Autonomic stimulating and direct actions of cyclohexylamine in isolated tissues

TOXICOLOGY AND APPLIEDPHARMACOLOGY 13,

Autonomic

Stimulating

339-345

(1968)

and Direct Actions in Isolated Tissues

of Cyclohexylamine

I. ROSENBLUM AND G. ROSENBLUM Department

of Pharmacology Albany

and Institute Medical College, Received

of Experimental Pathology Albany, New York 12208 May

and Toxicology,

7, I968

Autonomic Stimulating and Direct Actions of Cyclohexylamine in Isolated Tissues. ROSENBLUM,I., and ROSENBLUM,G. (1968). Toxicol. Appl. Pharmacol. 13, 339-345. The effects of cyclohexylamine have been studied in isolated tissues. It caused contraction of the smooth muscle of the guinea pig ileum by activation of cholinergic atropine-sensitive receptors but not strychnine-sensitive presynaptic receptors. The contractions of the ileum were not enhanced by physostigmine. It also contracted the rat urinary bladder by activation of hexamethonium-sensitive receptors. Contraction of rat vas deferens could be blocked by either phenoxybenzamine, phentolamine, or atropine, indicating activation of alpha adrenergic receptors as well as cholinergic receptors of that tissue. Contraction of a skeletal muscle, the frog rectus abdominis, could not be blocked by d-tubocurare demonstrating a noncholinergic stimulation by cyclohexylamine. This action on skeletal muscle occurred in a calcium-free medium and after muscle depolarization with excess potassium. Although the contraction could be reversed in part, by addition of acid to the medium, it was not due entirely to a pH effect of cyclohexylamine. Among the first experiments with cyclohexylamine were those of Barger and Dale (1910), who demonstrated that the agent elevated blood pressure in cats; but because of short supply, they made no further characterization of the compound. Since then, many compounds of a similar type, including cyclohexylalkylamines, have been shown to have pressor activity (Gunn and Gurd, 1940; Fellows et al., 1950; Kosegarten et al., 1967). It is generally agreed that all thesecompounds causetheir vasopressorresponse by a sympathomimetic action although other effects of some of these agents do not mimic the actions of endogenous catecholamines. Thus, in the experiments of Gunn and Curd (1940), cyclohexylethylamine did not produce the inhibition of cat intestine or uterus that is characteristic of epinephrine. Indeed, it is of some historical interest that Barger and Dale (1910) stated in speaking of cyclohexylamine, “the possibility is open that this is not of the sympathomimetic type.” Our earlier experiments have shown that the hypertensive effect of cyclohexylamine is mediated through activation of alpha adrenergic receptors (Rosenblum and Rosenblum, 1968). That study also indicated that cyclohexylamine produced cardiovascular effects uncharacteristic of a sympathomimetic amine thus suggestingother additional pharmacologic properties. The present report concerns these other effects. 339

