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DOI: 10.1369/jhc.4A6266.2004
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Journal of Histochemistry and Cytochemistry
Volume 52 (8): 1101-1106, 2004
Copyright ©The Histochemical Society, Inc.

Carbonic Anhydrase in Mammalian Vascular Smooth Muscle

John T. Berg, S. Ramanathan, M. Gabriella Gabrielli and Erik R. Swenson

Department of Pharmacology, University of Hawaii, Honolulu, Hawaii (JTB,SR); Department of Comparative Morphology and Biochemistry, University of Camerino, Camerino, Italy (MGG); and Department of Medicine, VA Puget Sound Health Care System, University of Washington, Seattle, Washington (ERS)

Correspondence to: Erik R. Swenson, MD, Pulmonary Section, S-111-Pulm, VA Puget Sound Health Care System, 1660 South Columbian Way, Seattle, WA 98108. E-mail: eswenson{at}u.washington.edu


   Summary
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 Materials and Methods
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 Literature Cited
 
Carbonic anhydrase (CA) is ubiquitously expressed and plays a pivotal role in acid–base balance, ion transport, and gas exchange. Limited observations by others, derived from functional, pharmacological, and histochemical studies, suggest that CA is present in vascular smooth muscle and is involved in vasoregulation. The present study, using measurements of bioactivity, inhibition characteristics, and immunohistochemical analysis, was undertaken to more fully evaluate CA in vascular smooth muscle. In isolated bovine aortic smooth muscle, which is devoid of erythrocytes, CA is present in low concentrations with a CO2 hydration activity (at 0C) of 3.5 ± 2.7 U/g. The I50 for acetazolamide inhibition is 0.07 ± 0.01 µM. Results with dorzolamide and bromopyruvate, selective inhibitors of the CA II and I isozymes, respectively, show that roughly 75% of the CA activity is accounted for by CA I, with 20% due to CA II. These results accord qualitatively with immunocytochemical staining with specific CA I and II antibodies, showing that both isozymes are present and that their staining co-localizes with cells positive for smooth muscle {alpha}-actin. These data establish the activity, inhibition, and isozyme pattern of carbonic anhydrase expression in mammalian vascular smooth muscle.

(J Histochem Cytochem 52:1101–1106, 2004)

Key Words: carbonic anhydrase • smooth muscle • vasculature • acetazolamide • dorzolamide • immunocytochemistry • bovine


   Introduction
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 Summary
 Introduction
Materials and Methods
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 Discussion
 Literature Cited
 
CARBONIC ANHYDRASE (CA) is involved in multiple aspects of systemic and cellular acid–base balance (Maren 1967Go). It rapidly catalyzes the reversible interconversion of CO2 and HCO3– in support of processes that include membrane ion transport, transepithelial fluid movement, intermediary metabolism, gas exchange, ventilation, bone turnover, neurotransmission, cell growth, and reproduction (Maren 1967Go; Swenson 2000Go). There are 14 known CA isozymes (Swenson 2000Go), attesting to the ubiquity and utility of this enzyme in biological processes. CA has been detected in virtually every tissue since its discovery in red cells by Meldrum and Roughton (1933)Go. Over the past 20 years, many tissues thought to lack CA have nevertheless been found to contain the enzyme in low concentrations and/or activity when suitable assays of high sensitivity were employed. It took over 40 years for CA to be detected in skeletal muscle (Register et al. 1978Go), where a low-activity isozyme, CA III, is present in the cytosol and high-activity isozymes (CA II and IV) are present in certain membrane fractions (Geers and Gros 1984Go), and more than a decade more for high-activity isozymes at very low concentration to be demonstrated in cardiac muscle (Bruns and Gros 1992Go; Swenson 1997Go).

Surprisingly, much less attention has been paid to the presence and role of CA in smooth muscle. In many histochemical studies of various organs, there have been observations of CA in vascular and non-vascular smooth muscle tissue but rarely was smooth muscle the primary object of these investigations. The first histochemical evidence of CA activity was reported by Jeffery et al. (1980)Go in systemic arterial smooth muscle, in which trace levels of CA III were detected and confirmed by Gabrielli et al. (1998)Go in renal arteries. Other non-vascular smooth muscle tissues, such as the prostate and uterus (Vaananen and Autio-Harmainen 1987Go), vas deferens (Parkkila et al. 1993Go; Ichihara et al. 1997Go), avian intestinal, and ureteral smooth muscle (Gabrielli and Menghi 1994Go; Gabrielli et al. 1998Go), have been found by histochemistry or immunocytochemistry to have several CA cytosolic and membrane-bound forms of CA.

