Cholera toxin stimulates adenosine3',5'-monophosphate synthesis and epithelial

wound closure in the rabbit corneaMarcia M. Jumblatt, J. A. Fogle, and Arthur H. Neufeld

Rabbit corneas were treated in vitro and in vivo with cholera toxin (CTX), a specific andirreversible activator ofadenylate cyclase. Tissue pieces incubated in vitro in the presence of 10fig/ml CTX for 15 min continuously synthesized adenosine 3' ,5'-monophosphate (cyclic AMP)at an increased rate for 3 hr in the absence of CTX in the medium. Corneas exposed for 10 minto CTX topically in vivo and after various time intervals incubated in vitro had an increasedability to synthesize cyclic AMP for at least 30 hr after topical treatment. Epithelialwounds, 6 mm in diameter, were made by brief exposure of corneas in vivo to filter disks soaked,in heptanol. Wounds in corneas pretreated with CTX closed at a faster rate and earlier thanwounds in corneas pretreated with inactivated CTX. We postulate that cyclic AMP mediatesthe initial events governing the rate of closure of an epithelial defect.

Key words: cornea, epithelium, wound healing, cyclic AMP, cholera toxin, rabbit

Adelenosine 3',5'-monophosphate (cyclicAMP) mediates several processes which mayfunction during the response of the cornealepithelium to stress. Cyclic AMP stimulatesthe active transport of chloride from stromato tears in frog,1 rabbit,2 and human3 corneas,and catecholamines or prostaglandins may lo-cally activate this response to dehydrate swol-len stromal areas. Furthermore, several in-vestigations indicate that adrenergic agents

From the Department of Ophthalmology, Harvard Med-ical School, and the Eye Research Institute of RetinaFoundation, Boston, Mass.

Supported in part by U.S. Public Health Service grantsEY-02360 and EY-02367, Biomedical Research Sup-port grant 5SO7RR05527 and Massachusetts LionsEye Research Fund, Inc. Dr. Neufeld is the recipientof a Research Career Development Award, EY-00114.Dr. Fogle is the recipient of a Bausch and Lomb Ex-ternal Disease Fellowship and a Heed FellowshipAward.

Submitted for publication Dec. 11, 1979.Reprint requests: Arthur H. Neufeld, Ph.D., Eye Re-

search Institute of Retina Foundation, 20 StanifordSt., Boston, Mass. 02114.

influence the mitotic rate of the corneal epi-thelium.4"10 Epinephrine and isoproterenoldecrease the rate or shift the diurnal rhythmof mitosis in mouse and rat corneal epithe-lium.5' 7' 8 Norepinephrine, released fromdegenerating nerve terminals after sympath-ectomy, depresses mitosis in rat5 and rabbit6

corneas and increases the level of cyclic AMPin this tissue.10 These adrenergic agents un-doubtedly stimulate /3-adrenergic receptorsin the cornea to cause the synthesis of cyclicAMP, which modulates the subsequent cellu-lar events."

The epithelial cells that participate in cor-neal wound healing cease mitotic activity,mobilize their glycogen stores, and slide cen-tripetally to cover the defect. 12~14 Cavanagh15

has suggested that these events are mediatedby relative changes in the cyclic AMP andguanosine 3',5'-monophosphate levels andhas presented evidence from experiments incell culture that changes in the ratio of cyclicnucleotides induced by adrenergic or cholin-ergic agonists are associated with changes in

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1322 Jumhlatt, Fogle, and Neufeld Invest. Ophthalmol. Vis. Set.November 1980

160

140

120

100

80

60

40

20

0772

30 60 90 120 150 180

Fig. 1. Effect of CTX on cyclic AMP synthesis in vitro. Pieces of cornea were incubated in thepresence (o) or absence (•) of CTX for 15 min (shaded area) and then placed in CTX-freeincubation medium. IBMX was always present. All points are the mean of three experiments,each determined in duplicate.

DNA and protein synthesis. Our own studieshave led us to suggest a central role for cyclicAMP in the early events of wound healing bythe corneal epithelium.10

To test whether increased synthesis of cy-clic AMP influences the early events in epi-thelial wound healing, we have studied theclosure of a standardized epithelial defect inrabbit corneas pretreated with cholera toxin(CTX), a specific and irreversible activator ofadenylate cyclase.

