Latvijas Universitāte
Medicīnas fakultāte
Medicīniskās bioķīmijas docētāju grupa
MAIJA DZINTARE
Slāpekļa oksīda koncentrācijas izmaiņas audos
dažādu farmakoloģisku preparātu ietekmē
Eksperimentāls pētījums
Changes of Concentration of Nitric Oxide in tissues
under action of different pharmacological aģents
Experimentat research
Promocijas darba kopsavilkums
Summarv of doctor thesis
Promocijas darba vadttājs: prof. dr. hab. biol.
NIKOLAJS SJAKSTE
Darbs veikts
Latvijas Organiskās sintēzes institūta Medicīniskās
ķīmijas nodaļas bioķīmijas grupā
Rīga, 2004
SUMMARY
INTRODUCTION
Discovery of the physiological activity of nitric oxide (NO) one of the
most important discoveries in biology and medicine made in late 20th century.
The numerous physiological activities of NO include vasorelaxation and signal
transduction in the nerve system.
NO is synthesized in organism by several enzymes that form family of NO
synthases (NOSs) (EC 1.14.23), all of them utilize L-arginine as the only
substrate. Three main NOS isoforms have evolved to function in mammals:
endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS).
The enzymes are encoded by different genes located in humans on Chromosome
7, 12 and 17 respectively.
The proposed thesis was dedicated to the eventual role of NO in the
mechanism of action of two groups of pharmacological agents. Halogenated
volatile anesthetics and anti-ischemic drug mildronate were in the focus of our
attention.
NO plays a wide variety of roles in the central nervous system. It was
discovered recently that NO is a neurotransmitter, and it is known to mediate Lglutmate and NMDA receptor-associated neurotoxicity. Very early, it was
supposed that halothane, one of the most widely used anesthetics, interferes with
the action of NO. Inhibitors of NOS acted synergistically with halothane and
decreased the minimal alveolar concentration (MAC) for anesthetics. It was
postulated that halogenated volatile anesthetics (HVAs) depress L-glutamate- and
NMDA-mediated neurotransmission. Several studies support this hypothesis.
According to the above hypothesis, halogenated volatile anesthetics should
decrease NOS activity (Johns et al., 1992) and NO production in neurons. On the
other hand, the vasomotor action of volatile anesthetics, also mediated by nitric
oxide, should lead to increase of the NO concentration in brain tissues. However,
some data in the literature indicated that, under HVA-induced anesthesia, NO
concentration in brain cortex should increase rather than decrease.
In order to establish whether the NOS-inhibiting action of halogenated
volatile anesthetics is reversed by increased NO production coupled to
vasodilatation produced by the anesthetics, we performed direct measurements of
NO content in tissues of rats under anesthesia by trapping NO in a stable complex
with iron and DETC and measuring its spectrum by means of electron
paramagnetic resonance (EFR) spectroscopy. This method was chosen for its
high resolution and ability to specifically detect the NO radical, but not its
metabolites as in many other methodical approaches. The study was performed
using experimental animals (rats), as brain and other tissue samples were
necessary for the measurements.
Mildronate [3-(2,2,2-trimethylhydrazine) propionate] is an anti-ischemic
drug developed in the Latvian Institute of Organic Synthesis and widely used in
some countries (Simkhovich et at, 1988; Dambrova et al., 2002). It is admitted
that the pharmacological effects of mildronate on ischemic myocardium are
produced by the inhibition of -butyrobetaine hydroxylase and reduction of the
fatty acid -oxidation (Shutenko et al., 1995; Dambrova et al., 2002). Via this
mechanism, mildronate inhibits the biosynthesis of L-carnitine and prevents the
accumulation of toxic acylcamitines in ischemic myocardium (Shutenko et al.,
1995; Simkhovich et al., 1998). However, this effect of mildronate can be
achieved after several day long treatments only. Meanwhile, some observations
indicate that mildronate elicits several fast effects related to vasorelaxation
(Simkhovich et al., 1988; Ratunova et al., 1989; Shutenko et al, 1995).
MiIdronate interferes with membrane receptors and secondary messenger
activity, triggers DNA replication, repair and methylation (Shutenko et al., 1995).
In addition, it has been found that administration of mildronate and Y~
buryrobetaine (GBB) composition abolished the physiological effects of nitric
oxide synthase (NOS) inhibitors (Kalvinsh, Veveris, 1999).
GOALS OF THE WORK
The goals of the work were:
1.
2.
to estimate the NO concentration in different organs of intact rats and changes
of it under action of halogenated volatile to establish the role of NO in
mechanism of action of halogenated volatile anesthetics,
to elucidate the eventual NO-dependent mechanism of action of anti-ischemic
drug mildronate and its analogues.
MATERIALS AND METHODS
Materials
Wistar male rats weighting 200 - 300 g were used in experiments. Animals
were purchased from the laboratory animal suppliers - Laboratory of
experimental animals of Riga Stradina University. All manipulations with
animals were performed in accordance with Republic of Latvia regulations;
permission from the Ethics Commission of the Latvian Council for Science was
obtained to perform this study.
Rats were anaesthetized by inhalation of an O2 mixture with volatile
anesthetics (1.5% for halothane (2 MAC); 1% for isoflurane (1 MAC), 2% for
sevoflurane (1 MAC), using a "Floutec" (Cyprane Ltd) rodent ventilator. In order
to keep body temperature constant during anesthesia, animals were covered with
a heating blanket. Body temperature during narcosis was constantly monitored
with a rectai thermometer and maintained at 36.6 - 37.5°C.
Other drugs: mildronate, GBB, neomildronate, GBB methyl - and
etlhylesters, LPS, inhibitors of NOS (N-nitro-L-Arg, AMT, 7-NI) were
administered i.p. as followed by experimental protocols (Fig. 1) LPS was
administered i.p. or intracerebroventricular. Different times after administration
of drugs animals were decapited.
Methods
EPR spectroscopy
The level of NO production in different rat organs was evaluated
quantitatively using EPR-spectroscopy method proposed by A.F. Vanin and
collaborators (Vanin and Kleschyov, 1998).
30 min before decapitation animals were administered the spin trap agents:
DETC (disodium salt, 400 mg/kg, i.p.) and ferrous citrate (40 mg/kg ferrous
sulphate + 200 mg/kg sodium citrate, s.c). The Fe-(DETC),2 complex formed in
tissues traps NO-, forms a Fe-(DETC)2-NO complex which exhibits a
characteristic EPR signal and allows the measurement of the formation of NO.
Tissue samples were immediately introduced in quartz tubes and frozen in liquid
nitrogen.
EPR spectra were recorded on a "Radiopan" SE/X2544 spectrometer
(Radiopan, Poland) operating at X-band. Quartz tubes were placed in a quartz
finger Dewar ER 167 FDS-Q (Bruker, Germany) filled with liquid nitrogen.
Operating conditions were as following: 25 mW microwave power (non
saturating conditions), 100 kHz modulation frequency, 5 G modulation
amplitude, receiver gain 0.5 x 104, and time constant 1 s.
