[Skip to Content]
Access to paid content on this site is currently suspended due to excessive activity being detected from your IP address 54.161.128.52. Please contact the publisher to request reinstatement.
[Skip to Content Landing]
Download PDF
Figure 1.
Time line for protocols determining the effects of intracameral and intravitreal H-7 [1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine] on pupillary and accommodative responses to pilocarpine in monkeys anesthetized with ketamine and pentobarbital sodium.

Time line for protocols determining the effects of intracameral and intravitreal H-7 [1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine] on pupillary and accommodative responses to pilocarpine in monkeys anesthetized with ketamine and pentobarbital sodium.

Figure 2.
Inhibition of anesthesia-induced miosis by topical H-7 [1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine] (150 mmol/L=1.1 mg). BL indicates pre–H-7 baseline. Data are given as the mean±SEM for the number of (n) monkeys anesthetized with ketamine and pentobarbital sodium. Significant difference between eyes: *, P<.05; **, P <.01.

Inhibition of anesthesia-induced miosis by topical H-7 [1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine] (150 mmol/L=1.1 mg). BL indicates pre–H-7 baseline. Data are given as the mean±SEM for the number of (n) monkeys anesthetized with ketamine and pentobarbital sodium. Significant difference between eyes: *, P<.05; **, P <.01.

Figure 3.
Accommodative responses to topical H-7 [1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine] (150 mmol/L=1.1 mg). A, Refraction; B, accommodation. BL indicates pre–H-7 baseline; dashed line, equality. Data are given as mean±SEM for the number of (n) monkeys. No difference between eyes.

Accommodative responses to topical H-7 [1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine] (150 mmol/L=1.1 mg). A, Refraction; B, accommodation. BL indicates pre–H-7 baseline; dashed line, equality. Data are given as mean±SEM for the number of (n) monkeys. No difference between eyes.

Figure 4
. Pupils, cynomolgus monkey, Figure 1 protocol. A, Baseline; B, 25 minutes after topical phenylephrine; C, 25 minutes after intracameral H-7 [1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine] (300 µmol/L) left eye, vehicle right eye; D, approximately 50 minutes after 1.5-mg/kg intramuscular pilocarpine hydrochloride.

. Pupils, cynomolgus monkey, Figure 1 protocol. A, Baseline; B, 25 minutes after topical phenylephrine; C, 25 minutes after intracameral H-7 [1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine] (300 µmol/L) left eye, vehicle right eye; D, approximately 50 minutes after 1.5-mg/kg intramuscular pilocarpine hydrochloride.

Figure 5.
Pupillary and accommodative responses to intracameral (IC) and intravitreal (IVIT) H-7 [1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine], topical phenylephrine hydrochloride), and intramuscular (IM) pilocarpine hydrochloride (1.5 mg/kg). Protocol per Figure 1. BL indicates baseline. Data are given as mean±SEM for the number of (n) monkeys. Significant difference between eyes: *, P <.05; **, P<.01; and ***, P <.001.

Pupillary and accommodative responses to intracameral (IC) and intravitreal (IVIT) H-7 [1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine], topical phenylephrine hydrochloride), and intramuscular (IM) pilocarpine hydrochloride (1.5 mg/kg). Protocol per Figure 1. BL indicates baseline. Data are given as mean±SEM for the number of (n) monkeys. Significant difference between eyes: *, P <.05; **, P<.01; and ***, P <.001.

Figure 6.
Effect of H-7 [1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine] on contraction of ciliary muscle strips. A, 10-µmol/L pilocarpine hydrochloride for 15 minutes, followed by 10-µmol/L pilocarpine hydrochloride plus increasing H-7 concentrations for 15 minutes each. B, Percentage of maximum pilocarpine response at increasing H-7 concentrations plus 10-µmol/L pilocarpine hydrochloride. Data are given as mean±SEM; the 5 ciliary muscle strips are from 4 rhesus monkeys.