Methods

Treatment with CTX in vitro. Male New Zea-land white rabbits (2 to 3 kg) were sacrificed byintravenous overdose with sodium pentobarbital.Corneas were immediately excised, cut into two tofour pieces, and preincubated at 37° for 30 min inincubation buffer, pH 7.4, containing 100 mMNaCl, 20 mM NaH2PO4, 6.9 mM dextrose, 4.5mM KC1, 0.8 mM MgCl2, 0.4 mM CaCl2, and 0.1mg/ml indomethacin. Following this preincuba-tion, corneal pieces were transferred to fresh buf-fer containing 0.5 mM 3-isobutyl-l-methylxan-thine (IBMX), either with or without 10 /ag/mlCTX. Fifteen minutes later, tissues were trans-ferred to toxin-free buffer containing IBMX andincubated for various intervals up to 3 hr for thedetermination of cyclic AMP levels.

Treatment with CTX in vivo. Rabbits wereanesthetized with intravenous sodium pentobarbi-tal and topical proparacaine HC1. CTX application

was limited to the anterior corneal surface by aplastic cylinder, 20 mm in height and 12 mm in-ternal diameter, sealed temporarily to the eyewith a fine bead of nontoxic silicone grease. A vol-ume of 200 jul of phosphate-buffered saline (PBS),pH 7.2, containing either 2 /ag of CTX or 2 fig ofinactivated cholera toxin (ICTX), dissociated byheating to 60° in the presence of 1 mM dithio-threitol for 15 min, was delivered to the well. Tenminutes later the toxin solution was aspirated fromthe well, the cylinder was removed, and the eyewas generously flushed with PBS.

In one group of animals, at 3, 24, 30, and48 hr after toxin treatment, corneas were excised,halved, and preincubated in vitro in incubationbuffer containing 0.1 mg/ml indomethacin for 30min at 37°. Following this preincubation, cornealhalves were transferred to fresh buffer containing0.5 mM IBMX and incubated for 20 min for thedetermination of cyclic AMP levels.

Determination of cyclic AMP levels. At the endof the in vitro incubation period, corneal pieceswere immediately transferred to glass hoinoge-nizers containing 250 fi\ of 0. IN KOH preheatedin a boiling water bath and homogenized by hand.Homogenates were neutralized with 250 jul of0.1N HC1 and centrifuged at 1650 x g for 15 minat 4° C. The supernatant was removed for radio-immunoassay of cyclic AMP,16 and the pellet wasdissolved in 1M NaOH for protein determinationby the method of Lowry et al.17

Assessment of epithelial wound closure. Threehours after in vivo treatment with CTX or ICTX,

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Cyclic AMP and epithelial wound closure 1323

epithelial wounds were produced in corneas ofrabbits by the heptanol de-epithelialization of Cin-tron et al.1" Briefly, 6 mm filter paper disks satu-rated with heptanol were placed centrally onproparacaine-pretreated corneas of anesthetized(sodium pentobarbital) rabbits. After 10 sec, thedisk was removed, and the eye was irrigated withPBS. The corneal epithelium in the wound areawas debrided with a cotton-tipped swab. A singledrop of Richardson's stain (1% azure II, 1% methy-lene blue in 1% borax, 1:1) demarcated the de-nuded area. At 0, 18, 24, 30, and 42 hr afterwounding, the stained lesion was photographed onKodachrome ASA 25 color slide film with a fixed-focus camera.

Wound areas were compared at each time pointby projecting the slides at constant magnificationwith a photographic enlarger, carefully tracing theperimeter of each wound on paper, and then cut-ting out and weighing each traced wound area. Toaccount for any irregularities in shape, subsequentweights of each wound area were compared to thatof the original wound area, and results were ex-pressed as percent of the original wound.

Materials. Indomethacin, IBMX, and dithio-threitol were obtained from Sigma Chemical Co.,purified CTX from Schwarz/Mann Div., Becton,Dickinson & Co., and components for the radio-immunoassay of cyclic AMP from New EnglandNuclear.

Results

Effect of CTX on cyclic AMP synthesis invitro. Exposure of corneas to 10 jU.g/ml CTXfor 15 min in vitro caused an increase in thesynthesis of cyclic AMP, which persisted inthe absence of CTX in the medium (Fig. 1).Following a lag period of 30 min, the cor-neas exposed to CTX accumulated increasingamounts of cyclic AMP in the presence ofIBMX for at least 3 hr. Corneas not exposedto CTX did not accumulate cyclic AMP dur-ing the 3 hr incubation, even in the presenceof the inhibitor of phosphodiesterase, IBMX.

Effect of topical CTX on cyclic AMP syn-thesis in vitro. Exposure of corneas to 10/ig/ml CTX for 10 min in vivo caused an in-crease in the synthesis of cyclic AMP whenthese tissues were excised and incubated invitro in the presence of IBMX (Fig. 2). By 3hr after treatment, corneas exposed to CTXsynthesized at least four times more cyclicAMP when incubated in the presence of

u 6 12 18 24 30 36 42 48Hr.