NO content in the samples was evaluated from the height of the third
component at g = 2.031 of the Fe-(DETC)2-NO complex.
Griess reaction was performed as described by Calapai et al. (2000).
Organ chamber studies - relaxation of ex vivo rats aorta rings was
performed as described (Munzel et a!., 1996).
[14-C]L-arginine - [14-C]L-Citrulline conversion test using the recombinant
NOSs was performed according Bredt and Schmidt (1996).
Enzymatic measurement of level of ATP (Biicher, 1947), lactate (Noll,
1974) and pyruvate (Czok and Lamprecht, 1974) were performed as indicated.
Significance of differences between the groups was evaluated according to
Student's /-test (Acaтиани, 1965). Significant differences were set at p < 0,05.
RESULTS
1. CONCENTRATION OF NITRIC OXIDE IN RAT TISSUES
To determine the NO level in rat organs, rats were injected with the NO
scavenger DETC and ferrous citrate and sacrificed 30 min later. Fig. 2 presents
NO spectra in the brain cortex in intact animals and after anesthesia with
halothane (1,5%; 30 min). The spectra have a typical shape of Cu-DETC
spectrum with a superposed NO peak. The intensity of the Cu-DETC spectrum
indicates the bioavailability of the tissues under study to DETC. We regarded the
height between the maximum of the peak at g = 2.031 as the size of the NO-Fe(DETC)2 signal (I) (Vanin et al., 1994) (Fig. 3).
The NO content was determined in brain cortex, cerebellum, liver, heart,
kidneys, blood and testes. There were differences in the level of NO in organs
(Fig.4). The highest NO content was found in brain cortex and blood: 46,0 ± 3,4;
33,6 ± 12,4 ng/g of tissue, respectively. The NO concentration was slightly lower
in cerebellum, liver and testes: 27,7 ± 2,6; 27,6 ± 4,7; 13,8 ± 1,1 ng/g of tissue,
respectively. The lowest NO content was in heart and kidneys: 4,8 ± 0,7; 3,3 ±
0,5 ng/g of tissue, respectively.
2. INFLUENCE OF ANESTHETICS ON NO CONCENTRATION IN RAT
TISSUES
2.1. Influence of halogenated volatile anesthetics on NO content in rat tissues
To study the influence of halothane anesthesia on NO content in rat brain,
rats were anesthetized with 1,5% halothane (Fig. 5). The average signal intensity
in the cortex of these animals will be referred to as 100% for further
determinations (100 ± 37; n=15). After 5 min of narcosis, the NO content in brain
cortex had already doubled and by 30 min it had increased six-fold, remaining at
this level for up to 60 min of anesthesia.
After improvement work technique we perform more detailed studies of
changes of NO concentration under action of halogenated volatile anesthetics.
Sevoflurane and isoflurane - anesthetics of second generation also were studied.
Control values of NO concentration in intact animals were slightly changed
during long period of time.
The 30-minute duration of anesthesia under halothane (1,5%) caused threefold increase of NO concentration in brain cortex - from 58,8 ±3,1 up to 177,9 ±
20,8 ng/g of tissue (Fig.6-A). NO content in the cerebellum decreased from 53,7
± 2,6 to 36,6 ± 7,3 ng/g of tissue. The 30-minute duration of anesthesia under
sevoflurane (2%) or isoflurane (1%) (Fig. 6-B) caused similar effect to that of
halothane: increased NO concentration in cortex from 46,0 ± 3,4 to 136,1 ± 33,9
and 105,0 ± 18,4 ng/g of tissue respectively, and decreased NO level in
cerebellum from 27,7 ± 2,6 to 11,3 ± 5,3 and 12,5 ± 0,9 ng/g of tissue
respectively. NO content in other tissues did not differ significantly of that of the
control group animals.
To ensure ourselves that the observed changes in EPR spectra were not due
to changes in DETC pharmacokinetics, but reflected the NO concentration per se,
we determined changes in NO metabolites (NO 2+ NO3) using the Griess
reaction (Table 1), We managed to determine the ion concentration in brain
cortex of intact animals (1288 ± 801 ng/g tissue), the concentration was on the
limit level of the method sensitivity, sevoflurane anaesthesia increased it to 6094
± 668 ng/g tissue, the difference was statistically significant. The result proved
validity of data obtained using EPR method in further series of experiments we
used exclusively EPR approach as more sensitive.
In order to exclude the possible impact of disturbances in energy
metabolism on NO synthesis provoked by HVAs following ischemisation of
brain tissue as reported by some authors, we compared ATP, pyruvate and lactate
concentration in brain tissue in control animals and rats under isoflurane and
sevoflurane anesthesia, with non-anesthetized rats serving as controls (Table 2).
Since the halothane effect on NO concentration was similar to that of isoflurane
and sevoflurane, a halothane-anesthetized group was not included in this study.
Isoflurane and sevoflurane anesthesia did not provoke any disturbances in energy
metabolism in our hands, as neither ATP concentration decrease nor increase of
lactate/pyruvate ratio were observed.
To clarify whether increase of NO content is caused by activity of NOS, we
used nonspecific inhibitor of NOS N-nitro-L-arginine. Administration of the N-nitro-L-arginine (50mg/kg, i.p., 30 min) (Fig. 7) in intact animals decreased the
NO content in cortex and cerebellum more than twofold: 35,6 ± 4,3 and 16,6 ±
1,9 (ng/g of tissue) respectively. NO level in other tissues was minimally
affected by the inhibitor. Administration of the N^nitro-L-arginine prior to
anesthesia attenuated the anesthesia-induced increase of NO content. In
halothane-treated animals NO content was similar to that of the control group
animals. In animals under isoflurane and sevoflurane anesthesia NO content was
close to that of non-anesthetized animals that received the same inhibitor (not
shown). Consequently the increase of NO concentration in cortex under
anesthesia is produced by activity of NOS.
Different direction of the NO concentration changes in brain cortex and
cerebellum indicated possible involvement of different NOS isoforms in the
observed effects. It is known that the nNOS content in the cortex and nNOS
activity measured in vitro are the lowest among brain compartments, the same
parameters in the cerebellum are the highest (Barjavel, Bhargava 1995; Singh et
al., 2000), Decrease of the NO content in the cerebellum could be easily
explained by inhibitory effect of HVA on nNOS and NO neurotransmitter
function (Johns et al., 1992, 1995; Pajewski et al., 1996; Zuo et al., 1996). This
seemed to be hardly possible in the case in the cortex. Involvement of different
NOS isoforms in the HVA-induced NO increase in brain cortex was tested using
isoform-specific NOS inhibitors.
Administration of nNOS inhibitor 7-Ni 30 minutes before the sevofiurane or
isoflurane anaesthesia did not prevent the NO increase in cortex during the
anaesthesia (Fig. 8; Table 3). However the increase was abolished by iNOS
inhibitor AMT, on the background of this inhibitor the NO concentration did not
exceed 23,0 ± 4,1 ng/g tissue for sevofiurane and 29,2 ± 7,8 ng/ g tissue for the
isoflurane.
