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1 From the Institut für Klinische Physiologie, and 2 Augenklinik Universitätsklinikum Benjamin Franklin, Freie Universität Berlin; and 3 Klinik und Poliklinik für Augenheilkunde, Charitè, Campus Virchow Klinikum, Humboldt Universität Berlin, Germany.
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Abstract |
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 Top Abstract IntroductionMethodsResultsDiscussionReferences |
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METHODS. Isometric tension measurements of bovine TM and ciliary muscle (CM)
were performed. Intra- and extracellular calcium buffering was
accomplished with EGTA and
1,2-bis(2-aminophenoxy)-ethane-N,N,N,N',N'-tetra-acetic
acid tetrakis/acetoxymethhyl ester (BAPTA-AM) followed by stimulation
of PKC with phorbolester (PMA) or 4
-phorbol. Calcium-independent
contraction was blocked using the highly specific ROCK inhibitor
Y-27632. Western blot analysis and immunoprecipitation was performed
using human TM cells.
RESULTS. In TM, carbachol induced partial contraction under conditions of
extracellular calcium depletion (22.1% ± 2.3% versus 100%, n
= 9). The membrane-permeable calcium chelator BAPTA-AM completely
blocked this response (1.1% ± 1.4% versus 100%, n = 9).
When calcium was completely blocked, PMA induced contraction in TM
(16.7% ± 5.9% versus 100%, n = 9) but not in CM (1.8%
± 2.5% versus 100%, n = 6). The inactive PMA analogue
4-phorbol did not induce contraction, indicating that activation of
PKC is involved in this contractile response. The ROCK inhibitor
Y-27632 completely blocked the calcium-independent PMA-induced
contraction in TM. Western blot analysis and immunoprecipitation
revealed the expression of the rho-A protein in human TM cells.
CONCLUSIONS. The data indicate that contrary to CM, the TM features calcium-independent contractile mechanisms linked to rho-A and PKC isoforms that do not require calcium for activation. ROCK inhibitors may allow specific modulation of the TM to enhance outflow facility, thus lowering intraocular pressure.
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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A possible group of compounds fulfilling these needs are the inhibitors
of protein kinase C (PKC). Recently, a role for PKC in the regulation
of smooth muscle contraction has been postulated.7
8
9
PKC
inhibitors such as H7 or chelerythrine are known to induce relaxation
in TM without affecting the CM.10
11
Additionally, PKC
inhibitors have been used successfully to lower IOP in an animal
model.5
Recently, we were able to show that PKC isoforms
show different tissue distribution in both human and bovine TM and CM
and that contractility in these tissues is differently regulated by
various PKC agonists and antagonists.11
One particular PKC
isoform (PKC-
) is highly expressed in TM and is known for its
involvement in the regulation of calcium-independent contraction in
various smooth muscle preparations.8
11
12
This form of
contractility is primarily based on pharmacomechanical coupling events
rather than calcium influx from the extracellular space or release of
calcium from intracellular stores. It has been suggested that the small
guanosine triphosphatase (GTPase) rho-A may be a target protein for
PKC-, and their interaction may contribute to the regulation
of calcium-independent contractility.13
14
In this study calcium-independent contraction existed exclusively in the TM, and this PKC-dependent modulation of contractility was specifically blocked by rho-A kinase inhibitor Y-27632. Based on these findings, we suggest a possible modulation of IOP through substances such as Y-27632 through relaxation of the TM. First evidence for such pressure-reducing properties in an animal model has been presented recently.15
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The effects of agents on contractility were measured isometrically with a custom-made force length transducer system, as described.16 Tissue strip contractions were expressed relative to the response obtained with a maximally effective carbachol concentration (10-6 M), which was tested in each tissue strip as a control. To determine the activity of a compound, the agent was added to the tissues at basal tension. The chamber solutions were kept at a stable temperature (37°C) and pH. The ionic concentrations (in millimolar) of Ringer’s solution were: 151 Na+, 5 K+, 1.7 Ca2+, 0.9 Mg2+, 131 Cl-, 0.9 SO42-, 1 H2PO4-, 28 HCO3-, and 5 glucose. Calcium-free solutions were buffered with EGTA (10-5 M) and/or BAPTA-AM (3 x 10-5 M). All solutions were gassed with 5% CO2 in air, which resulted in a pH of 7.4.
