Full text of DOI:10.1038/sj.bjp.0704025

British Journal of Pharmacology (2001) 133, 5 ± 12
ã
2001 Nature Publishing Group All rights reserved 0007 ± 1188/01 $15.00
www.nature.com/bjp
The polycationic aminoglycosides modulate the vasoconstrictive
e€ects of endothelin: relevance to cerebral vasospasm
1
1Grant Wickman, 1,2Mourad A. Nessim, David A. Cook & *,1Bozena Vollrath
1Department of Pharmacology, University of Alberta, Edmonton AB, Canada, T6G 2H7
1
The vasoactive peptide endothelin (ET) has been implicated in the pathogenesis of cerebral
vasospasm following subarachnoid haemorrhage. In these studies we investigated the involvement of
protein kinase C (PKC) in sustained vasoconstriction induced by ET-1 in canine cerebral arteries.
We also examined the ability of the aminoglycoside antibiotics to reverse the e€ects mediated by ET-
1 in canine cerebrovascular smooth muscle cells (CVSMC).
2
The ETA receptor antagonist, BQ-123, showed a competitive inhibition of the ET-1 responses.
3
The vasoconstrictor action of both ET-1 (0.5 nM) and phorbol myristate acetate (PMA) (160 nM)
was reversed by a selective PKC inhibitor, Ro-32-0432.
In cerebral arteries precontracted with ET-1 the aminoglycosides caused a concentration-
4
dependent relaxation. The EC50s for the relaxation were as follows: 0.54+0.05, 0.63+0.01,
1.88+0.46 and 2.3+0.92 mM for gentamicin, neomycin, streptomycin and kanamycin, respectively.
5
Gentamicin caused a concentration-dependent decrease of the PMA-induced responses in
calcium free medium.
PKC activity was elevated in CVSMC exposed to ET-1 (170%) and PMA (167%) for a period of
time (60 min) corresponding to maximum tonic contraction induced by these agents in arterial rings.
The administration of the aminoglycosides to CVSMC, in concentrations corresponding to the
6
7
EC50s from contractility studies, reduced the e€ects of both ET-1 and PMA on PKC activity to the
levels not di€erent from controls.
8 These results show that the aminoglycosides are able to inhibit sustained vasoconstriction
induced by ET-1, an e€ect which is due, at least in part, to the inhibition of PKC.
British Journal of Pharmacology (2001) 133, 5 ± 12
Keywords: Cerebral vasospasm; endothelin; aminoglycosides; protein kinase C
Abbreviations: CSF, cerebrospinal ¯uid; CVSMC, cerebrovascular smooth muscle cells; DAG, diacylglycerol; ET, endothelin;
PKC, protein kinase C; PMA, phorbol-12-myristate-13-acetate; SAH, subarachnoid haemorrhage
Introduction
Delayed cerebral vasospasm remains one of the major causes
of morbidity and mortality in patients with subarachnoid
haemorrhage (SAH), and at present there is no satisfactory
treatment which can prevent or reverse cerebral vasoconstric-
tion in humans (Cook & Vollrath, 1995). Although the
pathogenesis of this condition is unclear, there is evidence
that one of the principal causative agents in vasospasm is
endothelin-1 (ET-1), a potent vasoactive peptide generated in
vascular endothelial and smooth muscle cells (Zimmermann
level of gene transcription by a number of factors, including
the vasoactive agents, haemoglobin, thrombin and prosta-
glandin F2a, released from the blood clot after SAH (Schini et
al., 1989; Ohlstein & Storer, 1992; Kasuya et al., 1993).
Together these ®ndings support the concept that release of
ET-1 may play a role in the development of vasospasm, and
suggest that inhibition of the e€ects of the vasoconstrictors
released from the blood clot following SAH could provide a
valuable means of therapeutic intervention.
&
Seifert, 1998). Levels of ET-1 are elevated in the
The molecular mechanism of ET-mediated sustained
cerebral vasoconstriction remains unclear. In vascular smooth
muscle this peptide interacts with ETA receptors which are
coupled via Gq protein to multiple cellular e€ectors,
including phospholipases C (PLC) and D (PLD), protein
kinase C (PKC), tyrosine kinases (Schi€rin & Touyz, 1998;
Goldie, 1999), and several classes of calcium channels
(Nakajima et al., 1996; Iwamuro et al., 1999).
It has been suggested that a major role in the intracellular
events leading to sustained contraction is played by PKC and
its serine/threonine phosphorylated substrates (Horowitz et
al., 1996). The concept of a role for PKC in cerebral
vasospasm is supported by the observations that phorbol
esters, potent activators of PKC, induce angiographic
cerebrospinal ¯uid (CSF) of patients with SAH, and the size
of these changes is correlated with the timing and severity of
the spasm (Seifert et al., 1995). In addition, the ETA receptor
antagonists, BQ-123 (Itoh et al., 1994; Josko et al., 1998) and
TBC 11251 (Wanebo et al., 1998), attenuate cerebral
vasospasm in animal models, although not all studies have
been positive (Hino et al., 1995). Further evidence in favour
of the concept of a role for ET-1 is provided by the
observations that synthesis of this peptide is stimulated at the
*Author for correspondence; E-mail: bozena.vollrath@ualberta.ca
2Current address: Emory Eastside Medical Center, 1700 Medical
Way, Snellville, GA 30078, U.S.A.

