- Email: [email protected]
Phenazine methosulfate induces a neurally-mediated contraction of the guinea-pig ileum
Life Sciences, Vol. 39, pp. Printed in the U.S.A.
PHENAZINE
1805-1812
Pergamon
M E T H O S U L F A T E INDUCES A N E U R A L L Y - M E D I A T E D OF THE GUINEA-PIG ILEUM MENACHEM
HANANI
and SHEMUEL
Journals
CONTRACTION
NISSAN
Laboratory of Experimental Surgery and Surgery Department Hadassah University Hospital, Mt. Scopus, Jerusalem, 91240, Israel (Received
in final
form August
12, 1986)
Summary Phenazine methosulfate (PMS) and related phenazines are widely used in b i o c h e m i s t r y and histochemistry and act as anti-bacterial agents, however, there is little information on their pharmacological actions. In t~e present paper the guinea-pig ileum was used as a model for studying the effects of PMS on nerve cells. PMS was found to contract intestinal muscle. This action appeared to be mediated by the activation of muscarinic receptors since it was blocked by atropine. Neostigmine potentiated the response to PMS. The nerve blocker tetrodotoxin prevented the effect of PMS and it is concluded that PMS causes the release of acetylcholine from nerve elements. The action of PMS on nerves is not mediated by nicotinic receptors. Receptors for serotonin, substance P or cholecystokinin also appear not to be involved. Of all the phenazines tested PMS was found to be the most potent and reversible.
Phenazines are electron carrier molecules used in a variety of reactions. Among the phenazines are neutral red, safranin O and phenazine methosulfate (PMS). PMS is employed in histochemical reactions (i), as a catalyzer in oxidation-reduction reactions, and with other phenazines it was found to act as an anti-bacterial agent (2). The m e c h a n i s m of this action is not known. In spite of their widespread use, no information is available on the pharmacological activity of phenazines and in the present work we have studied the effects of PMS on the isolated guinea-pig ileum. This preparation is a reliable model for testing the effects of a wide spectrum of drugs and putative neurotransmitters on smooth muscle and nerve cells (3). Methods Seventy one guinea-pigs of either sex, weighing 300-700 g have been used in this study. The animals were stunned by a blow on the head and bled. Non-distal ileum (15-20 cm from the ileo-cecal valve) was used in all the experiments. Longitudinal segments 1.5 cm in length were suspended in iO ml organ baths containing Krebs solution at 37°C. The solution was bubbled with 95% oxygen and 5% carbon dioxide, pH was 7.2-7.3. The resting muscle was 0.5-0.8 g and force was recorded with an isomertric force tranceducer (Statham model UC-2).
Copyright
0024-3205/86 $3.00 + .00 (c) 1986 Pergamon Journals
Ltd.
1806
ACh Release
by Phenazine
Methosulfate
Vol.
39, No.
19, 1986
The following drugs were used, phenazine methosulfate (PMS),atropine sulfate, dimethylphenylpiperazinium iodide (DMPP), hexamethonium bromide, d-tubocurarine, tetrodotoxin, carbamylcholine chloride (carbachol), safranin O, neutral red, phenosfranin, substance P, cholecystokinin octapeptide, a1~ from Sigma, neostigmine methysulphate (from Teva), proglumide (from A.H. Robbins). All the drugs were dissolved in distilled water. Fresh solutions were prepared weekly and were kept frozen until used. Results The basic response. PMS contracted the intestinal segments in dependent manner. The threshold for contraction was about 10 -6 M maximal response was about 4xlO -5 M. Fig. 1 shows an example of the PMS. At the higher concentrations the response usually reached a peak declined to a stable level of contraction.
10"6
5x10~
A
10-5
A
2.5gI
a dose and half effect of and then
2.~10~ A
~
/ ~
A
A
FIG.
,llOOs
4
1
Responses of the longitudinal muscle of the guinea-pig ileum to phenazine methosulfate (PMS). The numbers above the recordings indicate molar concentrations of PMS. The response threshold was at about 10 -6 M. Note the change of vertical scale in the last two recordings. The pharmacological mechanism. A major excitatory m e c h a n i s m in the intestine is the release of acetylcholine (ACh), which acts on muscarinic receptors on the smooth muscle (4). To test whether the effect of PMS is mediated by astivation of muscarinic receptors, the tissue was incubated with atropine (5xlO -v M) for 15 min before PMS was added to the bath. This caused a significant inhibition of the PMS response as can be seen by the shift of the dose-response curve to the right by about 0.7 log units (Fig. 2). This result was reproduced in 20 preparations. Higher atropine concentrations were also tested and caused further shifts in the curve. In
another
series
of
experiments
PMS was added
in
the
presence
of
the
Vol.