340

ROSENBLUM

AND

ROSENBLUM

METHODS

Tissues, consisting of rat “stripped” vas deferens, urinary bladder, and guinea pig ileum were obtained after sacrifice by decapitation. Frog rectus abdominis muscle was dissected from doubly pithed animals. These tissues were arranged for isometric recording using Statham force-displacement transducers, and transducer signals were recorded on an Offner Dynograph. The mammalian tissues were immersed in oxygenated (95 % O2 and 5 % CO*) Ringer-Locke USP XIV solution consisting of the following salts (in g/liter quantities): NaCl (9.0), KC1 (0.42), CaC12 (0.24) MgCl,.6HzO (0.20), NaHCOs (0.5), and dextrose (0.5). This bath solution was maintained at 37” zt 1.O”C. Frog rectus abdominis muscles were placed in oxygenated (95% O2 and 5% COJ solution consisting of g/liter quantities of NaCl (6.0), KC1 (0.075), CaC12 (0. IO), and NaHCOs (1 .O). For a calcium-free solution, CaClz was omitted from this solution and sodium ethylenediaminetetraacetic acid (Na EDTA) (0.0074) was added to chelate “free” calcium. Depolarization of the muscle membrane with potassium was accomplished by using KC1 (2.98) in place of the usual amount of the salt. All experiments were at ambient temperature. The following drugs were used in addition to cyclohexylamine: acetylcholine chloride, atropine sulfate, hexamethonium chloride, phenoxybenzamine hydrochloride, phentolamine hydrochloride, physostigmine sulfate, procaine hydrochloride, strychnine sulfate, and d-tubocurarine chloride. Each was dissolved in 0.9 % NaCl with the exception of phenoxybenzamine, which was dissolved in absolute ethyl alcohol. They were added as 0.1 ml/l00 ml of bath solution. All concentrations of these drugs are expressed as weight of base. Cyclohexylamine was diluted in 0.9% NaCl and added in volumes of 0.1 ml to 1.0 ml/l00 ml of bath solution. Graded amounts of cyclohexylamine were added to the bath solution without washing between doses to produce cumulative dose-response curves (Van Rossum, 1963). Where blocking drugs were used, they were added 30 minutes before the doses of cyclohexylamine. The effectiveness of most blocking drugs was evaluated by using the appropriate agonist. The blocking action of atropine was challenged in the guinea pig ileum and rat vas deferens with 1 pg/ml of acetylcholine. The blocking effect of hexamethonium was challenged in the rat urinary bladder with 10 pg/ml of nicotine. The blocking action of phenoxybenzamine and phentolamine in the rat vas deferens was challenged with 5 pg/ml of norepinephrine and the blocking action of d-tubocurarine in the frog rectus abdominis was challenged with 10 pg/ml of acetylcholine. In these experiments, the response was recorded as a change in tension and it is expressed as grams of tension per tissue. The initial tension applied to ileum and vas deferens was 2 g while 5 g was applied to urinary bladder and rectus abdominis. The equilibration period for the tissues was 1 hour. Statistical comparisons were made by single-tail t test (Goldstein, 1964). RESULTS Guinea Pig Ileum

Cyclohexylamine (CHA) caused contraction of this smooth muscle (Table 1). The response could be blocked by atropine (0.4pg/ml), but not by hexamethonium (8 pg/ml).

CYCLOHEXYLAMINE

ON

SKELETAL

AND

SMOOTH

341

MUSCLE

Strychnine (2 pg/ml) blocked only the lowest (50 pg/ml) dose of CHA. The presence of physostigmine (0.01 pg/ml) did not affect the response to CHA, and higher concentrations of physostigmine could not be used because they produced contractions of the ileum. TABLE RESPONSE

OF GUINEA

DoseCHA” (tLg/mU

Control

400 200

2.9 zk 0.3 2.1 z!Y0.4

PIG ILEUM

Atropine

100

1.3 ZlI0.3

50

0.6 ?c 0.1

(0.4 pg/ml)

1

TO CYCLOHEXYLAMINE

IN GRAMS

Hexamethonium (8 tdml)

2.6 zt 0.3 0.6 zt O.lb 0.2 + 0. lb Ob

3.4 2.9 1.4 0.6

OF TENSION

Physostigmine Strychnine (0.01 pg/ml) (2 t4idmO 2.8 & 0.1 2.3 -c 0.1 1.450.1 0.4 * 0.1

zt 0.4 It 0.3 + 0.2

!I 0.1

3.4 2.0 0.9 0.2

rfr 0.2 * 0.2 IO.1 f O.lb

’ CHA iscyclohexylamine. bMeanvalue+ SE(12= lo), differssignificantlyfrom control (12= 10)at P = 0.05or less. Rat Vas Deferens CHA also caused graded contractions of the vas deferens (Table 2). Blockade of contraction could be obtained with atropine (0.4 pg/ml) and with two types of alpha adrenergic receptor blocking drugs, phenoxybenzamine (10 pg/ml) and phentolamine (1 tLg/ml). TABLE RESPONSE

DoseCHA” ~~dml) 800 400 200 100

50

OF RAT

VAS DEFERENS

TO CYCLOHEXYLAMINE

Atropine Control 3.1 2.5 1.4 0.9 0.4

zt 0.2 + 0.2 i 0.1 IO.07 * 0.05

2

(0.4 m/ml)