Human TM Cell Cultures
Human TM cells were isolated by methods based on those of Grierson
et al.17
and Siegner et al.18
with tissue
obtained from donor eyes (Department of Ophthalmology, Charitè,
Campus Rudolf Virchow, Berlin). Tenets of the Declaration of Helsinki
were followed, informed consent was obtained, and institutional human
experimentation committee approval was granted for the studies. In
brief, TM strips were carefully dissected under microscopic view. A
fine wire probe (0.5 mm) was used to cannulate Schlemm’s canal, thus
aiding visualization of the TM. The strips were placed under glass
coverslips in 35-mm plates and were incubated in Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 20% fetal calf serum (FCS),
100 U/ml penicillin, and 100 µg/ml streptomycin (all cell culture
material was obtained from Biochrom, Berlin, Germany). The cells were
maintained in a 95% air-5% CO2 atmosphere at
37°C and were passaged by the trypsin-EGTA method after having
reached confluence. Only well-characterized human TM cells from early
passage (passages 3–7) were used for Western blot and
immunoprecipitation experiments. Histologic characterization was
performed by Elke Lütjen–Drecoll, (Department of
Anatomy, Universität Erlangen, Nürnberg, Germany) and
showed typical immunostaining as described previously.19
Western Blot Analysis and Immunoprecipitation
Western blot analysis was performed as previously described in
detail.11
Confluent human TM cell monolayers were washed
three times with ice cold phosphate-buffered saline (PBS)-Tween,
scraped, and lysed in buffer A (1% NP40, 20 mM Tris [pH 8.80], 137
mM NaCl, and 10% glycerol) containing protease inhibitors (Complete;
Protease, Boehringer–Mannheim, Mannheim, Germany). After a preclearing
centrifugation step (14,000 rpm for 5 minutes at 4°C), the whole cell
lysate was subjected to sodium dodecyl sulfate–polyacrylamide gel
electrophoresis (SDS-PAGE; 25 µg protein per lane, 7.5% gel) and
immunoblotting. Nitrocellulose filter membranes (Polyscreen; NEN Life
Science Products, Boston, MA) were blocked in 5% bovine serum albumin
(BSA) for 2 hours at room temperature and consequently incubated
overnight with rho-A primary antibody (Santa Cruz Biotechnologies,
Santa Cruz, CA) diluted in 2% BSA-PBS (1:2000). The blots were
visualized using a peroxidase-conjugated secondary antibody (Dianova;
Jackson ImmunoResearch, West Grove, PA) diluted in PBS-Tween (1:20.000;
1 hour at room temperature) and a chemiluminescence kit (ECL, Amersham,
Amersham, UK) according to manufacturers instructions. The images were
digitalized using an image analyzer (Fujifilm; LAS 1000; Fuji, Tokyo,
Japan) and software (Aida 2.1; Raytest, Berlin, Germany).
For immunoprecipitation, protein A bound primary antibody (prewashed Sepharose A beads incubated with antibody for 1 hour at 4°C) was incubated with precleared whole-cell lysate overnight under gentle rotation at 4°C. After several washing steps (six times with lysis buffer A), the beads were centrifuged (2000 rpm for 2 minutes) and boiled in 50 µl 1x Laemmli buffer for 5 minutes. The supernatant was subjected to reducing SDS-PAGE, immunoblot assay, and signal detection, performed as described earlier. Blocking peptide was used in fivefold excess to primary antibody during the immunoprecipitation to verify specificity of the protein bands.