39, No.
19, 1986
ACh Release
by P h e n a z i n e
Methosulfate
1807
acetylcholinesterase inhibitor neostigmine (5xlO -8 M); this increased the response to PMS both in amplitude and duration (Fig. 3). Neostigmine iteslf, at this concentration had no contractile effect. These results indicate that the action of PMS is mediated by ACh.
,o0
~
If
2c
01 6
10-5
10-3 PMS (M)
FIG.
2
Effect of atropine on the response to PMS. Control responses (squares) were obtained in normal Krebs solution. The responses to PMS are expressed as a percentage of its own m a x i m u m contraction, w~ch was obtained by extrapolation. In the presence of atropine ( 5 x l O M) the curve was shifted to the right (triangles). The symbols represent the mean, and the bars the standard error of the mean (n=9).
To gain information on the site of PMS action, the nerve blocker tetrodotoxin (TT~8was added to the bath 15 min before PMS application. At a concentration of 5×10 M TTX reduced the response to PMS by a factor of 20 or more (Fig. 4). At TTX concentrations higher than 10 -7 M there was a complete block of the PMS response and therefore the residual responses seen in Fig. 4C and 4D are not due to direct action on the muscle. Dose-response curves for the effect of TTX on the response to PMS could not be constructed because in the presence of TTX the responses at most PMS concentrations were not measurable. These results show that PMS acts on neural receptors. The following experiments were designed in an attempt to determine whether PMS acts via an established receptor mechanism. A predominant type of receptor on enteric nerves is the nicotinic cholinergic (5). As a control, the nicotinic agonist DMPP has been used. As seen in Fig. 5, both DMPP and PMS contracted the muscle. The nicotinic antagonist d-tubocurarine (10-5 M) depressed the response to DMPP, but had no effect on the PMS response. This result was reproduced in five preparations. Another nicotinic antagonist, h e x a m e t h o n i u m (5xlO -4 M), had the same action as d-tubocurarine (n=5).
1808
ACh Release
by Phenazine
Methosulfate
A
A
PMS
Neo
FIG.
A
PMS
Vol.
39, No.
19, 1986
2min
3
Neostigmine enhances the response to PMS. A: control response to PMS (2.5xi0 -5 M). B: after the application of neostigmine (Neo, 5xlO -8 M) the response to the same PMS concentration was increased in both amplitude and duration.
A
B /
FIG.
,2rain
D
ilg
4
The nerve blocker tetrodotoxin (TTX) inhibits the responses to PMS. A,B: control responses to PMS (2xlO -5 M and 10 -4 M respectively). C,D: responses to the same PMS concentrations in the presence of TTX (SxlO -8 M).
l A
Serotonin (5-HT) is another putative neurotransmitter which activates neural receptors in the gut, releasing ACh (6). The possibility that PMS activates 5-HT receptors was also tested. In the control experiment both 5-HT and PMS contracted the muscle (Fig. 6). A single application of 5-HT produced strong desensitization to 5-HT. Still, during this desensitization the response to PMS was not altered (Fig. 6), this result was repeated in six preparations. It thus seems unlikely that PMS activates serotonergic receptors. Peptides have also been suggested as excitatory neurotransmitters which evoke ACh release from enteric nerves. These actions of the peptides substance P ($P) and cholecystokinin (CCK) are well documented and the possibility that receptors for these receptors mediate the effect of PMS was tested. Fig. 7 shows that during strong desensitization to SP the response to PMS was not altered. Similar
Vol. 39, No. 19, 1986
ACh Release by Phenazine Methosulfate
1809
results were obtained during desensitization to CCK (not shown). The CCK antagonist proglumide (i ~ ) reduced the response to CCK by 60-70%, but had no effect on the response to PMS (Fig. 8). These results indicate that PMS does not act by activating receptors for these peptides.
A
~
B
l,,i~[
FIG.