Effect of H-7 [1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine] on contraction of ciliary muscle strips. A, 10-µmol/L pilocarpine hydrochloride for 15 minutes, followed by 10-µmol/L pilocarpine hydrochloride plus increasing H-7 concentrations for 15 minutes each. B, Percentage of maximum pilocarpine response at increasing H-7 concentrations plus 10-µmol/L pilocarpine hydrochloride. Data are given as mean±SEM; the 5 ciliary muscle strips are from 4 rhesus monkeys.

Figure 7.
Effect of H-7 [1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine] on contraction of rhesus monkey ciliary muscle strip. A, Ciliary muscle strip at baseline tone, then exposed to 10-µmol/L pilocarpine hydrochloride, then to 10-µmol/L pilocarpine hydrochloride plus 300-µmol/L H-7. Note the response to pilocarpine and its inhibition by H-7. B, Same ciliary muscle strip after a 90-minute washout; 10-µmol/L pilocarpine hydrochloride elicits no contraction. C, Same ciliary muscle strip after an additional 60-minute washout; 10-µmol/L pilocarpine hydrochloride elicits sluggish contraction in both vectors.

Effect of H-7 [1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine] on contraction of rhesus monkey ciliary muscle strip. A, Ciliary muscle strip at baseline tone, then exposed to 10-µmol/L pilocarpine hydrochloride, then to 10-µmol/L pilocarpine hydrochloride plus 300-µmol/L H-7. Note the response to pilocarpine and its inhibition by H-7. B, Same ciliary muscle strip after a 90-minute washout; 10-µmol/L pilocarpine hydrochloride elicits no contraction. C, Same ciliary muscle strip after an additional 60-minute washout; 10-µmol/L pilocarpine hydrochloride elicits sluggish contraction in both vectors.

Figure 8.
Effect of H-7 [1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine] on cell contractility. Swiss 3T3 cells on silicone rubber film (A) prior to and (B) after 5.5-minute incubation with 300-µmol/L H-7. Note reduction in film wrinkling (arrowheads in A) after exposure to H-7. C, Wrinkle amplitude vs H-7 exposure time.

Effect of H-7 [1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine] on cell contractility. Swiss 3T3 cells on silicone rubber film (A) prior to and (B) after 5.5-minute incubation with 300-µmol/L H-7. Note reduction in film wrinkling (arrowheads in A) after exposure to H-7. C, Wrinkle amplitude vs H-7 exposure time.

