الفهرس 
Material and methodsOnly 14 pages are availabe for public view
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Abstract
Many of the generally accepted mechanisms by which vasopressin
increases water permeability of responsive epithelial cells are
precisely the same as some of the many effects ascribed to local
anesthetics in other cells systems, such as cyclic AMP synthesis,
calcium mo’~ementSt cytoskeletal disruption, intramembranous’particles
aggregatioll and membrane fluidization. Thus, it was of interest to
determine I:he ’possible interac-tion of these drugs on vasopressininduced
wa::er transport, and if present, the site or sites of this
inter£erenl::e. As an experimental model, we used the isolated toad
urinary bIll.dder and measured osmotic water transport across paired
bladder sa,s using the technique described by Bentley.
When added to the serosal bath 60 minutes before the hormone, none of
the anesthEtics tested affected baseline water flow. Procaine at the
concentration of 50 mM inhibited the water flow induced by vasopressin
20 mU/ml by 43%. However with 0.5 and 5 mM procaine, J was enhanced
v
by 25 and 45% respectively. Dibucaine at a concentration of 10-4M and
10-5M respectively inhibited and enhanced J by 56 and 42%.
v
Similarly, 5.10-
4
M tetracaine inhibited J by 35%, while at a cone env
tration of 5.10-
6
M, the drug increased J by 38% and had no signifiv
cant effect when used at an intermediate concentration of S.IO-SM.
Lidocaine,lt a concentration of 5.10-4M had a marked stimulatory
e£fect on vllsopressin-induced water flow of 95% but we were unable to
find a c o nc rn t r a t Lo n of the drug that would inhibit J . Thus, low
v
c o nc e n t r a t Lnn a of anesthetics enhance Jv while higher concentrations
inhibit Jv· Next, we tried to determine the effect of varying vasopressin
concentlation on the stimulatory action of 5 roM procainew
Vasopressin-induced water flow was 1.4, 2.5 and 45 times greater res-
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67.
Many of t he generally accepted mechanisms by which vasopressin
increases water permeability of responsive epithelial cells are
precisely the same as some of the many effects ascribed to local
anesthetics in other cells systems, such as cyclic AMP synthesis,
calcium movements, cytoskeletal disruption, intramembranous’particles
aggregation and membrane fluidization. Thus, it was of interest to
determine the possible interaction of these drugs on vasopressininduced
wa~er transport, and if present, the site or sites of this
interferen:e. As an experimental model, we used the isolated toad
urinary bl.tdder and measured osmotic water transport across paired
bladder sa”s using the technique described by Bentley.
When added to the serosal bath 60 minutes before the hormone, none of
the anesthEtics tested affected baseline water flow. Procaine at the
concentration of 50 mM inhibited the water flow induced by vasopressin
20 mU/ml by 43%. However with 0.5 and 5 mM procaine, J was enhanced
v
by 25 and 45% respectively. Dibucaine at a concentration of
10-5M respeotively inhibited and enhanced J by 56 and 42%.
v
-4 10 M and
Similarly,
;.10-
4
M tetracaine inhibited J by 35%, while at
v a cone entration
of ;.10-
6
M, the drug increased J by 38% and had no signifiv
cant effect when used at an intermediate concentration of S.JO-SM.
Lidocaine, Lt a concentration of 5.10-4M had a marked stimulatory
effect on Vcsopressin-induced water flow of 95% but we were unable to
find a concEntration of the drug that would inhibit J • Thus, low
v
concentraticns of anesthetics enhance J while higher concentrations
v
inhibit Jv . Next, we tried to determine the effect of varying vasopressin
concentration on the stimulatory action of 5 mM procaine.
Vasopressin-induced water flow was 1.4, 2.5 and 45 times greater res-
pectively in 5 mM procaine treated bladders as compared to controls
when the vlsopressin concentration was reduced from 20 to 0.5 and
O. I mU/ml. Thus with the latter hormonal concentration which is
minimally, and almost not, effective 5 mM procaine had a formidable
amplifying effect.
These marked effects of serosally added tertiary amine local anesthetics
were absent or blunted when the drugs were added to the mucosal
side. Inde~d. only the higher concentration of procaine did produce
a 29% inhibition of vasopressin-stimulated water flow as opposed to
the 43% inhibition observed when the drug was added to the serosal
bath. It should also be noted that the water flow produced by 0.5 mU/
ml vasopres,in was not affected by 5 mM procaine added to the mucosal
side while chis concentration of the drug added to the setosal side
enhanced t h- effect of that concentration of vasopressin by a factor
of 45.
