Journal of the Autonomic Nervous System, 35 (1991) 211-218 211 © 1991 Elsevier Science Publishers B.V. All rights reserved 0165-1838/91/$03.50

JANS 01196

Selective pharmacological blockade of parasympathetic and enteric ganglia

V.I. Skok 1 S.D. Groisman 2 L.V. Melni tchenko 1, V.V. Gersanich 1 and V.E. Gmiro 3

1 Bogomoletz Institute of Physiology, Kiev, 2 Institute of Physiology, Kiec State University, Kiev, and 3 Institute of Experimental Medicine, Leningrad, U.S.S.R.

(Received 26 November 1990) (Revision received and accepted 10 May 1991)

Key words: Parasympathetic ganglion; Enteric ganglion; Ganglion-blocking agent; Neurochemistry

Abstract

The blocking effects of two newly synthetized compounds, diisopropyldecylammonium iodide (IEM-1194) and 2,2,6,6-tetrameth- yldecylpiperidinium chloride (IEM-1559), on insulin- and pentagastrin-induced gastric secretion in chronic dogs, on stress-induced changes in gastric mucosa in rats, on vagus-induced effects in heart and intestine, on arterial blood pressure and on synaptic transmission through isolated ciliary and superior cervical ganglia in cats were studied. The effects observed were compared with those produced by hexamethonium (C6), a conventional ganglionic blocking agent. Both IEM-1194 and IEM-1559 inhibited gastric secretion and acid output for a much longer time than C 6 did and effectively protected gastric mucosa against stress-induced erosions and hemorrhages. IEM-1194 blocked the vagus-induced decrease in heart rate and increase in duodenal motility for a longer time than did C 6 and also, in contrast to C6, did not reduce the arterial blood pressure. The blockade of synaptic transmission through isolated ciliary and superior cervical ganglia produced by IEM-1194 and IEM-1559 was characterized by lower ECs0 and was more prolonged than that produced by C 6. In addition, both IEM-1194 and IEM-1559 were more potent blocking agents in ciliary ganglion than in superior cervical ganglion. It is suggested that IEM-1194 and IEM-1559 are selective blocking agents for parasympathetic and enteric ganglia versus sympathetic ganglia.

Introduction

Ganglionic blockers were commonly used in the past for treatment of various disorders of the autonomic nervous system [8]. Later they were almost completely abandoned because of wide use of more selective blockers of neuro-effector synaptic transmission. However, at present an

Correspondence: V.I. Skok, Autonomic Nervous System Physi- ology Department, Bogomoletz Institute of Physiology, Kiev 24, U.S.S.R.

increasing interest in ganglion blockers is again observed. This is due to recent findings of great diversity among the neurons of autonomic ganglia and plexuses in their pharmacology which pro- vides a possibility for their highly selective phar- macological blockade.

Furthermore, nicotinic acetylcholine receptors (AChRs) in nerve cells which are most important for synaptic transmission through autonomic gan- glia and have long been considered as a biochem- ically homogeneous receptor group, appear to be of rather variable molecular structure [1,3,14].

212

This observation provides a new insight into the origin of the diversity in pharmacological proper- ties within the group of neuronal AChRs.

One of the advantages of the blockade of autonomic ganglia, if compared with the neuro- effector blockade, is that in the latter many in- trinsic interneuronal functional connections in the ganglia and plexuses as well as their neuro-effec- tor connections remain intact.

It was shown that the organic ions of general formula CH3-(CHe)n-NRIR2R3, with n = 9 and R 1, R e and R 3 being isopropyl or aromatic radi- cals of equivalent size, predominantly block parasympathetic rather than sympathetic ganglia in mammals [6]. The molecular mechanism un- derlying their blocking effect is thought to be a combination of their non-reversible binding to AChR with a reversible blockade of the AChR open ionic channel [7; see 13,14].

The aim of the present work was to study the effects produced by this group of compounds in parasympathetic, enteric and sympathetic ganglia and to test their parasympathetic versus sympa- thetic selectivity. A preliminary report has been published [11].

Materials and Methods

The blocking effects of the compounds on gas- tric secretion in dogs, on stress-induced changes in gastric mucosa in rats, on the level of arterial blood pressure and on the vagus-induced effects in heart rate and in the intestinal motility in cats, and on synaptic transmission through isolated sympathetic and parasympathetic ganglia in cats, were studied.

