
in solution, there was a slight depressant effect on the
force and velocity of contraction. The potentiation of immobilized isoproterenol
action by cocaine was shown on all
12
muscles tested. Effect of reserpinization on cocaine potentiation
The effects of immobilized catecholamines and cocaine were tested on cats treated with reserpine. The positive inotropic effects of immobilized catecholamines and cocaine potentiation were not altered in papillary muscles from treated cats. Studies with immobilized ['Hicatecholamines
[8H ]Catecholamines determine
bound
whether
or
not
to
glass
beads
were
tested
catecholamines
were
displaced
to
from the beads by enzymes present on the surface of the
papillary muscles.
Groups of
10
['H Icatecholamine-labeled
glass beads of uniform size were isolated, and representative
samples
were counted
The effects observed with the immobilized catecholamines on isolated cat papillary muscle indicate that these agents, when bound, have a substantial and persistent positive inotropic effect. These studies add further scope to previously reported effects of immobilized catecholamines on the frequency of contraction in canine hearts in vivo and on chicken-embryo hearts and heart cells in vitro (1). An independent study by Dr. W. F. Riker, Cornell University Medical College, has shown that direct application of "isoproterenol glass beads" to the exposed sinoatrial region of isolated perfused guinea-pig hearts accelerated the spontaneous sinus rate. The rate appeared to increase in relation to the area of pacemaker tissue in contact with beads; the maximum increase observed was 129% (+77 beats/min) of the control. The effect developed to a peak over periods of 2-8 min and persisted as long as the beads remained in contact with the pacemaker tissue. On removal of the beads, the rate returned promptly to control. Due to the heterogeneous nature of commercially available glass beads, we found it more satisfactory to react untreated glass beads by the procedures described in this paper. The resulting aryl amine glass has been, in our experience, very homogeneous and allows reproducibility in the preparation of the aryl amine-catecholamine derivative. It is of interest to note that we have produced a succinyl derivative of the glass and norepinephrine by this procedure. The norepi-
(toluene-2-5 diphenyloxazole [PPO]-
Triton X-100 medium); a group of 10 beads represented about 1000 cpm. If these same beads were then ground into a fine
TABLE 2. Effects of immobilized ['H]isoproterenol incubation with papillary muscles on bead-amine release
powder, the observed cpm increased to more than 9000, the
-3
0
4
8
Blank 10 [8H]Isoproterenol beads Same 10 isoproterenol beads after grinding 10 Beads removed after 30 min from muscle Same 10 beads after grinding Muscle after 30 min of incubation with beads Control muscle (no beads) 100 Al of Kreb's solution after 30 min of incubation with beads
12 1620 2428 3236 4044 48
cpm of 3H Exp. 1 Exp. 2 43 46 1010 992 9552 9273 1123 975 9754 9400 42 43
47 45
43
47
MINUTES
FIG. 4. added
at
An example of the effects of immobilized isoproterenol (A),
cocaine (B).
and
the additional
effects
produced
by
1
jAM
Large (about 500 jAm) and fairly uniform-sized beads were selected. [3H] Beads were placed on and removed from the papillary muscle with forceps.
Proc. Nat. Acad. Sci. USA 70
(1973)
nephrine is bound through the amine group in a manner similar to the agarose-norepinephrine derivative previously reported (10). We have been unable to demonstrate biological activity of the glass-succinyl norepinephrine beads on papillary muscles when the beads were washed extensively to remove any free norepinephrine before use. Due to the variable size and shape of the glass bead population and because of the random orientation, position, and contact of the beads on the muscle (Fig. 2), the exact dose response relationships cannot, of course, be stated. Such a study might become possible with larger, nonporous beads of uniform size and shape. By use of such beads of known surface area of contact, it should then be feasible to calculate the number of drug molecules that are in contact with individual muscle cells relative to the overall changes in contracticility of the muscle. It is of interest that the application of one isoproterenol glass bead to a papillary muscle gave the same increase in force and velocity of contraction as that produced by a 0.1 AM solution of isoproterenol on the same muscle. Based on radioactive-binding studies, one bead binds about 200 pmol of isoproterenol, with roughly 10% of the agent on the surface of the bead. If this amount were displaced from the glass, it would yield only a 4 nM solution, inadequate