Paralleling the histochemical work, others have investigated CA inhibitor effects in non-vascular and vascular smooth muscle. Carmignani et al. (1981)Go showed that acetazolamide inhibits agonist-mediated ileal and vas deferens constriction. Faraci et al. (1990)Go found that acetazolamide increases choroid plexus blood flow despite a decrease in CSF formation. Acetazolamide inhibits neural-mediated bronchoconstriction (Sun et al. 1993Go) and reduces the glomerular filtration rate by efferent glomerular vasodilatation (Leyssac et al. 1994Go). Pickkers et al. (1999)Go demonstrated that CA inhibitors block norepinephrine-mediated mesenteric artery constriction and showed that extracellular acidosis generated with in vivo use of CA inhibitors is not critical in vasorelaxation because the vessels were studied in vitro under fixed acid–base conditions. In follow-up in vivo studies, Pickkers et al. (2001)Go showed that acetazolamide is vasodilating when infused into the human forearm. Lastly, in animal models we showed that acetazolamide blocks hypoxic pulmonary vasoconstriction (Deem et al. 2000Go; Hoehne et al. 2004Go) and by doing so can prevent high-altitude pulmonary edema (Berg et al. 2004Go).

Despite the cited literature, no biochemical and immunocytochemical studies have focused solely on smooth muscle vascular CA. We undertook the present study in bovine aortic smooth muscle using CA activity, sensitivity to CA inhibitors, and immunohistochemical (IHC) localization to determine which isozymes and in what quantities are present in mammalian vascular smooth muscle.


   Materials and Methods
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 Summary
 Introduction
 Materials and Methods
Results
 Discussion
 Literature Cited
 
Smooth Muscle Isolation
Bovine aortas were obtained from a local source (Schenk Packing; Stanwood, WA). Each aorta was trimmed of excess fat and connective tissue and cut lengthwise to expose the lumen. The aortas were then rinsed with cold saline (0.9% NaCl) and pinned to a dissecting board with the luminal surface exposed. A scalpel was used to scrape away the endothelial layer and the tissue was rinsed again and blotted dry. The smooth muscle was then separated from the underlying adventitia and stored at –70C for subsequent bioassay and immunocytochemistry (see below).

Immediately before assay the smooth muscle was thawed, weighed, minced, and homogenized in distilled water (1:5 w:v) at 4C using a tissue homogenizer (Tissue Tearor; Fisher Scientific, Pittsburgh, PA) at a high setting for two 60-sec pulses. The homogenate was then centrifuged (4000 rpm at 4C) for 20 min and the supernatant used for CA activity and inhibitor determination.

Determination of CA Activity
We used a modified method of Maren (1960)Go to determine CA bioactivity. Briefly, for uncatalyzed reactions, a mixture containing 0.4 ml barbital buffer (25 mM barbituric acid and 25 mM sodium barbital), 0.2 ml 0.04% phenol red, and 0.4 ml H2O was bubbled with 100% CO2 at 0–1C and timed for color change (from red to bright yellow). For catalyzed reactions, 0.4 ml of aorta supernatant was substituted for 0.4 ml H2O. For inhibition studies, 10 µl of appropriate concentrations of inhibitors was used such that the final concentration ranged from 0.01 µM to 0.1 mM. The final reaction volume was constant at 1.0 ml. CA activity in enzyme units was calculated as units = (time uncatalyzed – time catalyzed) ÷ (time catalyzed).