6
G. Wickman et al
Aminoglycosides modulate the effects of endothelin
vasospasm (Sako et al., 1993), and that the PKC inhibitors
improve the outcome in experimental models of cerebral
vasospasm (Minami et al., 1992). Furthermore, the level of
DAG exhibits a prolonged increase during experimental
cerebral vasospasm, suggesting a critical involvement of PKC
(Asano & Matsui, 1999). Since ET-1 activates both PKC and
PLD in a variety of cells (Eskildsen-Helmond et al., 1997), it
is conceivable that sustained cerebral vasoconstriction
induced by this peptide is due, at least in part, to a prolonged
generation of DAG and thus activation of PKC. We have
recently shown that cerebral vasoconstriction induced by
haemoglobin, released from haemolyzed blood after SAH,
and prostaglandin F2a, can be inhibited by neomycin, a
nonselective inhibitor of PLC which has been widely used to
assess the involvement of inositol 1,4,5 trisphosphate (IP3) in
cellular responses (Gergawy et al., 1998). A number of studies
have shown that in addition to PLC, neomycin inhibits other
targets, including PKC (Hagiwara et al., 1988), PLD
(Rumenapp et al., 1997) and a variety of calcium channels
(Perrier et al., 1992; Langton et al., 1996; Pichler et al., 1996).
The ability of neomycin and related agents to inhibit a wide
range of intracellular events, some of which probably
contribute to the vasospastic activity by means of their
involvement in the signalling pathways activated by vasocon-
strictors, suggests that these compounds, or agents with
similar characteristics, could be of potential bene®t in the
treatment of cerebral vasospasm. Therefore, the present
studies were conducted to explore whether PKC may be
involved in a sustained vasoconstriction induced by ET-1 in
cerebral arteries, and to determine whether the structurally
related aminoglycoside antibiotics, gentamicin, neomycin,
kanamycin and streptomycin, a€ect the contractile action of
ET-1 and, if so, what mechanism might be involved in this
action.
preparations to 60 mM KCl were induced. The responses
were recorded isometrically using force displacement trans-
ducers and a Grass 7D polygraph (Vollrath et al., 1994). To
obtain concentration responses to ET cumulative concentra-
tions of these agents were added to organ baths as soon as
the maximum response to the preceding concentration had
been obtained. In the experiments in which the e€ects of the
aminoglycoside antibiotics, gentamicin, neomycin, kanamycin
and streptomycin were measured, the increasing cumulative
concentrations of these agents were added after a plateau
tension in response to 0.5 nM ET-1 or 160 nM of phorbol-12-
myristate-13-acetate (PMA) had developed. The relaxation of
the preparations exposed to the aminoglycosides was
expressed as the percentage of maximum tension produced
by the vasoconstrictors. Similar protocol was employed in the
experiments in which the e€ects of PKC inhibitor, Ro-32-
0432, were examined. The presence of an intact endothelium
was examined by evaluating the relaxant responses to
bradykinin, in an arterial preparation that was precontracted
with 1 mM 5-hydroxytryptamine (5-HT). In addition, the
relaxing responses to the aminoglycoside antibiotics were
analysed in the presence of nitric oxide synthase (NOS)
inhibitor (L-NAME). In some experiments, the aminoglyco-
sides (5 mM) were added 30 min before the exposure of
cerebral artery preparations to the agonists. The relaxation of
the preparations exposed to the aminoglycosides was
expressed as the percentage of maximum tension produced
by the vasoconstrictors. The EC50 values for the aminoglyco-
sides were determined by ®tting an exponential curve to the
data using Microsoft Excel.
Cerebrovascular smooth muscle culture
Cells were prepared as described previously (Vollrath et al.,
1995). Brie¯y, canine basilar arteries were isolated under
sterile conditions and placed in a Petri dish containing
Dulbecco's modi®ed Eagle's medium (DMEM). The
adventitia was removed mechanically, the vessels were cut
into segments approximately 5 mm in length along the
longitudinal axis, and the endothelium was removed by
gently scraping the inner surface of the segments. The
tissue was then chopped into 1 to 2 mm pieces and the
explants were transferred to 25 cm2 culture ¯asks containing
1 ml of DMEM supplemented with 10% foetal calf serum,
Methods
Recording of isometric tension
Mongrel dogs of either sex, weighing about 20 kg, were used
in these studies. Protocols for the humane treatment of
animals, according to the Declaration of Helsinki, and as
approved by the University of Alberta Animal and Ethics
review Committee were followed in all experiments. The
animals were killed with an intravenous (i.v.) overdose of
pentobarbitone, and the brain with cerebral arteries attached
was removed and placed in oxygenated Krebs-Henseleit
solution of the following composition (in mM): Na+ 130,
penicillin (100 u ml71
)
and streptomycin (100 mg ml71).
After the explants had adhered, the volume was made up
gradually over the next 4 days to 4 ml per ¯ask, and
thereafter the culture medium was changed weekly. When
the primary cultures were almost con¯uent the cerebrovas-
cular smooth muscle cells (CVSMC) were transferred to
75 cm2 ¯asks and then routinely subcultured at a split ratio
of 1 : 3.
27
K+ 5, Ca2+, 2.5, Mg2+, 1.2, Cl7 12.2, HCO3 25, SO4 1.2,
27
H2PO4
1.2 and dextrose 11. Each artery was cut into
sections about 3 mm in length and these preparations were
suspended in tissue baths containing Krebs-Henseleit solution
at 378C, gassed with 95% oxygen and 5% carbon dioxide.
The arterial rings were equilibrated for 1 h. Isometric tension
was measured using a strain gauge transducer, attached to a
preampli®er and recorded on a potentiometric chart recorder.