1.9 It O.lb 0.9 i 0.08b 0.2 f omb ;:

IN GRAMS

Phenoxybenzamine (10 fdml) 2.0 i- 0.2b 0.9 i O.lb 0.2 I0.3b Ob Ob

OF TENSION

Phentolamine (1 pg/ml) 1.7 -c 0.3b 1.3 I 0.2b 0.7 E O.lb

’ CHA iscyclohexylamine. b MeanvaluesI SE(n = lo), differssignificantlyfrom control (n = 10)at P = 0.05or less. Rat Urinary Bladder Contraction of the bladder muscle by CHA could not be blocked by phenoxybenzamine (10 pg/ml) or atropine (10 pg/ml). Blockade of contraction could however be produced by hexamethonium (40 pg/ml) (Table 3).

342

ROSENBLUMAND

ROSENBLUM

TABLE RESPONSE OFRAT URINARYBLADDERTO Dose CHA” hM-4 800 400 200

Control 8.1 * 0.9 5.5 * 0.4 1.1 * 0.2

3

C~CLOHEXYLAMINEIN

Atropine (10 kcdml)

Hexamethonium (40 wdml)

7.9 * 0.6 5.1 f 0.4 1.0 f 0.2

4.5 f 0.4b 2.4 zt 0.3b 0.7 f 0.1

GRAMSOFTENSION Phenoxybenzamine (10 e/ml) 7.4 + 0.8 4.9 + 0.5 1.3 f 0.3

’ CHA is cyclohexylamine. b Mean value f SE (n = lo), differs significantly from control (n = 10) at P = 0.05 or less.

Frog Rectus Abdominis The rectus abdominis muscle was contracted by graded doses of CHA (Table 4). The contractions were not blocked by either d-tubocurare (10 pg/ml) or procaine (1 mg/ml). The response to CHA could still be elicited in calcium-free solution and in TABLE RESPONSE OF FROG RECTUSABDOMINISTO Dose CHA”

hdml) 1600 800 400 200

Control 11.1 It 0.8 8.640.7 4.9 f 0.6 1.5 rto.4

4 CYCLOHEXYLAMINEIN

GRAMSOFTENSION

Procaine

d-Tubocurare

Ca2+ Free

Double Ca2+

K+ (40 mM)

(1 mg/ml)

(10 e/ml)

13.0 * 0.4 10.3f0.3 4.2 + 0.4 1.2f0.3

13.0 + 1.1 11.9&0.9b 6.0 + 0.4 0.8 f 0.1

7.1 & 0.8b 5.0*1.0b 1.5 f 0.2b 0.4 f O.lb

12.7 f 0.9 lO.lhO.5 6.2 f 0.5 2.3 5 0.2

10.7 7.3 3.9 1.1

f f * It

0.8 1.2 0.8 0.3

g CHA is cyclohexylamine. b Mean value + SE (n = lo), differs significantly from control (n = 10) at P = 0.05 or less.

the presence of twice the usual amount of calcium. Depolarizing the muscle with excess potassium reduced the contractions produced by CHA significantly (P < 0.05) but did not abolish them.

CHA (1.5mg/ml)

10”

CHA HCI (I. 5 mg/ml)

FIG. 1. Partial reversal by hydrochloric acid (HCl) of tension developed in frog rectus abdominis muscle by addition of cyclohexylamine. At the point of the arrows, 1.5 mg/ml of cyclohexylamine was added to each muscle bath. During the peak of contraction, sufficient HCI was added to one muscle (panel B) to give a pH of approximately 7 while the control muscle (panel A) was not treated with HCl. The fmal pH values for each bath solution are shown in parentheses. Note the rapid relaxation of tension produced by the addition of HCl.