In all measurements, 10 µl of octanol was added to the reaction vessel before addition of enzyme to prevent frothing. The CA inhibitors studied were acetazolamide, a nonspecific CA inhibitor which at 1 mM fully inhibits all known CA isozymes but at 10 µM is ineffective against CA III, dorzolamide, a selective CA II inhibitor at ≤1 µM concentration (Conroy and Maren, 1995Go), and bromopyruvate at 20 mM (Conroy and Maren 1985Go), a selective CA I inhibitor. To abolish all CA activity, the aortic smooth muscle supernatant was also boiled for 5 min at 100C. The results with dorzolamide give the fraction of CA activity due to CA II and the results with bromopyruvate give the fraction of activity due to CA I. The difference between total activity and that after dorzolamide and bromopyruvate is taken as that attributable to CA III because all other known CA isozymes are inhibited by dorzolamide and acetazolamide at 1 µM.

Immunocytochemical Localization and Detection of CA Isozymes in Bovine Aorta
Tissue samples from bovine aorta were used. Immediately after isolation, pieces of tissue were fixed in Bouin's solution (4 hr at room temperature) and stored in 70% ethanol during shipping from Seattle, WA to Camerino, Italy, for analysis. On arrival, tissues were dehydrated through graded ethanol concentrations, cleared in xylene, and embedded in paraffin at 56–58C. Sections were serially cut at 5-µm thickness, placed on gelatin-coated slides, and dewaxed. Identification of the different tissue components was achieved by staining some sections with toluidine blue, iron hematoxylin, and Gomori's trichomic technique (Pearse 1985Go).

Rehydrated sections were processed for CA I and CA II immunostaining according to the avidin–biotin–peroxidase system (Hsu et al. 1981Go) as described previously (Gabrielli et al. 2001Go). Reagents from Vector Laboratories (Burlingame, CA) were used. Briefly, after inactivation of endogenous peroxidase activity (0.3% H2O2 in methanol for 30 min) and blocking of endogenous avidin-binding activity, the sections were treated for 15 min with normal rabbit serum diluted 1:5 with 1% bovine serum albumin (BSA; Sigma-Aldrich Chemie, Deisenhofen, Germany) in 0.1 M PBS, pH 7.6. Primary antibodies to detect CA I and CA II were polyclonal anti-human CAI and CAII (The Binding Site; Birmingham, UK). Incubation with anti-CA I or anti-CA II (1:200 in PBS plus 1% BSA) was performed overnight at RT. Biotinylated rabbit anti-sheep immunoglobulin (1:200) was used as a secondary antibody and allowed to bind for 45 min, followed by incubation with avidin–peroxidase complex (1:100) for 45 min. Peroxidase activity was developed using 3-3'-diaminobenzidine tetrachloride (DAB kit; Vector Labs), with and without nickel enhancement. Unless otherwise specified, PBS was used to wash sections after each step of the procedure (three times for 5 min each) and dilutions. After immunostaining, some sections were counterstained with toludine blue or Gomori's trichromic technique. Controls were carried out by omitting primary antibody.

For identification of CA-reactive cells, some sections were subjected to a double immunostaining with both CA I (CA II) antiserum and monoclonal anti-{alpha}-smooth muscle actin (clone 1A4; Sigma). At the first step, incubation with the primary antibody (anti-CA I or anti-CA II, 1:100) was followed by treatment of sections with an appropriate biotinylated secondary antibody (1:200) and then with avidin–FITC conjugate for 1 hr (1:50; Sigma). After washing in PBS containing 0.1% Triton X-100, tissues were incubated for 3 hr with anti-{alpha}–smooth muscle actin (1:200), followed by incubation with TRITC-conjugated goat anti-mouse IgG for 1 hr (1:75; Sigma). Sections were washed, mounted in glycerol–PBS (1:1), and observed under an Ar/Kr Bio-Rad MCR-600 confocal laser scanning microscope (Bio-Rad; Herfordshire, UK) connected to a Nikon Diaphot-TMD-EF inverted microscope equipped with a Plan Apo oil immersion objective (x10; NA 1.4). The standard BHS block (exciter filter 488 DF 10) and the YHS one (exciter filter 568 DF 10) were employed for FITC and TRITC, respectively. The image acquisition was carried out with Bio-Rad COMOS software. In the merged image, a yellow color was interpreted as co-localization of the two markers. Control sections were processed as above, with the alternate omission of one of the primary antibodies.