The resting tension was set at 1.0 g, which is optimal for
inducing maximum contraction, and the preparations were
allowed to equilibrate for 60 min, during which time the
bathing medium was renewed every 15 min. After the
equilibration period, contractile responses of the ring
Measurements of PKC activity
PKC activity was measured in the membrane and cytosolic
fractions of serum-starved and antibiotics (penicillin and
streptomycin) free (48 h) CVSMC exposed to ET-1 (0.5 nM)
or PMA (160 nM) for 60 min. The aminoglycoside antibiotics
were added 30 min after initial exposure to the agonists, in
the concentrations equal to the EC50s calculated from
contractility studies. After incubation, the cells were scraped
British Journal of Pharmacology vol 133 (1)

G. Wickman et al
Aminoglycosides modulate the effects of endothelin
7
in ice-cold phosphate bu€ered saline (PBS) (145 mM NaCl
and 10 mM sodium phosphate; pH 7.4), and centrifuged at
2006g for 10 min. The pelleted cells were homogenized in
ice-cold 25 mM Tris-HCl bu€er (pH 7.2) containing (mM)
EGTA 4, EDTA 2, dithiothreitol 2.5 and leupeptin 20 mM.
Homogenized cells were then separated into cytosolic and
particulate fractions by centrifugation at 15,0006g for
60 min at 48C. The supernatant was then assayed for soluble
PKC activity which was measured using a PKC enzyme assay
system (Pierce). The membrane pellets were resuspended in
the homogenization bu€er and the PKC was solubilized via
sonication in ice-cold homogenization bu€er containing 0.4%
Triton X-100. The standard reaction mixture (15 ml) con-
tained Tris at pH 7.4 100 (mM), ATP 10 mM, MgCl2 50 mM,
CaCl2 0.5 mM, 0.01% Triton X-100, phosphatidylserine (PS)
(1 mg ml71), PMA (1.6 mM), the peptide substrate (pseudo-
substrate peptide labelled with a ¯uorescent dye), and a
sample containing the endogenous PKC (10 ml). The samples
were incubated at 308C for 30 min. The reaction mixture was
then applied to the separation units containing the anity
membranes (Pierce), which speci®cally bind the phosphory-
lated peptide. The bound phosphorylated substrate was
eluted from the anity membranes using a bu€er containing
15% formic acid, and its absorbance was measured at
570 nm. PKC activity was estimated using a puri®ed PKC
from rat brain (0.02 units ml71) as a standard and was
measured as picomoles phosphate transferred per minute per
miligram protein. Protein concentration was determined by
the method of Bradford (Bradford, 1976) using bovine serum
albumin (BSA) as a standard.
patients (Seifert et al., 1995), and it may therefore be
considered relevant in the pathophysiological setting. To
elucidate the endothelin receptor subtype responsible for the
ET-1-induced contraction, the responses to ET-1 were studied
in cerebral arterial rings, in the presence or absence of a
selective ETA receptor antagonist BQ-123, administered in
the IC50 concentration (0.1 mM). As shown in Figure 1,
exposure to this agent resulted in a competitive antagonism
of the ET-1 responses.
To study whether PKC was involved in the sustained phase
of tonic contraction induced by ET-1, we examined the e€ects
of a selective, cell permeable PKC inhibitor, Ro-32-0432,
which displays 10 fold greater selectivity for the classical
PKC isoforms (IC50 about 25 nM) over PKCe
(IC50=180 nM) (Wilkinson et al., 1993). Ro-32-0432
(56 nM) administered to the preparations in which a plateau
tension had developed in response to 0.5 nM ET-1, produced
about 60% relaxation; a complete blockade was obtained at a
360 nM of the inhibitor (Figure 2). In contrast to ET-1
induced contraction, which consisted of both phasic and
tonic components (Figure 3a), PMA caused only a sustained
tonic contraction, slow in onset but progressive in the rise of
tension, and it took about 50 ± 60 min before a maximum
tension developed (Figure 3b). Ro-32-0432 (56 nM) was less
e€ective in the preparations precontracted with PMA
(160 nM), and the maximal relaxation was obtained with a
concentration of the inhibitor equal to 360 nM (Figure 2).
In the study in which the e€ects of the aminoglycoside
antibiotics were examined, neomycin, gentamicin, streptomy-
cin and kanamycin were administered in increasing cumula-
tive concentrations (0.01 ± 10 mM) to ring preparations of
canine basilar artery in which a tonic contraction to ET-1
(0.5 nM) or PMA (160 nM) had developed. Representative
traces of the e€ects of gentamicin on the responses of tissues
pretreated with ET-1 or PMA are shown in Figure 3. The
relaxation of the preparations exposed to the aminoglycosides
was expressed as the percentage of maximum tension
produced by the vasoconstrictors. Cumulative concentra-
tion-e€ect curves for the aminoglycosides are shown in
Figure 4a. The most e€ective agents were gentamicin and
neomycin, both of which produced half-maximal relaxation
Chemicals and other reagents
Endothelin-1 (ET-1), PMA, bradykinin (BK), 5-HT, NG-
nitro-L-arginine methylester (L-NAME) and sulphate salts of
gentamicin, neomycin, kanamycin and streptomycin were
obtained from Sigma. Sodium sulphate (15 mM) was used as
a vehicle control for the aminoglycosides and was shown to
be devoid of any e€ect on the muscle tension. Ro-32-0432 ([2-
{8-[(Dimethylamino)
methyl]-6,7,8,9-tetrahydropyridol[1,2-
a]indol-3-yl}-3-(1-methylindol-3-y)maleimide, hydrochloride]
was purchased from Calbiochem. PKC assay kit was
obtained from Pierce. Bovine calf serum and Dulbecco's
modi®ed Eagle's medium (DMEM) were obtained from
GIBCO Canada. All other reagents were of the highest
available quality and were obtained from Sigma, Calbiochem
or Fischer Scienti®c.