2.2. Effect of halogenated volatile anesthetics on background of stimulate
iNOS with lipopolisaharide (LPS)
Sensitivity of the HVA-produced increase of NO synthesis to iNOS
inhibitor raised the question about possible involvement of this enzyme in the
above phenomenon, HVAs are known to enhance expression of the iNOS gene in
vitro (Zuo, Johns, 1997) however it is unlikely that this effect can manifest itself
during 30 minutes long anaesthesia. However in some cases increase of iNOS
copies in neurons was observed in 15 minutes after stimulus, probably, from preexisting mRNAs (Holtz et al., 2001). We hypothesized that HVAs could enhance
activity of pre-existing iNOS copies in the brain cortex. Inducible NOS is found
in neurons, glia, macrophages and brain blood vessels (Petrov et al., 2000). The
enzyme is involved in several physiological and pathological processes (Heneka,
Feinstein, 2001; Ferrini et al., 2001; Madrigal et al., 2001), it is always expressed
to some extent. We have tried to increase the iNOS gene expression and the
enzyme copy number by injecting LPS. In the case if the HVAs-produced NO
increase was due to iNOS, the effect should have been manifold enhanced in
LPS-treated animals.
Indeed, LPS caused drastic increase of NO concentration in all tissues
studied in animals (Fig. 9; Table 4). To ensure ourselves that NO detected after
LPS injection in brain was due to increased synthesis in the tissue, but did not
reflect drastic increase of NO inside the brain blood vessels, we determined NO
concentration in the blood additionally. Although LPS injection drastically
increased DETC-trapped NO in blood, from 33,6 ± 12,4 in intact animals to
463,0 ± 46,4 ng/g tissue after LPS injection, it could not contribute much to NO
levels observed in brain cortex (157,4 ± 7,6 ng/g tissue) and cerebellum (143,1 ±
10,5 ng/g tissue), taking into account volume of blood vessels in the brain tissues.
AMT administered an hour or 30 minutes before the sacrifice abolished the
LPS effects, in most organs, except liver NO concentration diminished beyond
the normal levels (Fig. 10; Table 5). In animals subjected both to LPS treatment
and sevofiurane anesthesia we observed a tendency for further increase of NO
concentration in brain cortex, however differences were not statistically
significant. Interestingly, significant increase was observed in heart and testes.
Combination of LPS with isoflurane anaesthesia neither increased NO in brain
cortex above the LPS-induced level.
To define more exactly that HVAs cause increase of NO concentration in
cortex above LPS induced level that confirm main role of iNOS in this
phenomenon, we perform experiments with intracerebroventricular (i.c.v.)
administration of LPS (2,5 mg/kg).
Sham operation for implantation of canula in the cerebral ventriculi did not
cause any changes in NO level in comparison with the control level (Fig. 1 l).
Anesthesia under sevoflurane in operated rats caused similar changes like in the
non-operated rats: NO concentration increased in brain cortex (66,6 ± 7,2 nm/g of
tissue) and decreased in the cerebellum (16,6 ± 1,3 nm/g of tissue).
NO concentration increased drastically in all the organs under study after
LPS (2,5 mg/kg; 4h) i.c.v. administration similarly as after LPS i.p.
administration (Fig. 12), 30 minutes long sevoflurane anesthesia caused further
increase of NO concentration above LPS induced level in the cortex (from 250 ±
13 to 339 ± 18 ng/g of tissue) and in blood (from 336 ± 21 to 587 ± 59 ng/g of
tissue). It means that iNOS could be involved in the NO concentration increase in
cortex during the HVA-induced anesthesia.
We tested influence of halothane, isoflurane and sevoflurane on purified
enzymes: nNOS, iNOS and eNOS by using [14C] arginine - [14C] citrulline
conversion reaction (Fig. 13). Isoflurane at high concentrations (40mM) inhibited
all NOSs, most effective - iNOS (70%). Halothane and sevoflurane exhibited
less activity on NOS isoenzymes.
2.3. Effect of intravenous Anesthetics on NO content in rat tissues
In order to reveal if the observed increased of NO concentration in brain
cortex was specific for HVA or whether it was linked to anesthesia per se we
studied the influence of intravenous anesthetics on NO production in the brain
cortex.
After 30 min, ketamine (100 mg/kg, i.p.) action did not produce any
changes in the cortex: 102,38 ± 8,42 arbitrary units (arbitrary units (a.u.) percents of NO content in cortex of intact rats) (Table 6, fig. 14-A) and
manifested a tendency to decrease 30 min later: 51,50 ± 22,68 a.u. In cerebellum,
NO content remained unchanged 30 min after ketamine administration: 70,63 ±
26,56 a.u., decreased 30 min later: 45,36 ± 5,83 a.u.
Anesthesia with pentobarbital (30 min, 60 mg/kg, i.p.) increased NO
content in brain cortex: 137,80 ± 12,38 a.u. and in cerebellum: 84,88 ± 15,98 a.u.
However the differences were not statistically significant. (Table 6, fig. 14-B).
Under chloral hydrate (300 mg/kg, i.p.) action, NO increased slightly in
brain cortex 30 mm after administration: 178,84 ± 49,24 au. No effect was
detected in cerebellum: 55,33 ± 8,73 a.u. (Table 6, fig. 14-B).
Thus, during anesthesia produced by three different reference drugs, we
did not observe any drastic increase in NO-Fe-(DETC)2 formation characteristic
of HVAs action. Thus this effect appears to be specific exclusively only for
HVAs.
3. NO-DEPENDENT EFFECTS OF MILDRONATE, NEOM1LDRONATE,
GBB AND GBB ESTERS
In order to explain fast effects of mildronate we proposed the hypothesis
about eventual involvement of NO-dependent mechanisms in action of the drug.
We started with the study of influence of the mildronate and its analogues on NO
concentration in tissues.
3.1. Effects of Mildronate, GBB, neomildronate and GBB esters on NO
concentration in different rat tissues
Administration of mildronate (50 mg/kg, i.p.) triggered an increase of NO
concentration in some tissues (Fig. 15-a). The most marked increase (79,5 ± 5,1
ng/g of tissue) was observed in the brain cortex 30 min after the drug
administration, later the NO level gradually decreased and reached the initial
level. In the cerebellum changes in NO level were similar: 30 min after
mildronate administration the NO content increases (60,4 ± 1,6 ng/g of tissue).
Later the NO content decreases and reached the initial level in 2 h (Fig. 15-b). In
the heart the maximal NO concentration (13,6 ± 2,1 ng/g of tissue) was also
observed 30 min after mildronate administration (Fig. I5-c).
Simultaneous administration with mildronate of the NOS inhibitor NM-nitroL-arginine (50 mg/kg; i.p.) prevented the NO level increase triggered by
mildronate (Fig. 16. table 8). On die contrary, 30 min after administration of the
two substances the NO level was decreased: in cortex tissue 19,6 ± 4,9; in
cerebellum 23,1 ± 10,6 (ng/g of tissue). Abolition of the mildronate effect by a
NOS inhibitor indicates involvement of NOSs in the observed NO concentration
increase. This fact enables us to exclude the nonenzymatic nature of the above
effect. Thus mildronate triggers a transitory increase of NO concentration in brain
cortex, cerebellum and heart; this increase is due to activity of NOS.