The effects on gastric secretion were studied in beagle dogs weighing 17-20 kg, with chronic gastric fistulas. The experiment started after a 16-18 h fast. Following a 30-min control period during which there was no background secretion, insulin (0.5 U / k g ) or pentagastrin (5 ~ g / k g ) were injected subcutaneously to stimulate gastric se- cretion. The insulin-induced gastric secretion was due to activation of vagal efferents caused by hypoglycemia [9].

The blocking compounds were injected intra-

venously 10 min before pentagastrin was applied and 30 min after insulin was applied. Four and eight 30-min samples of gastric juice were col- lected with pentagastrin- and insulin-induced se- cretion, correspondingly; their volume was mea- sured, and they were analyzed for acid (electro- titration method) and pepsin contents (Hunt method).

The effects of blocking agents on stress-in- duced changes in gastric mucosa were examined in 12 white rats weighing 100-150 g. The animal was put into a space-limiting metal cartridge with a diameter somewhat larger than that of the animal body (the procedure was not painful for the animal). The transparent perspex bottom al- lowed the animal to see all around. The ganglion-blocking agents were injected intraperi- toneally (except for the control group of animals) immediately before the animal was put into the cartridge.

The rat was left in the cartridge for 24 h in the lit cage with other rats, moving freely and with access to food and water. This was usually fol- lowed by the appearance of changes of three types which could be observed in gastric mucosa of the experimental animals killed immediately after the experiment: (1) erosions, small cut-like changes; (2) small dot-like hemorrhages less than 1 mm in diameter; and (3) hemorrhages 2-3 mm or more in diameter.

The effects produced by the blocking agents on the level of arterial blood pressure and on the vagus-induced effects in heart and intestine were studied in acute experiments on cats anesthetized with hexenal (50 mg/kg , intramuscular injection), urethane and chloralose (250-500 m g / k g and 30 m g / k g intravenous injection, correspondingly). The blood pressure was measured with a tenso- metric transducer. The left vagus nerve was cut at the neck, and its caudal portion was stimulated with a 30-s series of 1 ms stimuli at 20 Hz. The ECG R - R intervals were measured with an elec- trical integrative circuit. The duodenal motility was recorded with a 2 cm balloon fixed 7-10 cm distal to the pyloric sphincter. To estimate the intensity of the motor response to vagal stimula- tion, the area in the tracings underneath a con- traction over a time twice as long as stimulation

period was measured. The arterial blood pressure was monitored~

The effects of blocking agents on synaptic transmission through isolated ganglia were stud- ied in ciliary and superior cervical ganglia of the cat. Both ganglia were taken from the same ani- mal in an acute experiment under nembuta l / hexenal anesthesia and were perfused in the com- mon bath at 35 o C with the physiological solution of the following composition (in mM): NaC1, 137.9; KC1, 4.0; MgC12, 0.5; KH2PO4, 1.0; NaHCO3, 12.0; glucose, 11.0. The solution was equilibrated with 95% 0 2 and 5% CO 2. Single electrical stimuli were applied to the oculomotor nerve and the cervical sympathetic nerve which contained preganglionic fibres to ciliary and su- perior cervical ganglia, correspondingly, through sucking pipette electrodes (see inset in Fig. 6). Similar electrodes were used to record action potentials from ciliary nerves and from internal carotid nerves containing postganglionic fibres of ciliary and superior cervical ganglia, correspond- ingly.

Two compounds, diisopropyldecylammonium iodide (IEM-1194) and 2,2,6,6,-tetramethyldecyl- piperidine chloride (IEM-1559), were tested for their ganglion-blocking activity. For comparison, hexamethonium dibromide, a classical ganglion- blocking agent, was also used. All compounds used were synthesized in the Institute of Experi- mental Medicine of the U.S.S.R. Academy of Medical Sciences.

Results

Effects of the ganglion-blocking agents on gastric secretion

Fig. 1 summarizes the results of studies into the effects produced by IEM-1559 and IEM-1194 on the insulin-induced (0.5 U / k g ) gastric secre- tion in three dogs. The effects of IEM-1559 were studied in 18 experiments, and the effects of IEM-1194 in 12 experiments. As shown in Fig. 1, 0.24 m g /kg IEM-1559 and 0.03 m g / k g IEM-1194 markedly reduced the juice volume and the acid output. The effect was also observed at 0.12 m g / kg IEM-1559 but at this dose it was insignifi-

213

*/o

150 [ ] Gastric juice volume (ml]

[ ] Output of acid [raM)

[ ] Output of pepsin [rag]

iiiii!!i!!i

ii iiiiii!