~ I Ig 2min
C ,
D~jQI~. ~~ A
A
DMPP
PMS
5
Comparison of the effects of PMS and the nicotinic cholinergic agonist ~MPP. A: control response to DMPP ( 5 x l O - M). B: control response to PMS (5xlO -b M). C: response to DMPP in the presence of the nicotinic antagonist dtubocurarine (10 -5 M). D: response to PMS in the presence of the antagonist. In contrast to the significant inhibition of the DMPP response by dtubocurarine, the PMS response was not affected.
~
~o' A
PMS
A
5-HT
A
A
5 - H T PMS
2rain
FIG. 6 Comparison of the effect of serotonin (5-HT) and PMS. A: control response to PMS (5xi0-5 M). B: a single application of 5-HT (5xi0-6 M) produced desensitization, as evidenced by the second 5-HT application. Under these conditions the response to PMS was not reduced. Responses to other phenazines. Three other phenazines in addition to PMS have been examined, safranin O, neutral red and phenosafranin. Safranin 0 caused contractions and was approximately as effective as PMS (Fig. 9A,B). The threshold for safranin O was also 10 -6 M, but complete dose-response curve could not be obtained because its effect declined after 3-4 applications and in some cases the contractions were even converted to relaxations. The mechanism of the sfranin O-induced contractions is similar to that of PMS; they were blocked by atropine and TTX. Neutral red also caused contractions of the ileum, but appeared to be approximately 10-20 times less effective than PMS (Fig. 9C,D). Its effects were also inhibited by atropine and TTX. The responses to safranin O and neutral red were not affected by prior to PMS. Also, during desensitization to safranin O the response to PMS was not altered. Phenosafranin, in contrast to the other phenazines caused muscle relaxation (Fig. 9E), which appeared to be nerve mediated as they were inhibited by TTX.
1810
ACh Release by Phenazine Methosulfate
Vol. 39, No. 19, 1986
FIG. 7
A
B
I
\
I ,~sg
I
2m;n
J~ ' i
~_j A PMS
t A SP
SP
A PM~
Comparison of the effects of PMS and substance P (SP). A:~eontrol response to8PMS (2.5xI0 -) M). B: SP (5×10M) caused strong contraction. A second application of SP at the same concentration shows significant desensitization to SP, but there was no reduction in the response to PMS.
I
J
~
2min
D
A CCK
The response to PMS is not blocked by the CCK antagonist proglumide. A: control response to CCK (2.5xi0 -9 M). 9: control response to PMS (SxlO -J M). In the presence of proglumide (I mM) the response to CCK was reduced (C), but the PMS response was not affected (D).
A PMS
Discussion The results of the present study demonstrate that PMS exerts well defined effects on the isolated guinea-pig ileum. PMS appears to activate nerves which release acetylcholine, causing muscle contraction. The detailed pharmacological mechanism underlying the PMS effects is not clear. The experiments show that PMS does not act on nicotinic cholinergic or on serotonergic receptors. Substance P or CCK receptors also appear not to mediate the actions of PMS. It is possible that PMS acts on other types of neural receptors which have not been examined. Another alternative is that it does not bind to a receptor but influences nerve membrane via a non-receptor mechanism.
Vol.
39, No.
19,
1986
ACh Release
A
by Phenazine
Methosulfate
1811
B
2min A
A
PMS
SO
C
D
A
PMS
E
A
NR
FIG.
A PS
9
Comparison between the effect of PMS to that of other phenazines. A: co n rtl o response to PMS (5xlO- 5 M); B: response of the same preparation to safranin O (SO, 5xi0- 5 M). C: control response to PMS (1.25xlO -5 M) in another preparation; D: response in that preparation to neutral red (NR, 2xlO -4 M). E: response to phenosafranin (PS, 5xlO -J M).