Administration of y-butyrobetaine (30 mg/kg) provoked similar transient
increase of NO content in heart and blood (Fig. 18; table 9).
A combination of mildronate and GBB in ratio 1:1 (neomildronate)
provoked a much more stable effect on NO content as compared to both its
components administred alone. Increase of NO concentration in the cortex (79,5
± 5,1 ng/g of tissue) and cerebellum (60,4 ± 1,6 ng/g of tissue) was observed 30
min after the neomildronate administration. Almost the same NO level was
maintained for 2 h (Fig 17-a; b). In other organs changes of the NO content were
not statistically significant (Fig. 17-c).
A modified neomildronate composition (30 mg/kg GBB and 15 mg/kg
mildronate) triggered a statistically significant NO concentration increase in
kidneys 15 and 30 min after administration (16,3 ± 3,6 ng/g and 8,4 ± 2,0 ng/g,
correspondingly) compared to 3,9 ± 0,7 in the controls (Table 8) and in testes
29,0 ± 4,2 ng/g. Increased concentration of mildronate over GBB (30 mg/kg
GBB and 120 mg/kg mildronate ) turned out to be more effective, and a
statistically significant increase of NO concentration was observed in brain
cortex, in kidneys ad in testes 15 min after administration of the composition.
Taken together the above data reveal the effect of transient increase of NO
synthesis in some organs and tissues after mildronate, GBB, neomildronate
administration. The effect seems to be more stable if mildronate is administred
together with GBB, but mildronate concentration is equal or increased as
compared to GBB concentration.
As a working hypothesis (prof. I. Kalvinsh hypothesis) we assume that the
GBB esters are involved in the pathway of activation of NOS by mildronate.
Cholinergic activity of the GBB esters (Hosein et al., 1970) permits us to suppose
that these esters act in a way resembling that of the acetylcholine action. We
assume GBB ester binds to its receptor, a specific enzyme hydrolyses the ester,
and the whole system can activate NOS in a process resembling that of
endothelium NOS activation via m-acetylcholine receptors. It is already known
that there is a certain similarity in physiological effects of GBB esters and
acetylcholine. So, both esters decrease systemic arterial pressure, cause
salivation, etc. (Hosein et al., 1970). Block of the GBB hydroxylation by
mildronate can increase the GBB pool, part of the "released" GBB can be
esterified and involved in the NOS activation process. To prove this hypothesis
we tested the influence of GBB methyl- and ethyl-esters on NO production.
After administration of GBB methyl-ester (GBQ-88) (0,3 mg/kg, i.e.,
about 150 nmol/kg, i.p.), that is close to physiological concentration of
acetylchoiine in blood), we observed similar transient increase of NO content in
all studied tissues (Table 9). After 30 min NO concentration in heart was 8,5 ±
1,5 (ng/g of tissue) and in blood 73,5 ± 9,2 ng/g of tissue (Fig. 18). These effects
are like GBB effects, but are reached with 100 times less concentration of GBB
methyl-ester.
GBB ethyl-ester (GBQ-87) (0,3 mg/kg, i.e., about 150 nmol/kg i.p.) cause
similar increase of NO concentration in all studied tissues as GBB methyl-ester
(Fig. 19). 30 min after administration of GBQ-87 NO concentration increased in
cortex (95,4 ± 15,8 ng/g), in cerebellum (55,2 ± 6,2 ng/g), in liver (121,6 ± 31,5
ng/g), in kidneys (12,1 ± 2,8 ng/g) and in heart (32,0 ± 5,1 ng/g).
These results apparently supported our hypothesis. To gain further
evidence of its validity, we studied interactions of mildronate, GBB and GBB
esters with the activities of purified NOSs, and their NO-dependent vasodilating
properties on rat aortic rings.
3.2. Effects of mildronate, GBB and GBB esters on recombinant NOSs
Mildronate, GBB, GBB methyl- and ethyl-esters in concentration up to 10
mM did not modify the NADPH consumption catalyzed by full-length nNOS
(Figure 20-A). They did not inhibit the conversion of [14C]L-arginine to citruiline
catalyzed by eNOS (Figure 20-B) and nNOS (Figure 20-C). We have
demonstrated lack of effects of these compounds on iNOS (Fig. 24), Further
studies using the oxygenase domain of nNOS (nNOSoxy) showed that these
compounds did not modify the optical properties of the heme, suggesting that
they did not bind to the active site (data not shown). Taken together, these data
exclude direct effects of mildronate, GBB and GBB esters on NO synthase
activities and indicate that another receptor-dependent mechanism must to be
sought.
3.3. Vasodilating activities of mildronate, GBB and GBB esters
The vascular effects of mildronate, GBB, neomildronate and GBB meihyland ethyl-esters (concentration range I0-9 to 10-4 M) were tested on rat aortic
rings preconstricted with phenylephrine. Under those conditions, mildronate
alone did not manifest any vasodilating activity up to 10-4 M (Fig 21-A). GBB
produced vasorelaxation at very high concentrations (EC50 = 10-4 M).
Interestingly, the vasodilating activity of GBB was two fold increased (Ec50 - 5 x
10-5 M) when the both compounds were added simultaneously (in a 1:1 ratio). Pretreatment of aortic rings with NOS inhibitor L-NAME (0,3 mM) abolished the
effects of GBB and GBB + mildronate mixtures, suggesting that the observed
effects were dependent upon NOS. Increase of the GBB efficiency in the
presence of mildronate also provided new elements in favor of a potential
involvement of GBB metabolites, namely GBB esters in mechanism of
mildronate action.
Indeed, both methyl- and ethyl-esters of GBB were found to be potent
vasodilator agents (EC50 close to 2.5 x 10-7 M). Pre-treatment of aortic rings with LNAME or removal of endothelium strongly reduced the vasodilator effects of
GBB esters (Figure 21 A and B).
Thus, GBB esters appear to be efficient vasodilators acting via NOS- and
endothelium-dependent mechanisms,
3.4. Effects of GBB and mildronate on NO production in LPS-treated
rats
Treatment with LPS (E. coli subtype O55:B5; 10 mg/kg; i.p., 4 h)
drastically increased nitric oxide concentration in all studied rat tissues (Fig 9,
table 4).
Administration of mildronate on background of 4 hours long LPS action
(see drug administration scheme in Fig 1) caused a significant (two-fold)
decrease of the nitric oxide level in brain cortex and cerebellum, the effect was
apparent when drug was administered 1 or 2 hours before sacrifice (134 ± 27 and
122 ± 26 ng/g tissue, respectively) Fig. 22, A and B). No statistically significant
changes were detected in other organs (not shown),
GBB appeared to be more effective. NO concentration was decreased
when the drug was administered 15 min before sacrifice, if it was injected 30 min
before sacrifice, the NO concentration decreased almost two times in alt tissues
under study (in brain cortex 115 ± 11 ng/g tissue, in the cerebellum 102 ± 12 ng/g
tissue in HverlO34 ± 146 ng/g tissue, in the heartl44 ± 19 ng/g tissue, in
kidneys216 ± 42 ng/g tissue, in testes 57 ± 6 ng/g tissue and in blood342 ± 59
ng/g tissue, Fig 23 A-G). However, the effect of GBB was not persistent; no
changes in the NO concentration were detected, if the drug was administered 2
hours before sacrifice.