Statistical analysis of results
All results are reported as means+s.e.mean, with number of
preparations used in parentheses. Statistical signi®cance was
assessed using one-way analysis of variance (ANOVA)
followed by Dunnet test when signi®cant probability was
reached. Values of P50.05 were considered to be signi®cant.
Figure 1 Concentration-e€ect curves for the vasoconstriction of
rings of basilar artery elicited by endothelin-1 (0.5 nM) in the absence
or presence of a selective antagonist of ETA receptor, BQ-123
(0.1 mM). Values are expressed as a percentage (% control) of the
response observed in the absence of the antagonist. Data points
represent means+s.e.mean (bars) for measurements with four to ®ve
individual ring preparations.
Results
ET-1 induced a concentration-dependent sustained contrac-
tion of cerebral arteries with an EC50 of 0.48 nM. This is in
the range of ET-1 concentrations observed following SAH in
British Journal of Pharmacology vol 133 (1)

8
G. Wickman et al
Aminoglycosides modulate the effects of endothelin
Figure 2 E€ects of the PKC inhibitor, Ro-32-0432, on vasocon-
striction induced in rings of basilar artery by endothelin-1 (ET-1)
(0.5 nM) and PMA (160 nM). The ring preparations were ®rst
exposed to ET-1 or PMA, and Ro-32-0432 was administered after
tonic response to the agents had developed. The responses are
expressed as the percentage of maximum tonic tension observed in
the absence of inhibitor (ordinate). Each bar represents mean+
s.e.mean of 4 ± 5 experiments. ***P50.001, compared with control
responses.
Figure 4 Cumulative concentration-e€ect curves for the relaxation
of rings of canine basilar artery produced by the aminoglycosides.
The preparations were ®rst exposed to ET-1 (0.5 nM) (a) or PMA
(160 nM) (b) and the aminoglycosides, gentamicin (genta), neomycin
(neo), kanamycin (kana) and streptomycin (strepto), were adminis-
tered in increasing cumulative concentrations to preparations in
which maximum tonic tension had developed. Ordinate: relaxation
expressed as a percentage of the maximum tension induced by ET-1.
Abscissa: concentration of the aminoglycosides on a logarithmic
scale. Data points represent the means+s.e.mean for experiments
conducted with ®ve or more ring preparations from three or more
animals.
Figure 3 Representative traces of the e€ects of gentamicin on the
responses of ring preparations pretreated with ET-1 (a) or PMA (b).
Arrows indicate time of administration of ET-1, PMA and
gentamicin. Each tracing illustrating the response of an individual
preparation is representative of three to ®ve independently conducted
experiments.
Vasoconstriction induced by PMA started within 5 ±
10 min, reached a maximum at about 60 min, and persisted
for several hours. As shown in Figure 4b, neomycin,
gentamicin and streptomycin induced about 80% of
maximum relaxation of the contractions induced with
PMA while kanamycin was less e€ective (about 52% of
maximal relaxation). To determine whether inhibition of
calcium in¯ux could contribute to the e€ects of the
aminoglycosides on contraction mediated by PMA, arterial
preparations were exposed to these agents in calcium free
medium. As shown in Figure 5, the PMA-induced
contraction was maintained in calcium free bu€er, and was
attenuated by gentamicin (Figure 5a) and Ro-32-432, an
inhibitor of PKC (Figure 5b), administered after a plateau
tension had developed.
of the contractions induced by ET-1 at 0.5 mM and 0.6 mM,
respectively. Streptomycin and kanamycin were less e€ective
and the EC50s for these agents were about 2 and 1.9 mM,
respectively (Table 1). This order of potency re¯ects the
number of positively charged amino groups; neomycin and
gentamicin which possess six and ®ve positively charged
amino groups, respectively, were most e€ective against
contractions caused by ET-1, while streptomycin and
kanamycin characterized by the presence of three and four
positive charges were less potent in producing relaxation. The
relaxation was maintained over a prolonged time of exposure
(several hours) to the aminoglycosides. In order to explore
the possibility that vasodilatory e€ects of the aminoglycosides
on ET-1 responses involve inhibition of PKC activity, we also
studied the e€ects of these agents on the contractions elicited
with PMA (160 nM).
In order to determine whether the vasorelaxant e€ects of
the aminoglycosides could arise from an e€ect mediated by
endothelium, responses to bradykinin were recorded as an
British Journal of Pharmacology vol 133 (1)

G. Wickman et al
Table 1 EC50 values (mM) for the aminoglycoside anti-
biotics against the vasoconstriction produced by endothelin-
1 and phorbol 12-myristate 13 acetate (PMA)
Aminoglycosides modulate the effects of endothelin
9
Direct measurements of the total PKC activity were
performed using cultured canine CVSMC stimulated with
ET-1 (0.5 nM) and PMA (160 nM), in the presence or
absence of the aminoglycosides. Since activated PKC
translocates to the plasma and/or nucleus membranes, a
process which is a hallmark of activation, the activity of this
enzyme(s) was determined in both cytosolic and membrane
(particulate) fractions. PKC activity was determined at the
time-point (60 min) corresponding to maximum tonic
contraction developed in response to ET-1 and PMA. The
data show that a major e€ect of ET-1 and PMA is to
elevate PKC activity in the particulate fractions of CVSMC
exposed to these agents. There were corresponding decreases
in the cytosolic fractions of cells treated with ET-1 and
PMA, suggesting that challenge with these vasoconstrictors
resulted in the PKC translocation. However, these changes
did not reach statistical signi®cance. The administration of
gentamicin, neomycin, kanamycin and streptomycin, in the
concentrations corresponding to the EC50 values determined
in contractility studies, signi®cantly reduced the e€ects of
ET-1 on PKC activity in particulate fractions to
111+11.5%, 107+13%, 122+15% and 129+12% of
control, respectively (Figure 6a). The e€ects of PMA on
PKC activity were signi®cantly reduced to 103+7%,
100+9%, 114+5.4% and 88.6+4.5% of control for
gentamicin, neomycin, kanamycin and streptomycin, respec-
tively (Figure 6b).