Fig. 2. Effect of topical CTX on cyclic AMP syn-thesis in vitro. Pieces of cornea from four to eightpaired eyes treated in vivo with CTX or ICTXwere incubated in the presence of IBMX for 20min. The data are presented as the mean ±S.E.M. of the ratio of the amount of cyclic AMPsynthesized in vitro by CTX- and ICTX-pretreatedcorneas.

IBMX than did corneas exposed to ICTX. In-creased synthesis of cyclic AMP persisted forat least 30 hr after treatment with CTX. By 48hr after treatment, no difference in the abilityof the paired corneas to synthesize cyclicAMP was detectable.

Effect of topical CTX on epithelial woundclosure. Heptanol de-epithelialization pro-duced discrete, circular, corneal woundswith distinct borders. Wounds in corneaspretreated with CTX 3 hr before heptanolde-epithelialization closed faster than didthose in corneas pretreated with ICTX. Atypical example of accelerated wound clo-sure, the left and right eyes of the same ani-mal, is shown in Fig. 3. The wounds ap-peared to close to a similar extent during thefirst 18 hr, but by 24 hr after wounding, thewound in the CTX-treated cornea was no-ticeably smaller than that in the ICTX-

. treated cornea. By 30 hr after wounding, thedifference in wound closure was quite appar-ent, and by 42 hr the wound on the CTX-treated side no longer stained, whereas thecontralateral wound was still evident.

In a control experiment, several animalswere pretreated with ICTX in one eye and anequivalent concentration of bovine serum al-

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1324 Jumblatt, Fogle, and Neufeld Invest. Ophthalmol. Vis. Sri.November 1980

Fig. 3. Comparison of corneal wound closure in the eyes of a rabbit. The eye depicted on theleft was pretreated with CTX; the eye on the right was pretreated with ICTX. Time in hoursafter wounding is indicated. Bright reflections are from photoflash unit.

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Volume 19Number 11 Cyclic AMP and epithelial wound closure 1325

bumin in the other eye, 3 hr before heptanolde-epithelialization. Wounds in these eyeswere observed to close at identical rates, andbilateral closure was completed 48 hr afterwounding.

The wound closure data from 13 animalsare summarized in Fig. 4. Closure rates wereidentical for the first 18 hr, at which time thewounds were 50% closed. By 24 hr, the per-cent of the wound remaining in the CTX-treated eyes was significantly less than that inthe ICTX-treated eyes and remained so untilthe defect closed completely. The closurerate for the wounds in the CTX- and ICTX-treated corneas during the period from 18 to30 hr was determined by linear regressionanalysis. During this time interval, woundsin corneas pretreated with CTX closed at therate of 0.85 mm2/hr, and wounds in corneaspretreated with ICTX closed at the rate of0.57 mm2/hr.

As shown in Fig. 4, for corneas pretreatedwith CTX the line generated by linear re-gression extrapolated to 100% of the originalwound at zero time, indicating that thesecorneas maintained the initial rate of woundclosure for at least 30 hr. For corneas pre-treated with ICTX, the line generated by lin-ear regression extrapolated to 89% of theoriginal wound at zero time, indicating that .between 18 and 30 hr these corneas hadslowed their rate of wound closure. Extrapo-lation of these lines to 0% of the remainingwound indicated a closure time of 34.6 hr forthe corneas pretreated with CTX and 40.5 hrfor the corneas pretreated with ICTX. Theexact time of closure was not experimentallydetermined, but by 42 hr wounds in cor-neas pretreated with CTX were completelyclosed, whereas those pretreated with ICTXwere not.

Eighteen hours after de-epithelializationwith heptanol, when the areas of remainingwound were approximately equal in botheyes, corneas were excised and incubated invitro for the determination of cyclic AMPsynthesis. In four animals, corneas which hadbeen pretreated with CTX in vivo synthesized2.5 ± 0.6 (mean ± S.E.M.) times more cy-clic AMP than contralateral corneas pre-

0 6 12 18 24 30 36 42 48

Hr.

Fig. 4. Effect of topical CTX on epithelial woundclosure. The percent of the original wound re-maining in corneas pretreated with CTX (o) orICTX (•) is shown as a function of time afterwounding. All data are expressed as the mean ±S.E.M. for nine to 13 animals. Linear regressionanalysis of the data at 18, 24, and 30 hr generatedthe dashed lines (r2 = 0.99 for each line), whichwere used to estimate the rate of closure (slope),the predicted time of wound closure (X-intercept),and the predicted percent of the original wound atzero time (Y-intercept).

treated with ICTX. This ratio, which was ac-tually determined 21 hr after toxin applica-tion, was similar to that shown in Fig. 2 at24 hr.