Ability of mildronate and GBB to attenuate the LPS effect on the NO
production in the tissues has raised the question about iNOS-inhibiting
capabilities of the compounds. However, in vitro assay of [ 14C]-arginine
conversion to citrulline could not reveal any inhibition of the purified iNOS
activity by mildronate and GBB (Fig 24).
DISCUSSION
1. CONCENTRATION OF NITRIC OXIDE IN RAT TISSUES
Intact animals
At first we undertook a study of NO concentration in rat tissues. Reports on
NO concentration in mammals tissues were not numerous, these studies were
based on indirect measurements of NO content in tissues or rather presented the
level of NO stable metabolite NO2 and NO3.
We used EPR method to determine the NO level in rat organs. Rats were
injected with the NO scavenger DETC and ferrous citrate and sacrificed 30 min
later. The NO content was determined in brain cortex, cerebellum, liver, heart
and kidneys. Fig. 1 and 2 presents NO spectra in the above organs. The spectra
all have a typical shape of Cu-DETC spectrum with a superposed NO peak. The
intensity of the Cu-DETC spectrum indicates the bioavailability of the tissues
under study to DETC. We regarded the height between the maximum of the peak
at g = 2.031 as the size of the NO-Fe-DETC signal (I) (Vanin and Kleschyov,
1998).
Apparently the observed intensities of the NO-Fe-DETC spectra in
different organs reflect the NO concentration therein. The Fe-DETC trap equally
distributes throughout the body (Vanin, Kteschyov, 1998), even disruption of the
blood-brain barrier does not influence its concentration in brain (Hooper et a!.,
1996). Both literature data (Mulsch et al., 1995) and analysis of the spectra shape
indicate a large excess of DETC over NO. It was reported that NO-Fe-DETC
complexes do not accumulate in tissues over time (Sato et al., 1994). Thus, the
observed spectra intensities cannot be due to specific accumulation of DETC in a
tissue or accumulation of NO-Fe-DETC complex over time. Special studies
indicate that Fe-DETC complex cannot interact with the nitrite ion in the cell
(Mulsch et al., 1992), although it is possible in vitro (Hirarnoto et al., 1997).
Further we will present data for each tissue studied and discuss possible reasons
of NO concentration differences in the organs.
Apparently, the NO content in the cerebellum is prone to seasonal changes.
Of course, no conclusion can be derived from this observation, the question
should be studied properly, Orcadian changes of NO content in brain and blood
serum were observed by several groups (Guerero et al., 1996; Pu et al., 1998;
Uludag et al., 1999), seasonal changes also seem to be probable.
The NO content in brain cortex was 46,0 ± 3,4 ng/g of tissue, in
cerebellum it was of about the same level (27,7 ± 2,6). Interestingly, the nNOS
content in the cortex and NOS activity measured in vitro are considered to be the
lowest among brain compartments, the same parameters in the cerebellum to be
the highest (Barjavel, Bhargava 1995; Singh et a!., 2000). Thus the produced NO
quantity in vivo does not depend on NOS copy number in the tissues, but sooner
on their activity.
NO plays an important ro!e in many brain functions including the effects
on long-term potentiation (Son et al., 1996), gonadotropin secretion (McCann,
1997), sexual behaviour (Nelson et al., 1995). NOS activity in brain is regulated
by sexual hormones (Singh et al., 2000). NO is a nerotransmitter, it is important
in mechanism of action of anaesthetics and ischemia-reperfusion injury in brain
(Sato et al., 1994, Sjakste et al., 1999a, 1999b, references therein). NO is
considered to be implicated in learning process (Scheweighofer, Ferriol, 2000).
Thus, the high NO content in brain might reflect its multiple functions.
The NO content in the brain cortex was rather resistant to the NOS
inhibitor administration. This could indicate that a part of NO is produced in nonenzymatic way, independently of NOS as a result of direct nitrite
disproportionation or its reduction (Zweier et al., 1999), On the contrary, NO
content in cerebellum was more susceptible to the inhibitor, the NO concentration
decreased drastically. Perhaps, the probable non-en2ymatic NO synthesis in brain
cortex could explain an apparent contradiction between comparable NO
concentrations in brain cortex and cerebellum despite much higher NOS
concentration in the latter.
Two types of NO spectra were observed in the liver, one representing high
NO concentration, the other - iow NO content. These discrepancies may be due
to reaction of the liver NOS on numerous environmental factors. In the liver,
constitutive NOS (cNOS) activity is normally detectable in Kupffer ceils,
whereas no cNOS is ever expressed in hepatocytes, However, hepatocytes,
Kupffer and stellate cells (the three main types of liver ceils) are prompted to
express an intense iNOS activity once exposed to effective stimuli such as
bacterial lipopolysaccharide and cytokines (Muriel, 2000). iNOS activity in liver
is susceptible to oxygen tension (Miralles et al., 2000). Insulin action, namely
glucose release from the hepatocytes is mediated by NO (Kahn et al., 2000), thusNO content in liver can depend on food intake. Dietary fat also augments the
iNOS activity in liver cells (Wan et al., 2000). Nitric oxide involvement in
multiple processes in the liver and susceptibility of NOS activity to numerous
factors could explain the high NO !eve! in this organ and a broad variation range
among individual animals. High sensivity of the liver NO concentration to iNOSspecific inhibitor AMT indicates the leading role of this isoform in maintenance
of the NO level in this organ (Table 3).
In the heart tissue the determined NO concentration was surprisingly low.
The low content of NO in the heart tissue seems to be striking, as all three types
of nitric oxide synthases (eNOS, nNOS, iNOS) were found in the heart. nNOS is
found in smooth muscle cells of blood vessels, eNOS in endothelium, but iNOS
is found in all celt types including cardiomyocytes (Papapetropulos et al., 1999).
NO regulates cardiac diastolic function (Paulus, Shah, 1999), it is involved in the
cardiac beta-adrenergic response (Balligand, 1999). NO regulates the cardiac
oxygen metabolism (Trochu et al., 2000). NO is involved in pathogenesis of
numerous heart disease, including arrhythmias (Paulus, Shah, 1999),
atherosclerosis, hypertension, diabetic myocardiopathies, heart failure (Kojda,
Harrison, 1999), vascular injury (Kibbe et al., 1999) ischemia-reperfusion injury
(Shen et al., 1998). NO is cardioprotective in ischaemic heart disease (Rakhit et
al., 1999). Despite involvement in numerous important processes, the NO content
in the heart is low.