Gentamicin Neomycin Kanamycin Streptomycin
Endothelin-1 0.54+0.05 0.63+0.1 2.34+0.9
1.88+0.46
(n=3)
1.43+0.35
(n=5)
(n=5)
1.54+0.3
(n=5)
(n=5)
1.63+0.3 4.51+0.48
(n=6) (n=4)
(n=5)
PMA
Figure 5 Representative traces of the vasoconstrictor responses
induced by PMA (160 nM) in calcium free medium. Gentamicin (a)
and Ro-32-0432 (b) were administered to the preparations in which
maximum tonic contraction to PMA had developed. (c) shows
representative traces of the e€ects of bradykinin on rings of basilar
artery with denuded endothelium. The rings were precontracted with
5-HT (10 mM). Bradykinin produced no relaxation suggesting that
endothelium had been removed from the preparations. Each trace,
which illustrates the response of an individual ring preparation, is
representative of three or four independent experiments.
indicator of the preservation of endothelial cells. Bradykinin
(1 pM ± 100 nM) produced a concentration-dependent vascu-
lar relaxation, in preparations which had been precontracted
with 5-HT, in the presence but not in the absence of
endothelium. In the arterial preparations in which bradykinin
had no relaxant e€ect, gentamicin retained its relaxing
activity thus indicating that the vasodilatory e€ect of this
agent was endothelium independent (Figure 5c). In another
series of experiments, rings were pre-incubated with an
inhibitor of nitric oxide synthase, NG-nitro-L-arginine
methylester (L-NAME) (100 mM), for 30 min. L-NAME did
not signi®cantly a€ect the contractions induced by ET-1, and
relaxation caused by gentamicin was identical for rings
incubated in the bu€er with or without this inhibitor (results
not shown).
Figure 6 E€ects of the aminoglycosides on protein kinase C (PKC)
activity in cultured canine cerebrovascular smooth muscle cells. PKC
activity was determined in the soluble and particulate fractions of
serum starved and (48 h) cells exposed to ET-1 (0.5 nM) (a) or PMA
(160 nM) (b) for 60 min. The aminoglycosides, gentamicin (genta),
neomycin (neo), kanamycin (kana) and streptomycin (strepto), were
administered 30 min after ET-1 application, at the concentrations
corresponding to their EC50s determined in contractility experiments.
Each bar represents mean value+s.e.mean of six experiments.
**P50.01 vs control; #P50.01 vs stimulation with ET-1 alone;
##P50.001 vs stimulation with ET-1 alone.
British Journal of Pharmacology vol 133 (1)

10
G. Wickman et al
Aminoglycosides modulate the effects of endothelin
Discussion
activity in cerebrovascular smooth muscle. This suggestion is
consistent with the observation that aminoglycosides prevent
protein phosphorylation mediated by phorbol esters in
cultured epithelial cells (Hagiwara et al., 1988).
The present studies demonstrate that PKC activation is a
major component of a sustained vasoconstriction mediated
by ET-1 in cerebral vessels. This is supported by the
observations that Ro-32-0432, a selective inhibitor of the
PKC isoforms (Wilkinson et al., 1993; Birchall et al., 1994),
reversed vasoconstriction induced by ET-1 in canine basilar
arteries. The inhibitory e€ects were evident with the
concentrations of this agent capable to inhibit the classical
and novel PKC enzymes, suggesting that both calcium-
dependent and independent PKC's may be involved in the
vasoconstrictor action of ET-1. Ro-32-0432 also reversed a
tonic contraction produced by PMA in calcium free medium,
indicating that the vasoconstrictor e€ects of PMA were
mediated by a calcium independent PKC isoform. The
contractile responses mediated by ET-1 were inhibited by
The precise mechanism by which aminoglycosides inhibit
PKC-mediated protein phosphorylation is unclear but it is
likely that inhibition of this enzyme is due to the interaction
of these compounds with acidic, negatively charged phos-
pholipids in the plasma membrane. The aminoglycosides are
polybasic due to their protonated amino groups and anionic
phospholipids of the plasma membrane are prime targets for
a charge interaction with these compounds (Lullmann &
Vollmer, 1982). The observation that potencies of aminogly-
coside antibiotics to inhibit PKC correspond to a number of
ionizable amino groups of compounds support this conten-
tion (Hagiwara et al., 1988). It is therefore conceivable that
the aminoglycosides could interfere with the association of
the ETA receptors selective antagonist, BQ-123 in
a
protein kinase
C
with phosphatidylserine,
a cofactor
competitive manner, indicating that the action of this peptide
is likely to occur in cerebral arteries via the ET-1 receptor
subtype coupled with PKC signaling pathway. The role of
PKC in sustained vasoconstriction was further supported by
direct studies of the PKC activity in CVSMC, which have
shown that ET-1 stimulates this enzyme in CVSMC, at the
time corresponding to maximum tonic contraction developed
in cerebral arteries after exposure to this peptide and PMA.