The few previous reports on the effects of phenazines do not provide a specific mode of action. The antibacterial action of PMS is probably effected intracellularly; i.e. it penetrates the infected cells. The large hydrophobic skeleton of PMS apparently enables the molecule to cross membranes easily, but the effects described in the present work may still be mediated extracellularly. There is only little information on the m e c h a n i s m underlying the cellular effects of PMS. In a study of the actions of PMS (in the presence of ascorbate) on red blood cells, it was found that it increases a Ca-dependent potassium conductance of the plasma membrane (9). This is believed to be mediated by electron transfer in which the PMS molecule participates. The site of action is presumably intracellular, but the details of the processes involved are not known. The effect described for the red blood cells still cannot explain the present results because an increase in K + conductance will hyperpolarize nerve membranes leading to inhibition. The experiments on the guinea-pig ileum clearly indicate that PMS causes transmitter release, which must be the result of depolarization or some other action which increases the intracellular concentration of calcium ions. There is evidence that oxidizing agents such as diamide cause transmitter release from nerve terminals (I0), ans effect which is probably mediated by an increase in calcium concentration. Such information is not available for PMS and obviously further work should be done to elucidate its mode of action.
Acknowledsement. This research was supported by a grant Research Fund of the Hebrew University and Hadassah.
from
the
Joint
1812
ACh Release by Phenazine Methosulfate
Vol. 39, No. 19, 1986
References 1.
A. G. E. PEARSE, Histochemistry , 3rd ed. vol. 2, p.916, Churchill Livingstone, Edinburgh (1972). 2. M. RABINOVITCH, J. P. DEDET, A. RYTER, R. ROBINEAUX, G. TOPPER and E BRUNET J. Exp. Med.155 415-431 (1982). 3. W. D. M. PATON, Brit. J. Pharmacol.12 119-127 (1957). 4. E. E. DANIEL, Handbook of Experimental Pharmacology , vol. 59/2, edited by G. Bertaccini, pp. 248-322, Springer, Heidelberg, (1982). 5. G. D. S. HIRST and H. C. MCKIRDY, J. Physiol. (London) 238 129-143 (1974) 6. M. DAY and J. R. VANE, Brit. J. Pharmacol. 20 150-170 (1963). 7. P. HOLZER and F. LEMBECK, Neurosci. Lett. 17 101-105 (1980). 8. W.M. YAU, G.M. MAKHLOUF, L.E. EDWARDS, and J.T. FARRAR, Can. J. Physiol. Pharmacol. 52 298-303 (1974). 9. J. GARCIA-SANCHO, A. SANCHEZ and B.HERREROS, Biochim. Biophys. Acta,556 118-130 (1979). iO. P. L. CARLEN, E. M. KOSOWER and R. WERMAN, Brain Res. 117 257-276 (1976) (1976).
PHENAZINE
1805-1812
Pergamon
M E T H O S U L F A T E INDUCES A N E U R A L L Y - M E D I A T E D OF THE GUINEA-PIG ILEUM MENACHEM
HANANI
and SHEMUEL
Journals
CONTRACTION
NISSAN
Laboratory of Experimental Surgery and Surgery Department Hadassah University Hospital, Mt. Scopus, Jerusalem, 91240, Israel (Received
in final
form August
12, 1986)
Summary Phenazine methosulfate (PMS) and related phenazines are widely used in b i o c h e m i s t r y and histochemistry and act as anti-bacterial agents, however, there is little information on their pharmacological actions. In t~e present paper the guinea-pig ileum was used as a model for studying the effects of PMS on nerve cells. PMS was found to contract intestinal muscle. This action appeared to be mediated by the activation of muscarinic receptors since it was blocked by atropine. Neostigmine potentiated the response to PMS. The nerve blocker tetrodotoxin prevented the effect of PMS and it is concluded that PMS causes the release of acetylcholine from nerve elements. The action of PMS on nerves is not mediated by nicotinic receptors. Receptors for serotonin, substance P or cholecystokinin also appear not to be involved. Of all the phenazines tested PMS was found to be the most potent and reversible.
Phenazines are electron carrier molecules used in a variety of reactions. Among the phenazines are neutral red, safranin O and phenazine methosulfate (PMS). PMS is employed in histochemical reactions (i), as a catalyzer in oxidation-reduction reactions, and with other phenazines it was found to act as an anti-bacterial agent (2). The m e c h a n i s m of this action is not known. In spite of their widespread use, no information is available on the pharmacological activity of phenazines and in the present work we have studied the effects of PMS on the isolated guinea-pig ileum. This preparation is a reliable model for testing the effects of a wide spectrum of drugs and putative neurotransmitters on smooth muscle and nerve cells (3). Methods Seventy one guinea-pigs of either sex, weighing 300-700 g have been used in this study. The animals were stunned by a blow on the head and bled. Non-distal ileum (15-20 cm from the ileo-cecal valve) was used in all the experiments. Longitudinal segments 1.5 cm in length were suspended in iO ml organ baths containing Krebs solution at 37°C. The solution was bubbled with 95% oxygen and 5% carbon dioxide, pH was 7.2-7.3. The resting muscle was 0.5-0.8 g and force was recorded with an isomertric force tranceducer (Statham model UC-2).