   Results
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 Materials and Methods
 Results
Discussion
 Literature Cited
 
CA Activity
CA activity was detected in homogenate from bovine aortic smooth muscle with an activity of 3.5 ± 2.7 U/g tissue (n=6). In comparison, bovine red cell CA activity in the same assay system was 1650 U/g. Analysis of hemoglobin in the supernatant (and therefore red cell contamination with their high CA content) showed that only 1% of the total CA activity could be accounted for by any red cells present in the supernatant. This was confirmed by the absence in the aorta of red cells among the smooth muscle cells (Figures 1a and 1b).



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  		Figures 1–4

Figure 1 Bovine aorta. Consecutive en face sections of the media were subjected to immunohistochemistry for CA I (a) and to Gomori's trichrome technique for morphological study (b). CA I immunostaining can be observed as thin deposits (a) in elongated cells interspersed among green-stained collagen fibers and elastic laminae. Magnification x280.

Figure 2 Immunohistochemistry for CA I in the media of the bovine aorta. A marked staining is specifically located in the cytoplasm of elongated cells and does not extend to the surrounding collagen fibers and elastic tissue. Magnification x1400.

Figure 3 Immunohistochemistry for CA II in the bovine aorta by avidin–FITC. Green fluorescence is closely restricted to bundles of thin cells. Magnification x800.

Figure 4 Confocal microscopy analysis of en face section of the bovine aorta. Double labeling with avidin–FITC conjugate for CA I (green) and TRITC conjugate for {alpha}-smooth muscle actin (red). Green fluorescence reveals CA I in thin elongated cells of the media (a). Red fluorescence specifically marks smooth muscle cells (b). The merged image (c) shows large overlapping (yellow) of CA I- and {alpha}-actin-reactive sites, supporting their co-localization in smooth muscle cells. Magnification x400.

 
CA Inhibition
The inhibition of bovine aortic smooth muscle by several CA inhibitors is given in Table 1. The I50 for acetazolamide was found to be 0.07 ± 0.01 µM, indicating a mix of CA isozymes because this I50 is intermediate between that for CA I and CA II (Maren 1967Go). Inhibition by 1 µM dorzolamide, 20 mM bromopyruvate, and 1 mM acetazolamide revealed that 70 ± 5% of the total CA activity is due to CA I and 20 ± 4% is due to CA II. The remaining approximate 5% of activity is due to CA III and/or other isozymes of CA.


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  		Table 1

Inhibition of bovine aortic smooth muscle carbonic anhydrase by nonselective and selective isozyme inhibitorsa

 
CA Immunocytochemistry
Analysis of bovine smooth muscle shows that both CA I and II isozymes are present, mainly in the media layer (Figures 1a and 2–4). The immunostaining of CA I (Figure 1a) suggests that it is located in thin elongated cells which, when observed in adjacent sections stained for morphological analysis (Figure 1b), appear to be obliquely oriented between the elastic laminae, with fine collagen fibers filling the spaces between cells. At higher magnification, the CA-positive cells showed marked staining throughout their cytoplasm, with different degrees of intensity in different cells. In addition, nuclei were also positive on occasion for CA I labeling (Figure 2). A similar distribution of immunoreactive sites was demonstrated with antibodies against CA II (Figure 3). Expression of CA in aortic smooth muscle cells was confirmed by double immunostaining and co-localization for CAI and {alpha}-smooth muscle actin antigens in the same tissue preparation in a number of cells positive for each antigen (Figure 4). Similar results were obtained for CA II (data not shown).


   Discussion
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
Literature Cited
 
Our main finding is the first demonstration of CA activity in mammalian vascular smooth muscle at very low but measurable catalysis of CO2 hydration. Several isozymes, including CA I, CA II, and CA III, are present, as demonstrated by direct biochemical measurement of bioactivity with selective CA inhibitors. This is further confirmed by CA I and II immunocytochemistry. By activity measurements, CA I is the most abundant isozyme, followed by smaller amounts of CA II activity and only a small fraction attributable to CA III. We show that smooth muscle CA activity is quite low in comparison to other cells and organs in which CA activity is in the range of 20–1000 U/g. The low activity may explain a previous report of undetectable CA activity in rabbit aortic vascular smooth muscle (Muhliesen and Kreye 1985Go).