It has been demonstrated that smooth muscle contraction
is initiated by phosphorylation of myosin light chain (MLC),
a process mediated by the calcium-calmodulin dependent
myosin light chain kinase (MLCK) (Somlyo & Somlyo,
1994). Nevertheless, other data have shown that MLC
phosphorylation does not participate in arterial narrowing
during sustained vasospasm following SAH (Sun et al., 1998).
These observations suggest that another mechanism may be
important for the development of vasospasm, and there is
evidence to suggest that this mechanism may involve the
PKC mediated phosphorylation (Minami et al., 1992; Sako et
al., 1993). How PKC might initiate vasospasm is unclear, but
an important step may be the phosphorylation of the actin
binding proteins, calponin and, indirectly, caldesmon, which
inhibit myosin ATP-ase activity (Horowitz et al., 1996; Walsh
et al., 1996). It has been reported that ET-1 stimulates
phosphorylation of calponin mediated by PKC in peripheral
blood vessels (Mino et al., 1995). Thus, it is conceivable that
activation of PKC by this peptide in cerebral arteries and
subsequent phosphorylation of actin thin ®laments proteins
play a role in the development of sustained vasoconstriction.
Another signi®cant observation to emerge from the present
studies is that gentamicin and the structurally related
aminoglycosides are capable of attenuating of ET-1 e€ects
in cerebral arteries and CVSMC. The vasodilatory e€ects
were highly signi®cant, endothelium independent, and
necessary for activity of this enzyme, and consequently
modulate its ability to respond to activation by DAG and
to phosphorylate target proteins. It is also likely that
aminoglycosides could interfere with the conversion of
phosphatidic acid, derived from phosphatidylcholine break-
down, to DAG, an e€ect believed to be responsible for
sustained vasoconstriction (Asano & Matsui, 1999). Ob-
viously, the elucidation of the molecular mechanism of PKC
inhibition will require additional studies.
Although our data provide a strong argument in favour of
a role of PKC in cerebral vasoconstriction, other possible
modes of action for the vasodilatory e€ects of the
aminoglycosides cannot be discounted. These agents exhibit
various actions on several targets. Neomycin and the
structurally related aminoglycosides inhibit inositol lipid-
speci®c PLC and a variety of calcium channels including
voltage-dependent L-type calcium channel (Langton et al.,
1996), and calcium release-activated channels known to be
stimulated by capacitative calcium entry (Kim et al., 1999).
Furthermore, we have shown in our previous studies, that
delayed administration of the aminoglycosides to the
cerebrovascular cells stimulated with haemoglobin, can be
very e€ective in decreasing the sustained elevation of
intracellular calcium (Gergawy et al., 1998). Since ET-1
stimulates PLC activity and calcium in¯ux mediated by a
number of voltage dependent and independent calcium
channels in smooth muscle cells (Nakajima et al., 1996;
Iwamuro et al., 1999), it is likely that relaxation of the
vasoconstriction induced with ET-1 may arise from multiple
actions of the aminoglycosides.
The present ®ndings support and extend our previous
studies with aminoglycosides in several key ways. First, the
®ndings provide evidence that the aminoglycosides are able to
reverse the vasoconstrictor action of ET-1 with EC50s similar
to those seen in our previous studies with haemoglobin and
PGF2a (Gergawy et al., 1998). These e€ects were both
signi®cant and maintained over prolonged time. Second, our
results demonstrate that delayed administration of the
aminoglycosides to the cerebrovascular cells exposed to ET-
1, can be e€ective in decreasing the sustained activation of
PKC activity implicated in the development of vasospasm.
The systemic administration of the aminoglycosides is not
likely to be useful, because these agents do not cross the
blood ± brain barrier. However, intracranial application of a
maintained over
a prolonged time of exposure to the
aminoglycosides. The ability of the aminoglycosides to
reverse the PMA-induced sustained contraction in the
calcium-free medium, indicate that the e€ects of the
aminoglycosides were indeed the consequence of PKC
inhibition. Further support to this notion was provided by
the observations that the aminoglycosides inhibited directly
PKC activity in CVSMC exposed to ET-1 and PMA.
Collectively these results suggest that the vasodilatory e€ects
of the aminoglycosides arise from the inhibition of PKC
British Journal of Pharmacology vol 133 (1)

G. Wickman et al
Aminoglycosides modulate the effects of endothelin
11
slow release formulation during surgical clipping of the
a realistic
for successful management of abnormal vasoconstriction,
ecacious compounds may need to interfere with common
signalling pathways shared by such factors. This carries with
it the implication that aminoglycosides which inhibit several
cellular actions, would be the promising agents in the search
for pharmacological means of reversing the development of
vasospasm and preventing cerebral ischaemia.
aneurysm (Shiokawa et al., 1998) remains
possibility.
Since we have shown in both our previous and the present
studies that the aminoglycosides inhibited the vasoconstric-
tion of multiple spasmogens implicated in the pathogenesis of
cerebral vasospasm, a clear advantage may exist over speci®c
antagonists or signal transduction inhibitors. It is now
recognized that a number of pharmacologically unrelated
factors are involved in the pathogenesis of cerebral
vasospasm. The implication of such observations is that,
This work was supported by the Alberta Heart and Stroke
Foundation.
References
ASANO, T.
&
MATSUI, T. (1999). Antioxidant therapy against
KIM, K.T., CHOI, S.Y. & PARK, T.J. (1999). Neomycin inhibits
cerebral vasospasm following aneurysmal subarachnoid hemor-
rhage. Cell. Molec. Neurobiol., 19, 31 ± 44.