Copyright
0024-3205/86 $3.00 + .00 (c) 1986 Pergamon Journals
Ltd.
1806
ACh Release
by Phenazine
Methosulfate
Vol.
39, No.
19, 1986
The following drugs were used, phenazine methosulfate (PMS),atropine sulfate, dimethylphenylpiperazinium iodide (DMPP), hexamethonium bromide, d-tubocurarine, tetrodotoxin, carbamylcholine chloride (carbachol), safranin O, neutral red, phenosfranin, substance P, cholecystokinin octapeptide, a1~ from Sigma, neostigmine methysulphate (from Teva), proglumide (from A.H. Robbins). All the drugs were dissolved in distilled water. Fresh solutions were prepared weekly and were kept frozen until used. Results The basic response. PMS contracted the intestinal segments in dependent manner. The threshold for contraction was about 10 -6 M maximal response was about 4xlO -5 M. Fig. 1 shows an example of the PMS. At the higher concentrations the response usually reached a peak declined to a stable level of contraction.
10"6
5x10~
A
10-5
A
2.5gI
a dose and half effect of and then
2.~10~ A
~
/ ~
A
A
FIG.
,llOOs
4
1
Responses of the longitudinal muscle of the guinea-pig ileum to phenazine methosulfate (PMS). The numbers above the recordings indicate molar concentrations of PMS. The response threshold was at about 10 -6 M. Note the change of vertical scale in the last two recordings. The pharmacological mechanism. A major excitatory m e c h a n i s m in the intestine is the release of acetylcholine (ACh), which acts on muscarinic receptors on the smooth muscle (4). To test whether the effect of PMS is mediated by astivation of muscarinic receptors, the tissue was incubated with atropine (5xlO -v M) for 15 min before PMS was added to the bath. This caused a significant inhibition of the PMS response as can be seen by the shift of the dose-response curve to the right by about 0.7 log units (Fig. 2). This result was reproduced in 20 preparations. Higher atropine concentrations were also tested and caused further shifts in the curve. In
another
series
of
experiments
PMS was added
in
the
presence
of
the
Vol.
39, No.
19, 1986
ACh Release
by P h e n a z i n e
Methosulfate
1807
acetylcholinesterase inhibitor neostigmine (5xlO -8 M); this increased the response to PMS both in amplitude and duration (Fig. 3). Neostigmine iteslf, at this concentration had no contractile effect. These results indicate that the action of PMS is mediated by ACh.
,o0
~
If
2c
01 6
10-5
10-3 PMS (M)
FIG.
2
Effect of atropine on the response to PMS. Control responses (squares) were obtained in normal Krebs solution. The responses to PMS are expressed as a percentage of its own m a x i m u m contraction, w~ch was obtained by extrapolation. In the presence of atropine ( 5 x l O M) the curve was shifted to the right (triangles). The symbols represent the mean, and the bars the standard error of the mean (n=9).
To gain information on the site of PMS action, the nerve blocker tetrodotoxin (TT~8was added to the bath 15 min before PMS application. At a concentration of 5×10 M TTX reduced the response to PMS by a factor of 20 or more (Fig. 4). At TTX concentrations higher than 10 -7 M there was a complete block of the PMS response and therefore the residual responses seen in Fig. 4C and 4D are not due to direct action on the muscle. Dose-response curves for the effect of TTX on the response to PMS could not be constructed because in the presence of TTX the responses at most PMS concentrations were not measurable. These results show that PMS acts on neural receptors. The following experiments were designed in an attempt to determine whether PMS acts via an established receptor mechanism. A predominant type of receptor on enteric nerves is the nicotinic cholinergic (5). As a control, the nicotinic agonist DMPP has been used. As seen in Fig. 5, both DMPP and PMS contracted the muscle. The nicotinic antagonist d-tubocurarine (10-5 M) depressed the response to DMPP, but had no effect on the PMS response. This result was reproduced in five preparations. Another nicotinic antagonist, h e x a m e t h o n i u m (5xlO -4 M), had the same action as d-tubocurarine (n=5).