to produce a response in the muscle, since 20 nM isoproterenol was the lowest dose at which a response could be evoked. Moreover, the radioactive-binding studies indicated that there was no detectable elution from the beads after 30 min of contact. Numerous investigators have found cocaine to be completely without effect on the responses of isolated or intact organs to isoproterenol (11-14). In confirmation of such findings, we were unable to show any effects of cocaine on papillary muscles activated by isoproterenol in solution. In contrast, cocaine potentiates the effects of immobilized isoproterenol. The potentiation of immobilized catecholamines by cocaine may give new credence to the arguments of Kalsner and Nickerson (15), Maxwell et al. (16), Bevan and Verity (17), Varma and McCullough (18), and Shibato et al. (19), that cocaine has a post-junctional site of action. Another explanation to account for these data could be that catecholamines were eluted from the glass, but the isoproterenol potentiation data negate this thesis. It could be argued that isoproterenol covalently bound to glass beads is no longer isoproterenol, but a new compound. It also is conceivable that the bound catecholamine on carbon chains linked to beads is reaching the prejunctional membrane site and is displaced from this site by cocaine, thus allowing the catecholamine derivative to swing over to the post-junctional site of action, although the length of the carbon chain would tend to argue against this. Papillary muscles from reserpinized cats serve as a control to indicate that the immobilized catecholamines and/or cocaine were not inducing the release of endogeneous norepinephrine. Our evidence seems to indicate that isoproterenol, epinephrine, and norepinephrine bound to glass beads are re-
Immobilized Catecholamines
1217
sponsible for the changes seen in cardiac muscle contracticility. Although it has not been completely ruled out that we are dealing with extremely small amounts of unbound catecholamine effectively pooled on the cell surface, this is an unlikely mode of action in view of the characteristics of the bound radioactive catecholamines and the duration of action of the immobilized amines. The potentiation of the action of the immobilized catecholamines by cocaine raises many questions about mechanisms of action of cocaine potentiation, especially in the case of immobilized isoproterenol. The immobilized catecholamines are new tools that may be useful in elucidating this and other adrenergic mechanisms. More importantly, the immobilized catecholamines provide a working example of how various drugs and hormones may be studied not only for their mode and mechanism of action, but also as a new approach to drug delivery. This work was supported in part by grants from the National Cancer Institute (CA 11683), the American Cancer Society (BC60N), and the National Heart and Lung Institute (HL-12373). J.E.D. is a postdoctoral fellow of the National Science Foundation. 1. Venter, J. C., Dixon, J. E., Maroko, P. R. & Kaplan, N. O. (1972) Proc. Nat. Acad. Sci. USA 69, 1141-1145. 2. Innes, I. & Nickerson, M. (1970) in The Pharmacological Basis of Therapeutics, eds., Goodman, L. S. & Gilman, A. (The MacMillan Co., New York), 4th ed., pp. 478-570. 3. Parmley, W. W. & Sonnenblick, E. H. (1971) in Methods in Pharmacology, ed. Schwartz, A. (Meridith Corp., New York), Vol. I, pp. 105-123. 4. Frohlich, A. & Loewi, 0. (1910) Arch. Exp. Pathol. Pharmakol. 62, 159-169. 5. Furchgott, R. F., Kirpekar, S. M., Rieker, M. & Schwab, A. (1963) J. Pharmacol. Exp. Ther. 142, 39-58. 6. McMillan, W. H. (1959) Brit. J. Pharmacol. 14, 385-391. 7. Inman, J. K. & Dintzis, H. M. (1969) Biochemistry 8, 40744082. 8. Cuatrecasas, P. (1970) J. Biol. Chem. 245, 3059-3065. 9. Cohan, A. L., Marlow, P. P. & Garner, G. E. (1968) J. Microscop. (Paris) 7, 331-342. 10. Lefkowitz, R. L., Haber, E. & O'Hara, D. (1972) Proc. Nat. Acad. Sci. USA 69, 2828-2832. 11. Maxwell, R. A., Daniel, A. I., Sheppard, H. & Zimmerman, J. H. (1962) J. Pharmacol. Exp. Ther. 137, 31-38. 12. Smith, C. B. (1963), J. Pharmacol. Exp. Ther. 142, 163-170. 13. Trendelenburg, U. (1966) Pharmacol. Rev. 18, 629-640. 14. Hardman, J. G., Mayer, S. E. & Clark, B. (1965) J. Pharmacol. Exp. Ther. 150, 341-348. 15. Kalsner, S. & Nickerson, M. (1969) Brit. J. Pharmacol. 35, 428-439. 16. Maxwell, R. A., Eckhardt, S. B. & Wastila, W. B. (1968) J. Pharmacol. Exp. Ther. 161, 34-39. 17. Bevan, J. A. & Verity, M. A. (1967) J. Pharmacol. Exp. Ther. 157, 117-124. 18. Varma, D. R. & McCullough, H. N. (1969) J. Pharmacol. Exp. Ther. 166, 26-34. 19. Shibata, S., Hattori, K., Sakurai, I., Mori, J. & Fujiwara, M. (1971) J. Pharmacol. Exp. Ther. 177, 621-632.