To study thE reversibility of local anesthetic’s effects, we used
tetracaine cnd lidocaine. During baseline period, experimental
bladders were treated with the anesthetic but with no ADH and no
effect was observed. Vasopressin 20 mU/ml was then added to both
experimental and control serosal baths and the usual inhibition
(tetracaine S.10-4M) or stimulation (tetracaine 5.10-6 and lidocaine
5.10-
4
M) was observed in the anesthetic treated bladders. After 30 min.
all serosal >aths were replaced with fresh Ringers containing ADH but
no anestheti: the observed effects were entirely reversible.
Procaine 5 rolf can also stimulate the hydrosmotic response in bladders
pretreated f••r 15 minutes with vasopressin. Interestingly enough, the
drug was 8;lso able to temporarily reverse the so-called intrinsic
inhibition, of vasopressin-induced water flow when added 60 minutes
after the hormone. However the high concentration of procaine did
not have any effect when used in the same experimental conditions.
Concerning serosal hypertonicity which can also stimulate water flow,
we showed that the lower concentration of procaine could not stimulate
the water I:low induced by 240 mM serosal Mannitol while, after a
30 minute la t e nc y ,
the higher concentration significantly inhibited J
Having demll’nstrated these properties of local anesthetics on the
hydrosmotici response to vasopressin, we still had to d e t erm In e at
which point: they act in the cascade of events set in motion by the
hormone.
Let us s t arc by examining the inhibition induced by the higher c on cen-ctrations
of drugs used. All these higher concentrations of local
anesthetics are able to inhibit the osmotic water flow induced by
10 M 8-brono-cyclic AMP. Similarly, the water flow induced by the
phosphodiesl-erase inhibitor methylisobuthylxanthine was inhibited by
procaine (6,1%), dibucaine (62%) and tetracaine (39%). Thus the site
of inhibiti”n can be assumed to be distal to the generation of cyclic
AMP and to :Its breakdown by phosphodiesterase. The next possible site
of action tel be investigated is the membrane-associated cytoskeletal
elements, i.e. microtubules and microfilaments. The possibility that
microtubule. are the target of local anesthetics was explored by using
the known mi~crotubules inhibitor, colchicine. Even after a presumably
complete dis!ruption of microtubules by a 4 hour incubation in serosal
baths containing 10-4M colchicine, dibucaine was still able to inhibit
VP-induced w.ter flow by a significant 52%.
On the contr.ry, in bladders incubated with both cytochalasin B, an
agent disr pting the microfilament system and the inhibitory concen-
tration of dibucaine, the inhibition produced by the local anesthet1C
was not si uificant, thus incriminating disruption of microfilaments
as its mod of action. Finally, as local anesthetics may disrupt
microfilam nts by a calcium-mediated effect, we tested the inhibitory
action of
0-4M dibucaine in bladders incubated in a Na-free Ringer’s
solution, procedure known to increase intracellular calcium concentration.
A: though dibucaine inhibited J by 36%, this effect did not
reach statlstical significance.
As to the ’ite of stimulation of VP-induced water flow by the lower
concentrations of local anesthetics, it can be deduced from their
effects on the hydrosmotic actions of cyclic AMP and MIX. Indeed,
none of the rugs tested could modify the response to 10-4M 8-bromocyclic
AMP. On the other hand, the hydrosmotic response to MIX was not
increased b dibucaine nor tetracaine but 5 mM procaine produced a
striking II % stimulation of J .
v In addition, at these lower cone entrations,
t e anesthetics could still stimulate VP-induced water flow
in the pres nce of colchicine or cytochalasin B.
To summariz, and conclude the present study, one could say that the
low concent: ations of local anesthetics acting from the serosal side
only, st i mu L, te the hydrosmotic effect of antidiuretic hormone by
enhancing vcsopressin-stimulated (and, in the case of procaine,
unstimulatec) adenylate cyclase activity and cyclic AMP formation.
On the- ethet hand, this mechanism of action cannot account for the
effects of tigher concentrations of the drugs that would rather act
at a step cl ser to the luminal membrane perhaps by displacing
membrane-boll d calcium and disrupting microfilaments.
The possib e clinical implications of this work remain to be seen as,
for exampl , many patients suffering a myocardial infarction are
treated wi h intra-venous lidocaine in order to prevent the
occurence l f ventricular arhytmias.