5 C ........

I E M - 1 5 5 9 IEM -1559 I E M - 1 5 5 9 IE M-1194 0 .06 mg /kg 0.12 m g / k g 0 2 4 m g / k g 0.03 m g / k 9

Fig. ]. Effect of [EM-]559 and ]EM-1194 (the i.v. doses indicated) on insulin-induced (0.5 U/kg, s.c.) gastric secretion

in the dog. For further details see text.

cant. Pepsin output was increased by 0.12 m g /k g IEM-1559 and by 0.03 m g / k g IEM-1194, but slightly decreased by a higher dose of IEM-1559. No effects were observed at a dose of IEM-1559 as low as 0.06 mg/kg.

Similar effects were produced by IEM-1194 on the gastric secretion stimulated by pentagastrin (5 /zg/kg), as shown in Fig. 2. The results were obtained from one dog in 7 control experiments, and with 6 .25 /zg /kg (4 experiments), 12 .5 /zg/kg

%

100

50

i~i!iii!i~]

ii!!iii i!} 0

[ ] Gastric juice volume (ml }

[ ] OuLpl~t of acid (mh4)

[ ] Output of pepsin [ma)

L 6.25 12.5 5 0

Dose of IEM-1194 ~Jg/kg, i,v.

Fig. 2. Effect of IEM-]194 (i.v. doses indicatedi' on pentagas- trin-induced (5 g/kg, s.c.) gastric secretion in the dog. For

further details see text.

214

*/o °o of rnasslCe haernorrage [ ] Er cs,ons per anlma! [ ] a f fected an~mql%

T O,o ot erosion affected *Io of polnt haerJ ,oFrage • an~r11Qls [ ] affected animals

[ ] P,!asstve haemorr0ges per {3nimal

100

50

o t__ I I I. i

C o n t r o l 2 m g / k g 4 m g / k g

IE iv l - 1 1 9 4 I E M - 1 1 9 4

Fig. 3. Effect of IEM- I |94 on experimental u|cerogenesis (stress-induced damages in gastric mucosa) in rats. For further

details see text.

(4 experiments) and 50 Izg/kg (5 experiments) of IEM-1194. At 12.5 /xg/kg dose, IEM-1194 markedly reduced juice volume, acid output and pepsin output, while somewhat weaker effects were observed at doses lower and higher than this. It can be concluded that both ganglion- blocking agents strongly reduce the insulin- and pentagastrin-induced gastric secretions and the acid outputs, whilst their effects on the output of pepsin are different, depending on whether in- sulin or pentagastrin is used to stimulate the secretion.

Effects of the ganglion-blocking agents on stress-in- duced changes in gastric mucosa

Gastric mucosa was examined in 12 rats after social stress experiments. The results obtained are summarized in Fig. 3. One can see that 2 mg / kg IEM-1194, if injected before the experi- ment, dramatically reduces both the number of hemorrhages per animal and the number of the animals affected. Marked reduction, although less strong, has been observed in the number of ero- sions per animal and in the number of the ero- sion-affected animals. There have been, however, no obvious changes in the dot-like hemorrhages of the mucosa.

For comparison, hexamethonium (10 mg/kg, i.v.) and pirenzepine (12.5 mg/kg, i.v.), the anti- ulcer drugs, were used instead of IEM-1194. The reduction in the number of erosions per animal and in the number of erosion-affected animals following the administration of either of these two drugs was 1.5-2.0 times less pronounced than in IEM-1194, whilst other types of stress-induced changes of gastric mucosa were not reduced. It can be concluded that IEM-1194 is much more effective than some conventional anti-ulcer drugs in decreasing the stress-induced changes of gas- tric mucosa.

Effect of the ganglion-blocking agents on the ua- gus-induced effects in the heart and intestine and on the arterial blood pressure

Intravenous injection of IEM-1194 was fol- lowed by a decrease in the contraction of the duodenum evoked by vagal stimulation, with EDs0 = 0.49 _+ 0.12 mg/kg (n = 3). The effect is demonstrated in Fig. 4A1, and in the inset of Fig. 5. Maximal blockade developed about 30 min after the administration of IEM-1194.

In order to know whether the above effect was due to the blockade of nicotinic or muscarinic AChRs, the ability of IEM-1194 to inhibit the atropine-sensitive duodenal contraction evoked by carbachol (5 mg/kg, i.v.) was tested (Fig. 4B1). No inhibition of the carbachol-induced contrac- tion was observed.