We did not measure the concentrations of the three isozymes, but given that CA I activity is approximately one tenth that of CA II, it is probable that the CA I concentration exceeds that of CA II in vascular smooth muscle by roughly 30-fold. Because immunocytochemical detection is only qualitative in nature, the roughly equal staining by both antibodies is not incompatible with large concentration differences in protein. We did not measure CA III activity directly but found that a small amount (5%) of the total activity was left uninhibited by concentrations of dorzolamide and bromopyruvate, which abolish all CA I and II activity. This remaining activity could be abolished by heating and denaturation or by acetazolamide at 1 mM. Although CA III is a sulfonamide-resistant isozyme, it can be ultimately inhibited by mM concentrations of permeant CA-inhibiting sulfonamides (Sanyal et al. 1982Go). The concentration of CA III in vascular smooth muscle may be on the same order of magnitude of CA I because its CO2 hydration catalysis rate is only 1% that of CA II and 10% of CA I. This probably explains why CA III has been readily detected in smooth muscle (with histochemical methods) by many workers (Jeffery et al. 1980Go; Vaananen and Autio-Harmainen 1987Go; Gabrielli and Menghi 1994Go; Ichihara et al. 1997Go; Gabrielli et al. 1998Go). Our results showing CA I and II isozymes by selective isozyme inhibition and immunocytochemistry are in accord with other immunocytochemical data in vascular smooth muscle that have shown CA I and CA II (Yamamoto et al. 2003Go).

The presence of both CA I and CA II in vascular smooth muscle, as well as CA III, is interesting in view of the fact that no other tissues have been shown to express all three cytosolic isoenzymes (Maren 1967Go). Very likely there may be exceptions to this, if the tissues express so little activity that it escapes detection by the particular activity assays or histochemical methods employed.

The role of CA in vascular smooth muscle and regulation of vascular tone is just beginning to be explored. It has been known for a long time that CA inhibitors are potent vasodilators in the central nervous system and eyes (Kiss et al. 1999Go). This has always been attributed to the concomitant metabolic acidosis and hypercapnia that arise with renal, red cell, and brain tissue CA inhibition, since hypercapnic acidosis in particular is a potent vasodilator in systemic blood vessels. More recently, however, studies done either in vitro or in situ to eliminate any systemic acidosis show that acetazolamide and other powerful CA-inhibiting sulfonamides relax preconstricted blood vessels (Pickkers et al. 1999Go; Deem et al. 2000Go) and vasodilate directly (Pickkers et al. 2001Go). The mechanism(s) involved in vasorelaxation by CA inhibition is unknown. Evidence in pulmonary vascular smooth muscle cells suggests that the reduction of hypoxic pulmonary vasoconstriction by acetazolamide (Deem et al. 2000Go; Hohne et al. 2004) is due in part to a decrease in intracellular calcium (Shimoda and Swenson 2004Go). However, changes in membrane potential and ion channel activity, or Ca2+ sensitivity of actin–myosin (Austin and Wray 2000Go), may also be possible, perhaps as a consequence of alkalinization of intracellular pH in isolated vascular smooth muscle cells with CA inhibition (Pickkers et al. 1999Go; Reber et al. 2002Go). This latter aspect of intracellular alkalinization of smooth muscle cells by inhibition of carbonic anhydrase may be related to the finding of vascular calcifications in mice that lack CA II (Spicer et al. 1989Go) because precipitation of calcium phosphate and sulfate is promoted by alkaline conditions.

A specific role for CA in the smooth muscle of the aorta is not obvious because the aorta and other large conduit vessels have few or no roles in the modulation of systemic vascular resistance. Nevertheless, smooth muscle cells in large conduit vessels may help to maintain normal compliance characteristics and elasticity of these vessels, which serve to store some of the energy of ventricular contraction and dissipate this during diastole to maintain forward flow.

In conclusion, we have shown definitive biochemical and immunocytochemical evidence of CA in vascular smooth muscle. Our data and those of others demonstrate that several CA isozymes are present and that, despite concentrations at low levels of tissue expression, this activity contributes to vasoregulation.


   Acknowledgments
 
Supported by grants from The Hawaii Community Foundation (Geist Award # 20010643; JTB) and the National Institutes of Health (HL-24163; ERS).

We thank Camille Bandet for assistance with performing the CA bioassay measurements.


   Footnotes
 
Received for publication January 27, 2004; accepted April 2, 2004


   Literature Cited
Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 

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