Pflugers Arch - Eur J Physiol (2004) 448: 547–551
DOI 10.1007/s00424-004-1284-3
SKELETAL MUSCLE
Francisco H. Andrade
.
Denise A. Hatala
.
Colleen A. McMullen
Carbonic anhydrase isoform expression and functional role
in rodent extraocular muscle
Received: 12 February 2004 / Revised: 16 March 2004 / Accepted: 22 March 2004 / Published online: 27 April 2004
# Springer-Verlag 2004
Abstract Carbonic anhydrase (CA) accelerates contrac-
tile function, particularly in fast-twitch skeletal muscles.
Since the extraocular muscles are considered to be
amongst the fastest skeletal muscles in mammals, this
study tested two hypotheses: (1) CA is expressed at higher
levels in rat extraocular muscles than in extensor digito-
rum longus (EDL, a fast limb muscle), and (2) inhibition
of CA activity increases twitch duration and force in the
extraocular muscles to a greater extent than in EDL. By
real-time quantitative PCR we determined that the
expression of CA3 isoform, typically high in skeletal
muscles, is significantly depressed in extraocular muscles.
Message levels for the CA2 and CA4 isoforms were
higher in the extraocular muscles, while CA5 expression
was equivalent in both muscles. Strong CA activity was
demonstrated by histochemistry in frozen EDL muscle
sections, in particular along the sarcolemma and in
capillaries. By contrast, extraocular muscle had very low
sarcolemmal or cytosolic CA activity. CA inhibition with
6-ethoxyzolamide (ETZ) reversibly increased twitch
duration and force in EDL muscle bundles. In the
extraocular muscles, ETZ did not alter twitch kinetics.
Based on these results, we reject our initial hypotheses and
conclude that CA does not influence the fast contractile
kinetics characteristic of the extraocular muscles.
Keywords Carbonic anhydrase
.
Gene expression
.
Muscle contraction
.
Oculomotor muscles
.
Skeletal
muscle
Introduction
The carbonic anhydrases (CAs) are ubiquitous enzymes
that catalyze the reversible hydration of CO
2
.CA
participates in acid-base balance and provides CO
2
for
use in gluconeogenesis and the urea cycle. Mammalian
skeletal muscles express at least four CA isoforms (CA2,
CA3, CA4 and CA5) in a fiber type- and structure-specific
manner [6]. In general, most CA activity in skeletal muscle
is found in capillaries and associated with the sarcolemma,
where it mediates the disposal of excess H
+
generated by
contractile and metabolic activity. There is also strong
evidence of significant CA activity in the sarcoplasmic
reticulum (SR) that may facilitate SR Ca
2+
fluxes by
providing H
+
for a Ca
2+
/H
+
countertransport system that
accelerates Ca
2+
release and uptake [15, 16].
The extraocular muscles are considered to be amongst
the fastest skeletal muscles in mammals, notable for
particularly short and shallow twitches [2, 3]. These small
muscles express mostly fast myosins, including an
ultrafast tissue-specific isoform, and contain abundant
mitochondria and SR [11, 18]. The unique contractile
characteristics of the extraocular muscles suggest the need
for very fast Ca
2+
kinetics to control the mechanical
transients. Therefore, to determine whether CA activity is
required to accelerate the contractions of the extraocular
muscles, this study tested two hypotheses: (1) CA
expression and activity is higher in the extraocular muscles
compared to an index fast limb muscle, and (2)
pharmacological inhibition of cellular CA activity slows
the isometric twitches and increase twitch force in the
extraocular muscle to a greater extent than in a fast limb
muscle. For the first hypothesis, we compared the
expression of four CA isoforms between extraocular
muscles and extensor digitorum longus (EDL, a proto-
typical fast-twitch limb muscle) from rats. We also
F. H. Andrade (*)
Department of Neurology, Case Western Reserve University,
11100 Euclid Avenue,
Cleveland, OH, 44106, USA
e-mail: fha@cwru.edu
Fax: +1-216-3683951
D. A. Hatala
Department of Ophthalmology, Case Western Reserve
University,
11100 Euclid Avenue,
Cleveland, OH, 44106, USA
C. A. McMullen
Department of Neurology, Case Western Reserve University,
11100 Euclid Avenue,
Cleveland, OH, 44106, USA