In kidneys the NO content was comparable to that in the heart and much
lover than in brain and iiver. This result is also striking as in the kidney, NO
plays.prominent roles in the homeostatic regulation of glomerular, vascular, and
tubular function. Differentia! expression and regulation of the NO synthase
(NOS) gene family contribute to this diversity of action (Kone, Baylis 1997).
Renal medulla is enriched in NOS when compared to renal cortex, the NO
produced by the enzyme from renal medulla exerts the arterial blood control
(Mattson, Wu, 2000). NO is a prominent vasodilatator in the renal vasculature,
probably by means of inhibiting of the phospodiesterase (Kurtz et al., 2000). The
neuronal NO-synthase in macula densa regulates the cyclooxygenase expression,
interfering in this way with the regulation of renin-angiotensin system by
prostaglandins (Harris et al., 2000). The type 2 angiotensin receptors trigger the
NO release (Carey et al., 2000) and modulate the angiotensin II-induced
constriction of the rabbit afferent arteriole (Kohagura et al., 2000). In the
juxtaglomerural complex NO interacts with O2" radicals, the interaction between
two radicals contributes to regulation of the tubuloglobelural feedback (Wilcox,
Welch, 2000). Both endothelial and neuronal nitric oxide synthases are active in
the kidneys. Endothelial NO increases in response to an increase in perfusion
pressure, whereas macula densa nNOS decreases upon a sustained increase in
distal delivery (Braam et al., 2000). Renal NO production is important in the
hypertension development (Persoon et al., 2000). Nevertheless comparatively
small NO amount is produced in this organ.
Our data are in good agreement with non-quantitative results obtained by the
same method (Mikoyan et al., 1997) and taken together, our data indicate the fact
that NO content in a given tissue cannot be predicted by NOS role in its functions
or number of NOS copies detected in the tissue. Possibility of non-enzymatic NO
synthesis should not be ignored, quantitative measurements of the synthesized
NO in a tissue should be performed in parallel with determinations of NOS
activity in vitro, assessment of NOS mRNA transcription and enzyme
biosynthesis.
LPS-induced NO synthesis in rats tissues
We have quantitatively determined increase of nitric oxide concentration
induced by the lipopolysaccharide administration. Use of the sensitive Fe-DETCNO trapping technique has enabled us to evaluate the NO increase in several
organs. Besides the well-established sites of the LPS-induced NO increase in
liver, kidney and blood (Yoshimura et al. 1996; Bergamini et al. 2001; Mailman
et al. 2001) detected by other approaches we report drastic relative increase of the
NO production in the heart, and comparatively moderate, but significant NO
production increase in brain and testes, In our experiments the NO concentration
was measured per gram of wet weight of the tissues, taking into account eventual
edema development after the LPS administration, the increase of NO content per
gram of dry weight would have been even more significant. The observed 50-fold
increase of the NO production in the heart appears to be the most important for
development of the circulatory sepsis complications. Excessive NO can be
produced by iNOS that is found in many cell types in the heart, including
cardiomyocytes (Papapetropulos et al. 1999), LPS activates the iNOS also in the
blood vessel adventitia (Kleschyov et al. 2000). Increase of NO in brain might be
also dangerous. The observed NO increase in brain tissues may be due to an
iNOS expressed in brain tissues (Heneka & Feinstein, 2001).
2. POSSIBLE MECHANISMS OF MODIFICATIONS OF NO
SYNTHESE BY HVAS
When we started our research about changes of NO concentration under
action of HVAs we came across two opposite points of view in the literature.
Some authors stated that inhibitors of NOS acted synergistically with halothane
and isoflurane and decreased the minimal alveolar concentration for anesthesia,
thus HVAs should decrease NO concentration in brain cortex. (Johns et al., 1992;
Pajewski et al, 1996), Supporters of this hypothesis postulate inhibition of NO
synthesis and blocking of its neutransmitter function during narcosis (Johns et al.,
1995; Zuo et al., 1996; Chen et al., 1999; Tao et al., 2000). Another point of view
was based on several reports about HVAs vasodilatating effects which are NOmediated and indicate possible increase of NO concentration in brain during
anesthesia (Harkin et al., 1997; Ogawa et al., 1997; Koenig et al., 1993; Smith et
al., 1995; Staunton et al., 2000). Our experimental data about increase of NO
concentration in brain cortex during narcosis confirm the second point of view
(Sjakste et al., 1999). Possible mechanisms of both processes are given in Fig. 25,
We observed increase of NO concentration in brain cortex during
development of anesthesia under halothane, isoflurane and sevoflurane. In
cerebellum the effect was opposite - we observed decrease of NO concentration.
In further experiments we tried to investigate mechanisms of these
phenomena The question was whether these effects were characteristic for
HVAs or coupled to anaesthesia process itself. Experiments with intravenous
anaesthetics gave the answer to this question. Ketamine, chloral hydrate and
pentobarbital evoked anaesthesia but did not produce increase of NO content in
brain tissue. Therefore increase on NO concentration in brain cortex and decrease
in cerebellum is characteristic of HVAs.
Brain cortex is the only tissue where the NO increase is observed during
anaesthesia. It additionally demonstrates that increase of NO content is closely
linked to the anaesthesia process. Fluctuations of NO concentration are linked to
changes in brain function during anaesthesia.
Experiments with NOS inhibitors demonstrated that increase of NO
concentration in cortex is produced by activity of NOS, because NOS isozyme
non-specific inhibitor N-nitro-L-arginine and specific iNOS inhibitor AMT
effectively abolish this increase.
Thus, the described effect is specific for action of HVAs, it is linked to the
anaesthesia process and it is produced by activity of NOS. It should be stressed
that this effect is not necessary for development of narcosis as inhibitors of NOS
acts synergistically with HVAs (Johns et al., 1992; Pajewski et al., 1996).
Moreover, inactivation of NO with scavenger decreases minimal alveolar
concentration for these anesthetics (Chen et al., 1999).
We assumed the possibility that observed increase of NO concentration is
produced due hypoxia in brain (Iadecola, 1997). Possible mechanism is given in
Fig. 26. It is known that anesthesia under halothane interferes with energy
metabolism of brain. Few seconds after administration of halothane consumption
of energetic metabolites (glucose, ATP, phosphocreatine) decreases (McCandless
and Wiggins, 1981). Brain hypoxia develops as a result. Hypoxia triggers release
of NO. It could happen as follows: ischemia triggers depolarization of neuron
membranes, neurotransmitter glutamate is released in synapses and opens NMDA
specific glutamate receptors-ionofors that enable Ca2+ accumulation in the
neurons. Ca2+ activates nNOS (Beckman, 1991; Cazeveille et al., 1993). A
"vicious circle" can be formed in this case. Anesthetized rats in our hands did not
suffer from hypoxia as indicated by by to measurements of concentrations of
ATP, lactate and pyruvate. Thus, hypoxia pathway did not explain our results.