1808
ACh Release
by Phenazine
Methosulfate
A
A
PMS
Neo
FIG.
A
PMS
Vol.
39, No.
19, 1986
2min
3
Neostigmine enhances the response to PMS. A: control response to PMS (2.5xi0 -5 M). B: after the application of neostigmine (Neo, 5xlO -8 M) the response to the same PMS concentration was increased in both amplitude and duration.
A
B /
FIG.
,2rain
D
ilg
4
The nerve blocker tetrodotoxin (TTX) inhibits the responses to PMS. A,B: control responses to PMS (2xlO -5 M and 10 -4 M respectively). C,D: responses to the same PMS concentrations in the presence of TTX (SxlO -8 M).
l A
Serotonin (5-HT) is another putative neurotransmitter which activates neural receptors in the gut, releasing ACh (6). The possibility that PMS activates 5-HT receptors was also tested. In the control experiment both 5-HT and PMS contracted the muscle (Fig. 6). A single application of 5-HT produced strong desensitization to 5-HT. Still, during this desensitization the response to PMS was not altered (Fig. 6), this result was repeated in six preparations. It thus seems unlikely that PMS activates serotonergic receptors. Peptides have also been suggested as excitatory neurotransmitters which evoke ACh release from enteric nerves. These actions of the peptides substance P ($P) and cholecystokinin (CCK) are well documented and the possibility that receptors for these receptors mediate the effect of PMS was tested. Fig. 7 shows that during strong desensitization to SP the response to PMS was not altered. Similar
Vol. 39, No. 19, 1986
ACh Release by Phenazine Methosulfate
1809
results were obtained during desensitization to CCK (not shown). The CCK antagonist proglumide (i ~ ) reduced the response to CCK by 60-70%, but had no effect on the response to PMS (Fig. 8). These results indicate that PMS does not act by activating receptors for these peptides.
A
~
B
l,,i~[
FIG.
~ I Ig 2min
C ,
D~jQI~. ~~ A
A
DMPP
PMS
5
Comparison of the effects of PMS and the nicotinic cholinergic agonist ~MPP. A: control response to DMPP ( 5 x l O - M). B: control response to PMS (5xlO -b M). C: response to DMPP in the presence of the nicotinic antagonist dtubocurarine (10 -5 M). D: response to PMS in the presence of the antagonist. In contrast to the significant inhibition of the DMPP response by dtubocurarine, the PMS response was not affected.
~
~o' A
PMS
A
5-HT
A
A
5 - H T PMS
2rain
FIG. 6 Comparison of the effect of serotonin (5-HT) and PMS. A: control response to PMS (5xi0-5 M). B: a single application of 5-HT (5xi0-6 M) produced desensitization, as evidenced by the second 5-HT application. Under these conditions the response to PMS was not reduced. Responses to other phenazines. Three other phenazines in addition to PMS have been examined, safranin O, neutral red and phenosafranin. Safranin 0 caused contractions and was approximately as effective as PMS (Fig. 9A,B). The threshold for safranin O was also 10 -6 M, but complete dose-response curve could not be obtained because its effect declined after 3-4 applications and in some cases the contractions were even converted to relaxations. The mechanism of the sfranin O-induced contractions is similar to that of PMS; they were blocked by atropine and TTX. Neutral red also caused contractions of the ileum, but appeared to be approximately 10-20 times less effective than PMS (Fig. 9C,D). Its effects were also inhibited by atropine and TTX. The responses to safranin O and neutral red were not affected by prior to PMS. Also, during desensitization to safranin O the response to PMS was not altered. Phenosafranin, in contrast to the other phenazines caused muscle relaxation (Fig. 9E), which appeared to be nerve mediated as they were inhibited by TTX.
1810
ACh Release by Phenazine Methosulfate
Vol. 39, No. 19, 1986
FIG. 7
A
B
I
\
I ,~sg
I
2m;n
J~ ' i
~_j A PMS
t A SP
SP
A PM~
Comparison of the effects of PMS and substance P (SP). A:~eontrol response to8PMS (2.5xI0 -) M). B: SP (5×10M) caused strong contraction. A second application of SP at the same concentration shows significant desensitization to SP, but there was no reduction in the response to PMS.