Discussion

De-epithelialization of the rabbit corneawith heptanol provides a simple and repro-ducible model for studying epithelial woundhealing.18 The wound heals in approximately48 hr and allows quantification by stainingand photography. The wound primarily in-volves the epithelial cells; the basementmembrane remains intact, and there is onlyminimal damage to stromal keratocytes. Cin-tron et al.'8 have reported that the normal

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1326 Jumblatt, Fogle, and Neufeld Invest. Ophthalmol. Vis. Set.November 1980

wound heals at a rate of 0.58 mm2/hr, and wefind that control wounds in corneas pre-treated with ICTX or with bovine serum al-bumin heal at essentially the identical rate(0.57 mm2/hr).

The precise cellular mechanism by which asuperficial defect closes is not clear. Slidingof the epithelium over the exposed basementmembrane is a major component in themechanism by which the wound heals to theendpoint that we used, the time when stain-ing is no longer observed. Epithelial cellsbegin their migration from the wound marginafter a lag period of at least 4 hr,14 and eachcell type (basal, wing, and squamous) con-tributes to closure of the defect.19 Buck14 hasobserved that the cellular layers move as amass and not as a monolayer.

There is, however, uncertainty about thedegree of proliferation of basal epithelial cellswhich participate in the sliding response. Inwounds in which the stroma is bared, a zoneof decreased 3H-thymidine incorporation isobserved adjacent to the wound. The cells inthis zone are primarily involved in slidingover exposed stroma and do not regain theirmitotic activity until the defect is covered.22

However, a superficial cautery burn healswithout an area of decreased proliferation ifthe stroma is not exposed.19 Thus the extentto which changes in cellular proliferationoccur during the healing of a heptanol-induced wound is not known.

The hypothesis that cyclic nucleotidesinfluence corneal epithelial wound healinghas been suggested,11' 15 but experimentalproof has not been obtained. Our interests instudying a role for cyclic AMP in initiatingthe response of epithelial cells to stress led usto these experiments in which CTX directlystimulates cyclic AMP synthesis in corneasundergoing wound healing.

CTX is a specific and irreversible activatorof adenylate cyclase. In its active form theexotoxin has two subunits, one of which bindsto GMi-gangliosides of the cell membrane,allowing the catalytic subunit to enter the celland activate adenylate cyclase.20 Thermal in-activation of CTX in the presence of dithio-threitol dissociates the complex, and the

catalytic subunit does not enter the cell. Thismechanism has been extensively studied andis considered quite specific.20'21* 22 Recently,CTX has been used as a tool to investigatecyclic AMP-stimulated events in turtle blad-der,23 choroid plexus,24 pineal gland,25 andcultured cells.26

The increased synthesis of cyclic AMP incorneas pretreated with CTX is consistentwith the mechanism described above. Briefexposure to corneas to CTX, either in vitro orin vivo, is sufficient to allow binding toGMi-gangliosides, and subsequent stimula-tion is not dependent on the continued pres-ence of CTX in the bathing medium. The lagperiod, which occurs before appreciable ac-cumulation of cyclic AMP, is related to thetime required for the catalytic subunit to be-come internalized and to irreversibly activateadenylate cyclase.

Between 18 and 30 hr after wounding, clo-sure of an epithelial defect in corneas pre-treated with CTX proceeds at a rate of 0.85mmVhr, which is approximately 50% fasterthan that in control corneas. Because the lin-ear regression of the data from corneas pre-treated with CTX extrapolates back to theoriginal wound size at zero time, we concludethat the initial rate of closure is prolonged forat least 30 hr. Closing of the wound in controlcorneas begins at this rate but after 18 hrslows to a rate of 0.57 mm2/hr.

The increased rate of wound closure incorneas pretreated with CTX may be due toan indirect or direct action of cyclic AMP. Anindirect stimulation of ongoing events by cy-clic AMP would have led to a greater initialrate of wound closure in CTX-treated cor-neas. However, we observed that the initialrates of closure are the same in CTX-treatedand untreated eyes. Therefore we hypothe-size that in response to the stress of wound-ing, an as yet unknown stimulus elevates thelevel of cyclic AMP which activates cellularprocesses contributing to wound healing. Inour experiments, use of CTX prolonged theincreased synthesis of cyclic AMP and thusprolonged the initial rate of wound closure.In untreated corneas, the initial rate was notmaintained after 18 hr.

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The mechanism by which cyclic AMP acti-vates cellular processes involved in woundclosure is not clear. Cyclic AMP stimulatesglycolysis and gluconeogenesis in many tis-sues,27 and perhaps increased utilization ofcorneal glycogen stores provides glucose forenergy production during wound healing.Furthermore, this cyclic nucleotide maypromote sliding of epithelial cells or depressproliferative activity while enhancing cellularmigratory activity.

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