BIRCHALL, A.M., BISHOP, J., BRADSHAW, D., CLINE, A., COFFEY,
J., ELLIOTT, L.M., GIBSON, V.M., GREENHAM, A., HALLAM, T.J.,
HARRIS, W., HILL, C.H., HUTCHINGS, A., LAMONT, A.G.,
LAWTON, G., LEWIS, E.J., MAW, A., NIXON, J.S., POLE, D.,
catecholamine secretion by blocking nicotinic acetylcholine
receptors in bovine adrenal chroman cells. J. Pharmacol. Exp.
Ther., 288, 73 ± 80.
LANGTON, P.D., FARLEY, R. & EVERITT, D.E. (1996). Neomycin
inhibits K+-induced force and Ca2+ channel current in rat
arterial smooth muscle. P¯ugers Arch. - Eur. J. Physiol., 433,
188 ± 193.
È
WADSWORTH, J.
&
WILKINSON, S.E. (1994). Ro-32-0432, a
selective and orally active inhibitor of protein kinase C prevents
T-cell activation. J. Pharmacol. Exp. Ther., 268, 922 ± 929.
BRADFORD, M.M. (1976). A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the
principle of protein-dye binding. Anal. Biochem., 72, 248 ± 254.
COOK, D.A. & VOLLRATH, B. (1995). Free radicals and intracellular
events associated with cerebrovascular spasm. Cardiovascular
Res., 30, 493 ± 500.
LULLMANN, H. & VOLLMER, B. (1982). An interaction of
aminoglycoside antibiotics with Ca2+ binding to lipid mono-
layers and to biomembranes. Biochem. Pharmacol., 31, 3769 ±
3773.
MINAMI, N., TANI, E., MAEDA, Y., YAMAURA, I. & FUKAMI, M.
(1992). E€ects of inhibitors of protein kinase C and calpain in
experimental delayed cerebral vasospasm. J. Neurosurg., 76,
111 ± 118.
ESKILDSEN-HELMOND, Y.E., BEZSTAROSTI, K., DEKKERS, D.H.,
VAN HEUGTEN, H.A. & LAMERS, J.M. (1997). Cross-talk between
receptor-mediated phospholipase C-beta and D via protein
kinase C as intracellular signal possibly leading to hypertrophy
in serum free cultured cardiomyocytes. J. Molec. Cell. Cardiol.,
29, 2545 ± 2559.
GERGAWY, M., VOLLRATH, B. & COOK, D. (1998). The mechanism
by which aminoglycoside antibiotics cause vasodilation of canine
cerebral arteries. Br. J. Pharmacol., 125, 1150 ± 1157.
MINO, T., YUASA, U., NAKA, M. & TANAKA, T. (1995). Phosphor-
ylation of calponin mediated by protein kinase C in association
with contraction in porcine coronary artery. Biochem. Biophys.
Res. Commun., 208, 397 ± 404.
NAKAJIMA, T., HAZAMA, H., HAMADA, E., WU, S.-N., IGARASHI,
K., YAMASHITA, T., SEYAMA, Y., OMATA, M. & KURACHI, Y.
(1996). Endothelin-1 and vasopressin activate Ca2+-permeable
non-selective cation channels in aortic smooth muscle cells:
Mechanism of receptor-mediated Ca2+ in¯ux. J. Mol. Cell.
Cardiol., 28, 707 ± 722.
OHLSTEIN, E.H. & STORER, B.L. (1992). Oxyhemoglobin stimulation
of endothelin production in cultured endothelial cells. J.
Neurosurg., 77, 274 ± 278.
PERRIER, M.L., SCATTON, B. & BENAVIDES, J. (1992). Dihydropyr-
idine and omega-conotoxin resistant, neomycin sensitive calcium
channels mediate the depolarization induced increase in internal
calcium levels in cortical slices from immature rat brain. J.
Pharmacol. Exp. Ther., 261, 324 ± 330.
PICHLER, M., WANG, Z., GRABNER-WEISS, C., REIMER, D.,
HERING, S. & GRABNER, M. (1996). Block of P/Q-type calcium
channels by therapeutic concentrations of aminoglycoside
antibiotics. Biochemistry, 35, 14659 ± 14664.
RUMENAPP, U., SCHMIDT, M., WAHN, F., TAPP, E., GRANNASS, A.
& JAKOBS, K.H. (1997). Characteristics of protein kinase C- and
ADP-ribosylation factor-stimulated phospholipase D activities
in human embryonic kidney cells. Eur. J. Biochem., 248, 407 ±
414.
GOLDIE, R.G. (1999). Endothelins in health and disease: an
overview. Clin. Exper. Pharmacol. Physiol., 26, 145 ± 148.
HAGIWARA, M., INAGAKI, M., KANAMURA, K., OHTA, H.
&
HIDAKA, H. (1988). Inhibitory e€ects of aminoglycosides on
renal protein phosphorylation by protein kinase C. J. Pharmacol.
Exp. Ther., 244, 355 ± 360.
HINO, A., WEIR, B.K.A., MACDONALD, R.L., THISTED, R., KIM, C.J.
& JOHNS, L.M. (1995). Prospective, randomized, double-blind
trial of BQ-123 and bosentan for prevention of vasospasm
following subarachnoid hemorrhage in monkeys. J. Neurosurg.,
83, 503 ± 509.
HOROWITZ, A., MENICE, C.B., LAPORTE, R. & MORGAN, K.G.