It appears that increase of NO concentration in brain cortex is mainly
linked with vasodilatating effect of HVAs. Vasodilatation is observed only in
brain cortex (Hansen et al., 1988), the same is valid for the increase of NO
concentration. Possibly vasodilatation and increased synthesis are linked in
another way, not like shown in Figure 25. It can be speculated that vasodilatation
is produced not by NO but by prostaglandins. Probably, HVAs induce
cyclooxigenase that catalyses biosynthesis of prostaglandins (Okamoto et al.,
2000). Moreover, the shear stress can activate eNOS and trigger synthesis of NO
(Rosenblum, 1998). Further we will see that these mechanisms (Fig. 27) are
improbable.
Literature data suggest that NOS mediates (nemely nNOS!) halothaneinduced cerebral microvascular dilatation (Staunton et al., 2000). In addition,
volatile anesthetics have been demostrated to increase intracellular calcium in the
cerebrocortical neurons, which induces NOS activity (Kindler et al., 1999).
Several recent studies performed using methodological approaches different from
ours indicate the increase of NO or its metabolite concentration in brain under
isoflurane anesthesia (Loeb et al., 1998; Matsuoka et al., 1999).
Thus question about concrete NOS isozyme responsible for increase of NO
concentration in brain cortex during anaesthesia was not resolved in this stage of
the study. Apparently this was not nNOS, eNOS and iNOS were likely
candidates. We have tried to answer this question by using NOS isoform-specific
inhibitors and stimulation of iNOS expression by lipopolisacharide (LPS). Our
data indicate that nNOS specific inhibitor can not affect increase of NO
concentration in brain cortex triggered by HVAs. However, iNOS specific
inhibitor AMT effectively attenuates increase of NO content triggered by
isoflurane or sevoflurane. Thus, experiments by using specific inhibitors exclude
participation of nNOS in this process and indicate probable role of iNOS.
Experiments by using LPS confirmed this idea. Series of experiments with
intraperitonial administration of LPS did not give unambiguous results,
nevertheless we can observe tendency of stimulation of NO synthesis induced by
LPS in narcotized animals. The question was solved in experiments with
intracerebroventricular administration of LPS. Sevoflurane anaesthesia
significantly enhanced the NO biosynthesis in brain cortex induced by LPS after
it intracerebroventricular administration. Thus, by increasing the number of iNOS
enzyme copies we can acquire marked increase of NO concentration in brain
cortex. HVAs are known to enhance expression of the iNOS gene in vitro (Zuo
and Johns, 1997), however it is unlikely that this effect can manifest itself during
30 min long anaesthesia. Some authors mention increase of number of iNOS
copies beginning with the 6th hour after of narcosis (Kapinya et al., 2002).
However in some cases increase of iNOS copies in neurons was observed in 15
minutes after stimulus, probably, from pre-existing mRNAs (Fig. 28) (Holtz et
at., 2001).
Similar conclusion is made by Okamoto and co-workers (2000), who
observed intensification of brain hyperemia caused by halothane on the LPS
background.
Summarizing data about HVAs effects on NO synthesis in brain cortex we
conclude that they cause increase of NO concentration, effect is specific for
narcosis evoked by this group of anesthetics this process is not necessary for
development of narcosis, mainly it is effectuated by iNOS. Apparently NO
content increased during narcosis causes vasodilatation in brain vasculature. Are
our data in contradiction with John's hypothesis? Certainly not! John's
hypothesis postulates the blocking of NMDA - nNOS - cGMP pathway. John's
group and some other groups in several publications try to prove this hypothesis.
By using arginine - citrulline conversion test in the cell or ex vivo tissue
homogenate systems for measurements of NOS activity contradictory data were
obtained. Some authors found that activity of NOS decreases (Tobin et al., 1994;
Galley et al,, 1995), others insists that anaesthetics do not alter activity of enzyme
(Rengasamy et al., 1995; Tagliente et al., 1997; Galley et al., 2001). John's group
even found increase of NOS activity under action of isoflurane (Rengasamy et
al., 1997). Data about HVAs action on cGMP content reproduce better - usually
a decrease is found (Rengasanmy et al., 1997; Galley et al., 2001). In later
publications this group concludes that, halothane and other HVAs act on cGMPdependent protein kinase la (Tao et al., 2000). It seems that our work gives new
arguments in favour of John's hypothesis. First, we found manifested decrease of
NO concentration in cerebellum of anesthetized animals, in tissues rich in nNOS.
Thus inhibition of nNOS is not excluded. Second, isoflurane, sevoflurane and
halothane can inhibit activity of biotechnologically obtained enzymes, the three
NOS isoforms. However this effect is achieved by high drug concentrations only
(ECjo = 20 mM). It might seem that these results have no physiological
significance, but careful analysis of literature reveals that local concentration of
HVAs in synapses reaches comparable level (Eckenhoff and Eckenhoff, 1998).
Hence, action of anesthetics on NOS systems in brain is manifold and different
depending on concrete NOS izozyme. Our results and data from literature about
HVAs interaction with nNOS and iNOS enzymatic systems in brain cortex are
systemized in Fig. 29.
3. EVENTUAL NO-DEPENDENT MECHANISM OF ACTION OF
ANTI-ISCHEMIC DRUG MILDRONATE AND ITS ANALOGUES
Our results provide evidence that GBB methyl and ethyl esters are potent
NO- and endothelium-dependent vasodilators. While mildronate alone elicits no
activity it sharply potentates the activity of GBB in endothelium- and NOSdependent responses. These data suggest that known fast anti-ischemic effects of
mildronate may be in part related to the stimulation of formation of NO by the
endothelium. Previously, it has been reported that mildronate has beneficial
effects on energetic parameters of ischemic heart and increases the survival of
rats after experimental myocardium infarction (Ratunova et al., 1989). None of
these effects could be explained by the inhibition of carnithine biosynthesis (Fig.
30). However, they may be, in part, due to an increase in NO formation. We have
demonstrated transient but significant increases in the formation of NO in blood
by pharmacologically relevant doses of mildronate, GBB and GBB methyl ester.
These changes in NO levels may reflect its production by the vascular
endothelium, including the endothelia! cells within the heart. Changes of NO
concentration in myocardium tissue appear to be less spectacular, however these
may be elusive due to very low basic NO level in the myocardium (Dzintare et
al., 2001).
The stimulation of NO production by mildronate, GBB and GBB esters is
clearly not related to a direct effect on NOSs activities as the above substances
modified neither the oxidation of L-arginine to citrulline nor the NADPH oxidase
activities of the purified enzymes, and did not interact with the heme active site
of the nNOSoxy. Our results suggest that some receptor-mediated mechanisms are
involved in the activation of the formation of NO in blood vessels.