I
J
~
2min
D
A CCK
The response to PMS is not blocked by the CCK antagonist proglumide. A: control response to CCK (2.5xi0 -9 M). 9: control response to PMS (SxlO -J M). In the presence of proglumide (I mM) the response to CCK was reduced (C), but the PMS response was not affected (D).
A PMS
Discussion The results of the present study demonstrate that PMS exerts well defined effects on the isolated guinea-pig ileum. PMS appears to activate nerves which release acetylcholine, causing muscle contraction. The detailed pharmacological mechanism underlying the PMS effects is not clear. The experiments show that PMS does not act on nicotinic cholinergic or on serotonergic receptors. Substance P or CCK receptors also appear not to mediate the actions of PMS. It is possible that PMS acts on other types of neural receptors which have not been examined. Another alternative is that it does not bind to a receptor but influences nerve membrane via a non-receptor mechanism.
Vol.
39, No.
19,
1986
ACh Release
A
by Phenazine
Methosulfate
1811
B
2min A
A
PMS
SO
C
D
A
PMS
E
A
NR
FIG.
A PS
9
Comparison between the effect of PMS to that of other phenazines. A: co n rtl o response to PMS (5xlO- 5 M); B: response of the same preparation to safranin O (SO, 5xi0- 5 M). C: control response to PMS (1.25xlO -5 M) in another preparation; D: response in that preparation to neutral red (NR, 2xlO -4 M). E: response to phenosafranin (PS, 5xlO -J M).
The few previous reports on the effects of phenazines do not provide a specific mode of action. The antibacterial action of PMS is probably effected intracellularly; i.e. it penetrates the infected cells. The large hydrophobic skeleton of PMS apparently enables the molecule to cross membranes easily, but the effects described in the present work may still be mediated extracellularly. There is only little information on the m e c h a n i s m underlying the cellular effects of PMS. In a study of the actions of PMS (in the presence of ascorbate) on red blood cells, it was found that it increases a Ca-dependent potassium conductance of the plasma membrane (9). This is believed to be mediated by electron transfer in which the PMS molecule participates. The site of action is presumably intracellular, but the details of the processes involved are not known. The effect described for the red blood cells still cannot explain the present results because an increase in K + conductance will hyperpolarize nerve membranes leading to inhibition. The experiments on the guinea-pig ileum clearly indicate that PMS causes transmitter release, which must be the result of depolarization or some other action which increases the intracellular concentration of calcium ions. There is evidence that oxidizing agents such as diamide cause transmitter release from nerve terminals (I0), ans effect which is probably mediated by an increase in calcium concentration. Such information is not available for PMS and obviously further work should be done to elucidate its mode of action.
Acknowledsement. This research was supported by a grant Research Fund of the Hebrew University and Hadassah.
from
the
Joint
1812
ACh Release by Phenazine Methosulfate
Vol. 39, No. 19, 1986
References 1.
A. G. E. PEARSE, Histochemistry , 3rd ed. vol. 2, p.916, Churchill Livingstone, Edinburgh (1972). 2. M. RABINOVITCH, J. P. DEDET, A. RYTER, R. ROBINEAUX, G. TOPPER and E BRUNET J. Exp. Med.155 415-431 (1982). 3. W. D. M. PATON, Brit. J. Pharmacol.12 119-127 (1957). 4. E. E. DANIEL, Handbook of Experimental Pharmacology , vol. 59/2, edited by G. Bertaccini, pp. 248-322, Springer, Heidelberg, (1982). 5. G. D. S. HIRST and H. C. MCKIRDY, J. Physiol. (London) 238 129-143 (1974) 6. M. DAY and J. R. VANE, Brit. J. Pharmacol. 20 150-170 (1963). 7. P. HOLZER and F. LEMBECK, Neurosci. Lett. 17 101-105 (1980). 8. W.M. YAU, G.M. MAKHLOUF, L.E. EDWARDS, and J.T. FARRAR, Can. J. Physiol. Pharmacol. 52 298-303 (1974). 9. J. GARCIA-SANCHO, A. SANCHEZ and B.HERREROS, Biochim. Biophys. Acta,556 118-130 (1979). iO. P. L. CARLEN, E. M. KOSOWER and R. WERMAN, Brain Res. 117 257-276 (1976) (1976).