Reagents
The following reagents were used for contractility measurements:
4-phorbol, (RBI; Sigma, Deisenhofen, Germany); phorbol-12-myristate
13-acetate (Biomol, Hamburg, Germany);
1,2-bis(2-aminophenoxy)-ethane-N,N,N,N',N'-tetra-acetic
acid tetrakis/acetoxymethhyl ester; BAPTA-AM; Sigma);
(+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)
cyclohexane-carboxamide dihydrochloride, monohydrate (Y-27632) was
kindly supplied by Yoshitomi Pharmaceutical, Osaka, Japan. All other
chemicals were of analytical grade and were purchased from Sigma.
Statistical Analysis
The results of contractility measurements were expressed as mean
values ± SEM. Statistical analysis was performed using analysis
of variance and Student’s t-test for paired observations
(% changes versus carbachol-contracted tissues). The unpaired
Student‘s t-test was used for comparison of values in TM
and CM. Significance was assumed when P < 0.05. The
number (n) refers to the number of experiments. Western blot
and immunoprecipitation experiments were repeated at least three times,
with cells from individual primary cultures showing identical results.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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-Phorbol on Contractility-Phorbol is a biologically inactive analogue of PMA, serving
as negative control. The experiments were performed with TM only,
because only this tissue showed a development of contractile force
under calcium-independent conditions. The PMA analogue had no effect on
baseline tension in TM (Fig. 5
, summarized data: 0.25% ± 0.42% versus 100%, n = 4,
P < 0.62 versus zero contraction).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Smooth muscle contractility is regulated by cytosolic calcium
concentration, either by calcium influx through voltage- and
receptor-operated membrane channels or by release of calcium from
intracellular calcium stores.13
There are similar
mechanisms in the TM.2
20
In recent years it has become
obvious that smooth muscle cell contractility is not only regulated by
changes of intracellular calcium and electromechanical coupling. Other
important signaling mechanisms such as membrane potential–independent,
pharmacomechanical coupling events seem to trigger the tonic (slow)
phase of smooth muscle contractility and are referred to as
calcium-independent mechanisms.8
13
21
Calcium-independent
contractility has been investigated in various smooth muscle
preparations.7
8
9
The involvement of PKC isoforms that are
activated in a calcium-independent fashion (namely PKC-), as well as
participation of the small GTPase rho-A in responses linked to myosin
light-chain kinase phosphorylation and dephosphorylation, has been
postulated by many investigators.7
8
9
12
13
22
It has been shown before that part of the carbachol-induced contraction in TM is still present under conditions in which all extracellular calcium has been removed with calcium buffers.23 It is of interest that CM did not contract under these conditions.23 The data indicate that this contractile force in TM relies on the release of calcium from intracellular stores. BAPTA-AM is a membrane-permeable intracellular calcium chelator that leads to a complete depletion of cytosolic calcium levels in TM strips exposed to extracellularly calcium-free environment. Under these conditions the carbachol-induced contraction was completely absent. That part of TM contractility appeared to be regulated in a different way than CM indicates that the development of TM-specific pharmacologic agents for the regulation of contractility should be possible.