c~c~or-mxyL.4~1~~ ON SKELETAL AND SMOOTH MUSCLE

343

Concentrations of CHA such as were used in these experiments made a solution with a final pH of greater than 9. In order to assessthe effect of pH upon contraction produced by CHA, the pH of the solution at the peak of contraction was reduced toward 7 by addition of HCl. Figure 1 shows that this reduction of pH lowers muscle tension but does not abolish the rise in tension caused by CHA. DISCUSSION Our previous experiments have demonstrated a hypertensive effect of CHA in cats which is due to stimulation of alpha adrenergic receptors (Rosenblum and Rosenblum, 1968). The present experiments on isolated tissues show additional effects of CHA on autonomic receptors which are cholinergic in nature. One such tissue, the rat vas deferens, probably contains both adrenergic and cholinergic receptors since catecholamines and choline acetylase have been found within it, and it is contracted by either norepinephrine or acetylcholine (Ohlin and Stromblad, 1963). CHA caused contraction of the vas deferens which could be blocked by phentolamine, phenoxybenzamine, or atropine. Blockade by phentolamine indicates that CHA caused contraction of the vas deferens by stimulation of alpha adrenergic receptors but because the response could also be blocked by atropine, stimulation of cholinergic “muscarinic” receptors must also have been involved. It has also been reported (Graham et al., 1968) that dibenamine-type compounds, like phenoxybenzamine, can block acetylcholine-induced contraction of the rat vas deferens. Additional evidence that CHA can activate cholinergic receptors comes from the observations with guinea pig ileum. The contraction of this smooth muscle by CHA could be readily blocked by atropine. No stimulation of “nicotinic” cholinergic receptors was demonstrable in this preparation since hexamethonium, a ganglionblocking drug, could not reduce the effect of CHA, nor could, in most cases, strychnine which is believed to be a presynaptic blocking agent (Neal, 1967). Physostigmine did not potentiate the response to CHA, indicating a direct action of CHA on cholinergic receptors independent of release of acetylcholine. It may also indicate that acetylcholine esterase does not act upon CHA to terminate its cholinergic actions. Stimulation of “nicotinic” cholinergic receptors by CHA does evidently occur in the rat urinary bladder. This tissue is innervated only by cholinergic motor nerves in the rat (Vanov, 1965) and is contracted by either acetylcholine or nicotine (Chesher and James, 1966). This latter nicotinic stimulation of ganglion cells of the bladder produces an “atropine-resistant” contraction (Chesher and James, 1966). In the present experiments, CHA caused contraction of the urinary bladder that could not be blocked by atropine, but it was blocked by hexamethonium in a concentration reported to block nicotinic stimulation of the guinea pig urinary bladder (Chesher and James, 1966). It has been shown that norepinephrine can cause slow contraction of the urinary bladder (Hukovic et al., 1965). Bladder contractions produced by CHA developed rapidly and were not blocked by phenoxybenzamine, evidently indicating that they were not mediated through stimulation of alpha adrenergic receptors. The effect of CHA on skeletal muscle was also studied, using the frog rectus abdominis. CHA produced graded contractions of the rectus abdominis that were not blocked by d-tubocurare, indicating that contractions did not involve an acetylcholine-

