In this study, in contrast to the CM, TM was able to contract
under calcium-independent conditions. The TM features
smooth-muscle–like properties and is actively involved in aqueous
humor outflow, in the sense that its contraction decreases outflow,
whereas relaxation increases this parameter.
2 Because the
conventional outflow through the TM accounts for 80% to 90% of the
total aqueous humor outflow rate, knowledge about the modulation of
contractility in this tissue is important. Smooth-muscle–relaxing
substances appear to be suitable candidates for glaucoma therapy with
the goal of reducing IOP through TM relaxation without adverse effects
on accommodation or pupil diameter, by circumventing the CM and by
possibly improving retinal hemodynamics through relaxation of vascular
smooth muscle cells or pericytes, i.e., vasodilation.
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-ε), 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 (α, βI, βII, and γ), the calcium-independent
(δ, ε, η, and θ), and the atypical isoforms (ζ and λ). We
have shown recently that the TM, contrary to the CM, features a high
expression of the PKC-ε, an isoform that has been linked to
calcium-independent contraction in various smooth muscle
preparations.
8 11 12 24 25 The involvement of PKC-ε 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 4α-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-β isoform can be inhibited through an orally
effective antagonist.
30 However, PKC-β 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-ε 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-ε,
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.
The authors thank Marianne Boxberger, Helga Höffken,
and Karin Oberländer for technical support.