IEM-1194 also reduced the decrease in the heart rate and a decrease in arterial blood pres- sure produced by vagal stimulation (Fig. 4A2,3; Fig. 5), the former effect being characterized by ED~0 = 0.07 m g /k g (n = 3). The time-course of both blocking effects resembled that in the intes- tine.

The level of arterial blood pressure either re- mained unchanged or slightly increased following the administration of large doses of IEM-1194 (Fig. 5). No significant increase in the level of the blood pressure was observed at the doses of IEM- 1194 comparable to ED~0 for the above effects in the heart and in the intestine.

If compared with IEM-1194, hexamethonium (0.2 mg/kg) exerted faster and more short-lasting blocking effects in the heart and in the intestine

215

(Fig. 5), with EDs0 = 0.19 + 0.03 m g / k g (n = 4)

in the hear t and EDs0 = 0.18 + 0.06 m g / k g (n = 3) in the intest ine. In contrast to IEM-1194, hex- a m e t h o n i u m at doses comparab le to ED50 for its blocking effects in the vagus- induced decrease in the hear t rate and in the cont rac t ion of the in tes t ine markedly reduced the level of the arte- rial b lood pressure (Fig. 5).

Effects of the ganglion-blocking agents on synaptic transmission in parasympathetic and sympathetic ganglia

The blocking activity of the compounds was es t imated from a decrease in the ampl i tude of postgangl ionic act ion potent ia ls evoked by pre- gangl ionic st imuli following bath appl icat ion of blocking compounds to the isolated ciliary gan- glion and super ior cervical gangl ion of the cat. At

A [EM-1194 0 3 m g / k g

Contro l {50 min 180 min

mm H20

ms

3 f;oo o

3 0 s

B Corbochol 5 ~g / kg

IEM-1194 0.5 m g / k g + Corbochol 5 Ng/kg

rnm H20 ~ . , 1320

mm Hg . . . . . . . . . . . .

2 min

Fig. 4. Effect of IEM-1194 on vagus-induced (A) and carba- chol-induced (B) contractions of duodenum (1), decrease in I heart rate (A, 2; the R-R intervals of the ECG are indicated), i and in the blood pressure level (A, 3; B, 2) in cat. The 30-s long stimulation periods and the injection of carbachol (arrow)

are indicated beneath.

*/,

. . . . o ~ - - ~ ' 0 o ~ -o 1 1001~

5oI~ \ ~o -~ .~ .x 3

°1- ' ,'0 ' ~o ' ~'o Min.

*1,

-120

LH~

stim. 30 s

1 O0 ~ , , o _ _ o / o.-.............._o ._._ o J~ 1

w ×

O~ i I l i I i lO ~'o ~o 4'0

Min

C 6 : 0.2 m g / k g ; I E M - 1 1 9 4 : 0 . 6 m g / k g

Fig. 5. Effect of hexamethonium and IEM-1194 on the arterial blood pressure (1), on vagus-induced increase in duodenal motility (2) and decrease in heart rate (3) in the cat. Abscissa: the time after the administration of the blocking drug. Ordi- nate: the level of the blood pressure and the amplitude of the corresponding response in % to their control values. The concentrations of the blocking drugs used are indicated be-

neath.

a concen t ra t ion of 1 × 10 -5 M, IEM-1194 re-

duced the postgangl ionic act ion potent ia l in the ciliary gangl ion to less than 50% of its control ampl i tude , while only slightly reducing its ampli- tude in super ior cervical gangl ion (Fig. 6).

At a higher concen t ra t ion (4 x 10 -5 M), IEM-

1194 blocked completely the t ransmiss ion in cil-

TABLE 1

Blocking activities of IEM-1194 and hexamethonium esti- mated from their effects on synaptic transmission through isolated superior cervical and ciliary ganglia in the cat. The concentration EC50 of the blocking drugs and the number of experiments (n) are indicated. For further details see text.