Cholinomimetic activity of GBB esters and compounds of similar structure has
been described long time ago (Hosein et al., 1970). Mildronate ethyl ester
EDIHYP is even considered to be a synthetic analogue of acetylcholine (Meerson
et al., 1991). Indeed, recent in vitro data obtained by other researcher team in our
Institute have shown that GBB methyl ester is a potent agonists for m-type of
acetylcholine receptors, GBB affinity to these receptors is much lower
(Dambrova et al., submitted, preliminary publication in Liepins et al, 2003). In
our experiments, mildronate alone elicited no vasodilating activity but five fold
increased the activity of GBB. Furthermore, GBB esters appeared to be one
hundred fold more active than GBB itself on NO- and endothelium dependent
relaxation of rat aortic rings. The synergistic effects of mildronate on GBB
activity may involve the esterification of GBB, as GBB esters trigger their
vasorelaxing effects at much lower concentrations than GBB itself. These results
suggest us that fast anti-ischemic action of mildronate could be mediated, at least
in part, by stimulation of NO production in the vascular endothelium through a
modification of the GBB/GBB esters pools. This stimulation of NO formation
might be rationalized in the following ways (Figure 31):
• administration of mildronate inhibits GBB hydroxylation and increases the
intracellular pool of GBB;
• a part of GBB is released from cells, and, after esterification, forms potent
cholmomimetics GBB esters;
• GBB esters, via acetylcholine receptors on endothelial cells could activate
eNOS.
Above data provide evidence for most steps of this hypothetical
mechanism, the increase of GBB esterification after mildronate administration
remaining the missing link. In this regard, the presence of GBB esters in living
tissues was described long time ago, although their physiological significance
remains poorly known (Hosein et al., 1970). As they decrease systemic blood
pressure, they display analogies with acetylcholine and activation of eNOS
activity might be one of the functions of these compounds. Interestingly,
carnitine has been found to produce endothelium-dependent vasorelaxation in
aortic rings (Herrera et al., 2002), an activity that might be mediated by
esterification, as carnithine esters also possess cholinergic activity (Hosein et al.,
1970).
Taken together, our data demonstrate the endothelium- and NO-dependent
vasodilating effects of GBB esters and provide new evidences for the
involvement of these compounds in the mechanisms of action of mildronate.
The magnitude of the GBB effect on the LPS-induced NO production is
comparable to that of N-acetylcysteine, a well-known antioxidant and radical
scavenger (Bergamini et al., 2001). The GBB and mildronate effects are not due
to the iNOS inhibition, as the substances could not inhibit the enzyme in vitro.
Rapid, but transient nature of the effect clearly indicates that it is not due to
decrease of iNOS gene expression. Probably, the substances interact with the NO
in direct or indirect way. The recently described ability of carnitine to interfere
with NO and superoxide interplay in the cells supports this idea (Koeck and
Kramser, 2003). Moreover, antioxidant activity of the GBB and some of its
analogues in vitro has been reported already (Shutenko et al., 1997). Pretreatment
with the GBB attenuates the hydrogen peroxide -induced metabolic
derangements in the myocardium (Akahira et al., 1997). It seems that we provide
novel evidence of antioxidant activity of the drugs. Mechanism of the NO -
decreasing activity of GBB and mildronate remains to be established. Apparently,
mildronate cannot be recommended for treatment of the sepsis complications, as
its NO-decreasing activity is restricted to brain cortex. On the contrary GBB
appears to be more effective. Our findings suggest that novel drugs for treatment
of sepsis complications could be sought among the GBB analogues.
CONCLUSIONS
1 Volatile halogenated anesthetics halothane, isoflurane and sevoflurane induce
NO concentration decrease in the cerebellum.
In brain cortex the volatile halogenated anesthetics halothane,
isoflurane and sevoflurane produce an opposite effect: a two-fold or three-fold
NO concentration increase, hi the case of sevoflurane the data of EPR
spectroscopy were confirmed by measurements of the NO stable metabolite
(nitrite and nitrate) concentration detection by Griess method, the latter
indicated increase of nitrite and nitrate concentration.
In liver, heart, kidney, testes and blood NO concentration does not
change under anesthesia induced by these drugs.
2. We are first to demonstrate significant differences in the NO concentration in
different rat organs, it is the highest in brain cortex (46,0 ± 3,4 ng/g tissue)
and in blood (33,6 ± 12,4 ng/g tissue), somewhat lower in the cerebellum
(27,7 ± 2,6 ng/g tissue), liver (27,6 ± 4,7 ng/g tissue) and testes (13,8 ± 1,1
ng/g tissue), the lowest NO concentration was observed in the heart (4,8 ± 0,7
ng/g tissue) and kidneys (3,3 ± 0,5 ng/g tissue).
3. In the course of anesthesia no changes were observed in ATP concentration
and lactate/piruvate ratio in rat brain cortex. Thus anesthesia by halogenated
volatile anesthetics does not influence the tissue energy metabolism in rat
brain cortex. The above increase of NO is not due to ischemia.
4. Intravenous anesthetics (kentamine, pentobarbital, chloral hydrate) do not
influence NO concentration in rat brain cortex.
5. The selective iNOS inhibitor 2-amino-5(6-dihidro-6-methyl-4H-l,3-tiazine
(AMT) inhibits the NO concentration increase in the rat brain cortex induced
by halogenated volatile anesthetics, but the selective nNOS inhibitor 7-nitroindazole (7-Ni) does not influence it. 7-Ni decreases the NO concentration in
the cerebellum.
Isoflurane and sevoflurane enhance the NO increase produced by
intraventricular administration of lipopolysaccharide.
These data indicate possible iNOS involvement in the NO concentration
increase during halothane, isoflurane and sevoflurane anesthesia.
6. [14C]arginine - [14C]citruIlJne conversion test reveals inhibiting activity of
all NOS isoforms by high concentrations of isoflurane (40mM).
7. Mildronate, gamma-butyrobetaine, GBB methyl- and ethyl-esters produce a
transient increase of NO concentration in several organs. The latter is due to
NOS activity as NOS inhibitors abolish the effect. GBB esters produce the
effect at 100 times lesser concentrations as compared to mildronate and GBB.
8. Experiments with rat aorta rings revealed that GBB methyl- and ethyl-esters
are potent NO and endothelium-dependent vasodilatators. Mildronate does not
possess vasodilating activity but it enhances the weak vasodilating effect of
GBB.
Mildronate, gamma-butyrobetaine, GBB methyl- and ethyl-esters do
not produce direct effects on NOS activities as revealed in experiments with
purified NOS isozymes.
9. Mildronate and gamma-butyrobetaine decrease the enhanced NO synthesis
induced by LPS beginning from IS minutes and up to one hour after
administration. "Hie drugs do not inhibit iNOS in vitro. Apparently mildronate
and gamma-butyrobetaine possess antioxidant activity.
The pronounced ability of the GBB to decrease the NO synthesis
triggered by LPS can be used for design of novel remedies aimed on treatment
of the circulation-related complications of sepsis.
10.According our hypothesis halogenated volatile anesthetics produce different
effects on NOS isoforms. To acieve the narcosis effect nNOS is inhibited. In
the same time iNOS activity is increased in the brain cortex. The
consequences of the latter effect are observed as overall NO concentration
increase of in brain cortex during anesthesia,
11.The obtained data enable to explain the fast physiological effects of
mildronate, GBB and GBB esters, namely the vasodilating activity of the
drugs. According to our hypothesis this happens as follows: administration of
mildronate inhibits hydroxylation of GBB and increases the GBB
concentration in tissues, a certain GBB fraction is esterified and forms potent
cholinomimetic compounds, the latter bind the M-cholinoreceptors and
activate eNOS.