The PKC family of isoforms consists of three groups: the
calcium-dependent (, ßI, ßII, and 
), the calcium-independent
(
, , 
, and 
), and the atypical isoforms (
and 
). We
have shown recently that the TM, contrary to the CM, features a high
expression of the PKC-, an isoform that has been linked to
calcium-independent contraction in various smooth muscle
preparations.8
11
12
24
25
The involvement of PKC- in
the calcium-independent contraction of the TM presented in this study
seems likely, because the PMA-induced contractility was completely
absent in CM. That the biologically inactive form of PMA 4-phorbol
failed to induce contraction suggests a PMA-induced effect highly
specific for PKC. Focusing on PKC regulation appears to be important,
because inhibitors of this enzyme have been used successfully in an
animal model to lower IOP.4
5
The main mode of action of
these compounds seems to be the initiation of relaxation of the TM or
by the modulation of actin microfilaments.5
10
11
It is
believed by some investigators that PKC inhibition ultimately leads to
disruption of actin filaments and thereby alters the organization of
cell–cell and cell–extracellular matrix adhesions in
TM.5
In our experiments, however, the main action of PKC
blockers was the initiation of relaxation. This observation is further
supported by findings of other groups investigating PKC in different
smooth muscle tissues. It has been shown that activation of PKC
enhances contraction by inhibiting myosin phosphatase
directly.21
26
27
The need for the modulation of more specific signaling pathways is justified, because most inhibitors of PKC are rather nonspecific, acting on a wide variety of intracellular enzymes.28 29 Currently, only the PKC-ß isoform can be inhibited through an orally effective antagonist.30 However, PKC-ß was not detected when bovine and human TM and CM tissues were screened for smooth-muscle–associated isoforms of PKC.11
A promising target for a highly specific modulation of
calcium-independent contraction in smooth muscle tissues appears to be
the small GTPase rho-A and its kinase, ROCK.31
The active,
GTP-bound form of rho-A activates a serine-threonine kinase, ROCK,
which in turn phosphorylates myosin light-chain phosphatase
(MLCP).21
This results in inhibition of MLCP and in
increased myosin phosphorylation—i.e., contraction. The identification
of this pathway is greatly facilitated by the use of the highly
selective ROCK inhibitor Y-27632. This pyridine derivative has been
used successfully in inhibiting smooth muscle contraction and in
reducing blood pressure in hypertensive rats without affecting blood
pressure in normotensive animals.31
Furthermore, Y-27632
inhibits the tonic (slow) phase of agonist-induced contractions in
smooth muscle.26
Our experiments clearly show that
specific inhibition of the rho-A/ROCK pathway blocks
calcium-independent contraction initiated through activation of PKC.
Furthermore, the rho-A protein was detected in human TM cells by
Western blot and immunoprecipitation analysis. Some investigators
suggest the PKC-mediated inhibition or activation of MLCP to be an
independent pathway of rho-A’s effects on
contractility.21
26
Contrary to this, our results suggest
an interaction of PKC- and the small GTPase rho-A in the modulatory
pathway influencing TM contractility. In our experiments, contractility
was measured in intact tissue strips rather than permeabilized smooth
muscle preparations as performed by other groups, which may explain the
varying results. In addition, the variable findings may also be the
result of differences in the tissue types or cells investigated.
Further experiments are needed to clarify the downstream effector
proteins of rho-A in the TM and the effects of Y-27632 on modulation of
TM contractility. The IOP-lowering properties of Y-27632 have been
demonstrated recently in an animal model, where it was administered
intracamerally, intravitreally, and topically.15
In summary, we have shown that in TM contractility is partly regulated
in a unique way that is independent of extracellular calcium and not
present in the CM. In addition to the established ways of initiating
contractile force through calcium-dependent mechanisms, the
TM presents an alternate route leading to contraction that involves
pharmacomechanical coupling events. Furthermore, this
calcium-independent contraction is most probably modulated by PKC-,
which does not require calcium for its activation, and rho-A. Both
proteins are strongly expressed in the human TM, suggesting that this
smooth-muscle–like tissue may be influenced by highly specific
compounds such as Y-27632. Modulating TM contractility downstream of
PKC with specific inhibitors of rho-A seems more promising than
nonspecific inhibition of a broad selection of PKC isoforms. The data
in this study indicate that the ROCK inhibitor Y-27632 may have
beneficial effects on IOP in primates.
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Acknowledgements |
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Footnotes |
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Commercial relationships policy: N.
Corresponding author: Hagen Thieme, Institut für Klinische Physiologie, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, Hindenburgdamm 30, 12203 Berlin, Germany. thieme{at}ukbf.fu-berlin.de
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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-smooth muscle actin in normal and glaucomatous human trabecular meshwork of different age groups J Glaucoma 1,165-173
S-, and phorbol ester-induced Ca2+-sensitization of smooth muscle FEBS Lett 440,183-187[Medline][Order article via Infotrieve]
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