Ganglion ECs0 (mol/1 x 10-5)

Hexamethonium IEM-1194

Superior cervical 11.6 4- 1.7 3.2 4- 0.2 ganglion (n = 10) (n = 3)

Ciliary 38.5 _+2.2 1.15 4-0.34 ganglion (n = 4) (n = 3)

2 1 6

googr, on

It I ~ ~. ~s I

I I% ~,. s

C 6

S C G C G

4 x 10-4M

Wash

J l mV [1 mV

50 ms

/EM - 1194

S C G C G

4 x 1 0 - S M

0 5 m V j 0 5 rnV

50 ms

Fig. 6. Effect of IEM-1194 and hexamethonium (C 6) on synaptic transmission through isolated superior cervical ganglion (s.c.g.) and ciliary ganglion (c.g.) in the cat. The concentrations of the blocking agents are indicated over the records obtained 30 rain after

the blocking agents were applied. Inset demonstrates the experimental procedure. For further details see text.

iary ganglion, while a postganglionic action po- tential of about 60% of its control amplitude remained in superior cervical ganglion. This is just the opposite to what was observed with hex- amethonium (Fig. 5). The values of ECs0 esti- mated from these results are shown in Table I. One can see from Table I that IEM-1194 is much more effective than hexamethonium in blocking both ciliary and superior cervical ganglia, and also is more effective in ciliary than in superior cervical ganglion.

In conclusion, IEM-1194 is a more effective ganglion-blocking agent than hexamethonium in a parasympathetic ganglion and, .in contrast to hexamethonium, is more effective in parasympa- thetic than in sympathetic ganglia.

Discussion

From the above results, we conclude that IEM-1559 and IEM-1194 are potent inhibitors of gastric secretion evoked by insulin or pentagas- trin. An interesting feature of their blocking ef- fects is that acid secretion is reduced more than gastric juice secretion, a result similar to what has been observed following surgical vagotomy and opposite to what results from the administration of atropine [10]. This result suggests that neural

pathways, other than those operated through muscarinic receptors, are involved in the insulin- and pentagastrin-evoked gastric secretion. The latter suggestion is probably also true for the stress-induced effect on gastric mucosa, as the protection by IEM-1194 is more effective than that by pirenzepine.

The pepsin output is enhanced by both IEM- 1559 and IEM-1194 when evoked by insulin, but is reduced if evoked by pentagastrin. The origin of this difference is not yet clear.

Although IEM-1194 blocks the vagus-induced e f fec t s - - a decrease in heart rate and an increase in intestinal mot i l i ty- -a t somewhat higher con- centration than hexamethonium, it does not de- crease the arterial blood pressure level, in con- trast to hexamethonium. The latter difference is consistent with the fact that IEM-1194, in con- trast to hexamethonium, is a more selective blocking agent in isolated parasympathetic gan- glion than in isolated sympathetic ganglion. It is also a selective blocking agent for the neurons of enteric ganglia which possess nicotinic AChRs (see [5]).

The mechanisms responsible for parasympa- thetic and enteric vs. sympathetic ganglion selec- tivity of IEM-1559 and IEM-1194 as well as for their higher ganglion-blocking potencies in hex- amethonium are not yet clear. As mentioned

above, both former compounds bind irreversibly to AChR and also block reversibly the open ionic channel of the AChR [7].

The unusually long duration of ganglionic blockade produced by IEM-1559 and by IEM- 1194 is due to binding of their decyl radicals [7]. Similar long-lasting blocking effects were de- scribed for tetraethylammonium derivatives with decyl radicals in their influence on nerve conduc- tion, although in concentrations higher than those used in the present work [4,12]. Thus the possibil- ity is not excluded that, in addition to their post- synaptic blocking action, the compounds tested also block conduction in preganglionic nerve fi- bres. This possibility is supported by the observa- tion that decyltransbutyl ammonium hydrochlo- ride (IEM-1678, 5 X 10 -5 M), a compound very close in its chemical structure to those used in this work, blocks conduction in rabbit cervical sympathetic nerve fibres in parallel with the blockade of synaptic transmission through supe- rior cervical ganglion (A. Bobrishev, unpublished observations). In its postsynaptic effect, the decyl radical probably can incorporate either into hy- drophobic site in AChR molecule, or into the nearby lipid membrane (see [2]). This implies that the AChRs in the neurons of parasympathetic and enteric ganglia may differ from those in the neurons of sympathetic ganglia either in hy- drophobic sites in their molecules, or in the nearby lipid membrane, as well as in sensitivity to the blocking compounds of their preganglionic nerve fibres.

In contrast to the effect of the above tetraeth- ylammonium derivatives upon the nervous con- duction which is thought to be exerted from in- side the nerve fibres [4,12], the effects of the compounds tested in this work upon the AChRs are exerted from the outside of the nerve cell [151.

It can be concluded that the parasympathetic and enteric vs. sympathetic ganglion-blocking se- lectivity of IEM-1559 and IEM-1194 character- izes them as a new class of ganglion-blocking agents.

217

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