(1996). Mechanism of smooth muscle contraction. Physiol. Rev.,
76, 967 ± 1003.
ITOH, S., SASAKI, T., ASAI, A. & KUCHINO, Y. (1994). Prevention of
delayed vasospasm by an endothelin ETA receptor antagonist,
BQ-123: change of ETA receptor mRNA expression in a canine
subarachnoid hemorrhage model. J. Neurosurg., 81, 759 ± 764.
IWAMURO, Y., MIWA, S., ZHANG, X.-F., MINOWA, T., ENOKI, T.,
OKAMOTO, Y., HASEGAWA, H., FURUTANI, H., OKAZAWA, M.,
ISHIKAWA, M., HASHIMOTO, N. & MASAKI, T. (1999). Activa-
tion of three types of voltage-independent Ca2+ channel in A7r5
cells by endothelin-1 as revealed by a novel Ca2+ channel blocker
LOE 908. Br. J. Pharmacol., 126, 1107 ± 1114.
JOSKO, J., HENDRYK, S., JEDRZEJOWSKA-SZYPULA, H., GWOZDZ,
B., HERMAN, Z.S. & GAWLIK, R. (1998). In¯uence endothelin
ETA receptor antagonist BQ-123 on changes of endothelin-1 level
in plasma of rats with acute vasospasm following subarachnoid
hemorrhage. J. Physiol. Pharmacol., 49, 367 ± 375.
SAKO, M., NISHIHARO, J. & OHTA, S. (1993). Role of protein kinase
C in the pathogenesis of cerebral vasospasm after subarachnoid
hemorrhage. J. Cereb. Blood Flow Metab., 13, 247 ± 254.
SEIFERT, V., LOFFLER, B.M., ZIMMERMANN, M., ROUX, S.
&
STOLKE, D. (1995). Endothelin concentration in patients with
aneurysmal subarachnoid hemorrhage: Correlation with cerebral
vasospasm, delayed ischemic neurological de®cits and volume of
hematoma. J. Neurosurg., 82, 55 ± 62.
SCHIFFRIN, E.L.
endothelin. J. Cardiovasc. Pharmacol., 32 Suppl 3: S2 ± S13.
SCHINI, V.B., HENDRICKSON, H. HEUBLEIN, D.M. (1989).
& TOUYZ, R.M. (1998). Vascular biology of
&
KASUYA, H., WEIR, B.K.A., WHITE, D.M. & STEFANSON, K. (1993).
Mechanism of oxyhemoglobin-induced release of endothelin-1
from cultured vascular endothelial cells and smooth muscle cells.
J. Neurosurg., 79, 892 ± 898.
Thrombin enhances the release of endothelin from porcine aortic
endothelial cells. Eur. J. Pharmacol., 165, 333 ± 334.
British Journal of Pharmacology vol 133 (1)

12
G. Wickman et al
Aminoglycosides modulate the effects of endothelin
SHIOKAWA, K., KASUYA, H., MIYAIMA, M., IZAWA, M.
&
VOLLRATH, B.A.M., WEIR, B.K.A., MACDONALD, R.L. & COOK,
D.A. (1994). Intracellular mechanisms involved in the responses
of cerebrovascular smooth muscle cells to hemoglobin. J.
Neurosurg., 80, 261 ± 268.
TAKAKURA, K. (1998). Prophylactic e€ect of papaverine
prolonged release pellets on cerebral vasospasm in dogs.
Neurosurgery, 42, 109 ± 115.
SOMLYO, A.P. & SOMLYO, A.V. (1994). Signal transduction and
regulation in smooth muscle. Nature, 372, 231 ± 236.
SUN, H., KANAMARU, K., MASAAKI, I., SUZUKI, H., KOJIMA, T.,
WAGA, S., KUREISHI, Y. & NAKANO, T. (1998). Myosin light
chain phosphorylation and contractile proteins in a canine two-
hemorrhage model of subarachnoid hemorrhage. Stroke, 29,
2149 ± 2154.
WANEBO, J.E., ARTHUR, A.S., LOUIS, H.G., WEST, K., KASSELL,
N.F., LEE, K.S. & HELM, G.A. (1998). Systemic administration of
the endothelin-A receptor antagonist TBC 11251 attenuates
cerebral vasospasm after experimental subarachnoid hemor-
rhage: dose study and review of endothelin-based therapies in
the literature on cerebral vasospasm. Neurosurgery, 43, 1409 ±
1417.
WALSH, M.P., HOROWITZ, A., CLEMENT-CHOMIENNE, O., AN-
DREA, J.E., ALLEN, B.G. & MORGAN, K.G. (1996). Protein kinase
WILKINSON, S.E., PARKER, P.J. & NIXON, J.S. (1993). Isoenzyme
speci®city of bisindolylmaleimides, selective inhibitors of protein
kinase C. Biochem. J., 294, 335 ± 337.
C
mediation of Ca2+-independent contractions of vascular
smooth muscle. Biochem. Cell Biol., 74, 485 ± 502.
VOLLRATH, B., CHAN, P., FINDLAY, M. COOK, D. (1995).
ZIMMERMANN, M. & SEIFERT, V. (1998). Endothelin and subar-
achnoid hemorrhage: An overview. Neurosurgery, 43, 863 ± 876.
&
Lazaroids and deferoxamine attenuate the intracellular e€ects
of oxyhemoglobin in vascular smooth muscle. Cardiovasc. Res.,
30, 619 ± 626.
(Received July 3, 2000
Revised January 4, 2001
Accepted February 8, 2001)
British Journal of Pharmacology vol 133 (1)