344

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AND

ROSENBLUM

like action (Van Maanen, 1950). Since contractions, though reduced, could still be produced in potassium-depolarized muscles and in the present of the membranestabilizing drug procaine (Bianchi and Bolton, 1967), it is suggested that the contractions did not depend on depolarization of the muscle membrane. CHA still caused contractions in calcium-free electrolyte solution containing Na EDTA. Since it is generally accepted the calcium is necessary for muscle contraction (Sandow, 1965), this observation indicates that another source of external calcium was made available for contraction. The source of this calcium may have been the stores bound in the sarcoplasm, which could have been released by CHA. Doubling the external calcium usually found in the electrolyte solution did not alter the response to CHA, emphasizing further an independence of external calcium. It is evident from these experiments that contraction of rectus abdominis by CHA resulted from a direct action on the muscle. One such action could be related to the shift in pH which high concentrations of CHA causes. This shift to an alkaline pH seems to be partially responsible for the rise in tension produced by CHA since a reduction in pH by addition of HCl leads to a lowering of muscle tension. The shift in pH may also account for the inability of procaine to block some of the effect of CHA. At a pH of 9.2, only about 36% of the procaine molecule is in the cationic form (Bianchi and Bolton, 1967), and it is this form that is said to be responsible for the membrane-stabilizing effect of the drug (Ritchie et al., 1965). It is concluded from these experiments that CHA has a number of actions in vitro. It can activate alpha adrenergic receptors and stimulate cholinergic “muscarinic” and “nicotinic” receptors in smooth muscle and can, by direct action, cause contractions of skeletal muscle. ACKNOWLEDGMENTS This work was supported in part by the Food and Drug Administration, Department of Health, Education, and Welfare, through Contract No. FDA 67-12. The authors wish to express their gratitude to Dr. Frederick Coulston, Director, Institute of Experimental Pathology and Toxicology, Albany Medical College, for his activities as consultant in these experiments. REFERENCES G., and DALE, H. H. (1910). Chemical structure and sympathomimetic action of amines. J. Physiol. (London) 41, 19. BIANCHI, C. P., and BOLTON, T. C. (1967).Action of local anestheticson coupling systemsin muscle.J. Pharmacol. Exptl. Therap. 157, 388. CHESHER, G. B., and JAMES, B. (1966). The “nicotinic” and “muscarinic” receptors of the urinary bladder of the guineapig. J. Pharm. Pharmacol. 18,417. FELLOWS, E. J., MACKO, E., and MCLEAN, R. A. (1950).Observationson severalcyclohexyland phenyl-alkyamines.J. Pharmacol. Exptl. Therap. 100,267. GOLDSTEIN, A. (1964).Biostatistics, p. 51. Macmillan, New York. GRAHAM, J. D. P., KATIB, H. Al, and SPRIGGS, T. L. B. (1968).The isolatedhypogastricnervevas deferenspreparation of the rat. Brit. J. Pharmacol. 32, 34. GUNN, J. A., and GURD, M. R. (1940). The action of someaminesrelated to adrenaline. Cyclohexylalkylamines.J. Physiol. (London) 97,453. HUKO~IC, S., RAND, M. J., and VANOV, S. (1965). Observationson an isolated, innervated preparation of rat urinary bladder. Brit. J. Pharmacol. 24, 178. BARGER,

CYCLOHEXYLAMINE ON SKELETAL AND SMOOTH MUSCLE

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KOSEGARTEN,D. C., DEFEO, J. J., and DEFANTI, D. R. (1967). Comparative cardioinhibitory effects of certain cyclohexanol and cyclohexylamine derivatives. J. Pharm. Sci. 56, 1104. NEAL, M. J. (1967). The effect of convulsant drugs on coaxially stimulated guinea-pig ileum. Brit. J. Pharmacol. 31, 132. OHLIN, P., and STROMBLAD, B. C. R. (1963).Observationson the isolatedvas deferens.Brit. J. Pharmacol.20,299. R~TCHIE,J. M., RITCHIE,B., and GREENGARD, P. (1965). The active structure of local anesthetics.J. Pharmacol.Exptl. Therap. 150, 152. ROSENBLUM, I., and ROSENBLUM, G. (1968). Cardiovascular responsesto cyclohexylamine. Toxicol. Appl. Pharmacol.12,260-264. SANDOW,A. (1965). Excitation-concentration coupling in skeletalmuscle.Pharmacol.Rez?. 17, 265.

VAN MAAHEN,E. F. (1950).The antagonismbetweenacetylcholine and the curare alkaloids, d-tubocurarine, c-curarine-I, c-toxiferine-II and 6-erythroidine in the rectus abdominusof the frog. J.Pharmacol.Exptl. Therap.99,255. VANOV,S.(1965).Responses of the rat urinary bladderin situto drugsandto nerve stimulation. Brit. J. Pharmacol.24, 591. VAN ROSSUM, J. M. (1963). Cumulative dose-response curves. II. Techniquefor the making of dose-response curves in isolated organsand the evaluation of drug parameters.Arch. Intern. Pharmaco&n. 143, 299.