1.
Yousufzai  SYKChen  ALAbdel-Latif  AA Species differences in the effects of prostaglandins on inositol triphosphate accumulation, phosphatidic acid formation, myosin light chain phosphorylation and contraction in iris sphincter of the mammalian eye: interaction with the cyclic AMP system. J Pharmacol Exp Ther. 1988;2471064- 1072
2.
Volberg  TGeiger  BCiti  SBershadsky  AD Effect of protein kinase inhibitor H-7 on the contractility, integrity, and membrane anchorage of the microfilament system. Cell Motil Cytoskeleton. 1994;29321- 338Article
3.
Kamm  KEStull  JT The function of myosin and myosin light chain kinase phosphorylation in smooth muscle. Annu Rev Pharmacol Toxicol. 1985;25593- 620Article
4.
Sommerville  LEHartshorne  DJ Intracellular calcium and smooth muscle contraction. Cell Calcium. 1986;7353- 364Article
5.
Sugiura  MInagami  THare  GMTJohns  JA Endothelin action: inhibition by a protein kinase C inhibitor and involvement of phosphoinositols. Biochem Biophys Res Commun. 1989;158170- 176Article
6.
Grunstein  MMChuang  STSchramm  CMPawlowski  NA Role of endothelin 1 in regulating rabbit airway contractility. AM J Physiol. 1991;260 ((4)) L75- L82
7.
Mobley  PLHedberg  KBonin  LChen  BGriffith  OH Decreased phosphorylation of four 20-kDa proteins precedes staurosporine-induced disruption of the actin/myosin cytoskeleton in rat astrocytes. Exp Cell Res. 1994;21455- 66Article
8.
Tian  BKaufman  PLVolberg  TGabelt  BTGeiger  B H-7 disrupts the actin cytoskeleton and increases outflow facility. Arch Ophthalmol. 1998;16633- 643Article
9.
Bárány  EH Simultaneous measurement of changing intraocular pressure and outflow facility in the vervet monkey by constant pressure infusion. Invest Ophthalmol. 1964;3135- 43
10.
Erickson-Lamy  KAKaufman  PLMcDermott  MLFrance  NK Comparative anesthetic effects of aqueous humor dynamics in the cynomolgus monkeys. Arch Ophthalmol. 1984;1021815- 1820Article
11.
Asseff  CFWeisman  RLPodos  SMBecker  B Ocular penetration of pilocarpine in primates. Am J Ophthalmol. 1973;75212- 215
12.
Harris  JE Problems in drug penetration. Leopoid  IHed.Symposium on Ocular Therapy. Vol 3. St Louis, Mo CV Mosby1968;96- 105
13.
Janes  RGStiles  JF The penetration of C14-labeled atropine into the eye: a comparison of methods of application. Arch Ophthalmol. 1959;6269- 74Article
14.
Kaufman  PL Accommodation and presbyopia: neuromuscular and biophysical aspects. Hart  WM  Jred.Adler's Physiology of the Eye. 9th ed. St Louis, Mo Mosby–Year Book Inc1992;391- 411
15.
Thompson  HS The pupil. Hart  WM  Jred.Adler's Physiology of the Eye. 9th ed. St Louis, Mo Mosby–Year Book Inc1992;412- 441
16.
Bito  LZDeRousseau  CJKaufman  PLBito  JW Age-dependent loss of accommodative amplitude in rhesus monkeys: an animal model for presbyopia. Invest Ophthalmol Vis Sci. 1982;2323- 31
17.
Kaufman  PLCalkins  BTErickson  KA Ocular biometry of the cynomolgus monkey. Curr Eye Res. 1981;1307- 309Article
18.
Poyer  JFKaufman  PLFlügel  C Age does not affect contractile responses of the isolated rhesus monkey ciliary muscle to muscarinic agonists. Curr Eye Res. 1993;12413- 422Article
19.
Poyer  JFMillar  CKaufman  PL Prostaglandin F2α effects on isolated rhesus monkey ciliary muscle. Invest Ophthalmol Vis Sci. 1995;362461- 2465
20.
Millar  CPoyer  JFGabelt  BTKaufman  PL Endothelin subtypes: effect on isolated rhesus monkey ciliary muscle. J Pharmacol Exp Ther. 1995;2751143- 1147
21.
Harris  AKWild  PStopak  D Silicone rubber substrata: a new wrinkle in the study of cell locomotion. Science. 1980;208177- 179Article
22.
Chrzanowska-Wodnicka  MBurridge  K Rho-stimulated contractility drives the formation of stress fibers and focal adhesions. J Cell Biol. 1996;1331403- 1415Article
23.
Westheimer  GBlair  SM Accommodation of the eye during sleep and anesthesia. Vision Res. 1973;131035- 1040Article
24.
Crawford  KGabelt  BTKaufman  PLBito  LZ Effects of various anesthetic and autonomic drugs on refraction in monkeys. Curr Eye Res. 1990;9525- 532Article
25.
Törnqvist  G Comparative studies of the effect of pilocarpine on the pupil and on the refraction in two species of monkey (Cercopithecus ethiops and Macaca irus). Invest Ophthalmol Vis Sci. 1964;3388- 398
26.
Kaufman  PLBárány  EH Loss of acute pilocarpine effect on outflow facility following surgical disinsertion and retrodisplacement of the ciliary muscle from the scleral spur in the cynomolgus monkey. Invest Ophthalmol. 1976;15793- 807
27.
Nardin  GFZimmerman  TJ Ocular cholinergic agents. Ritch  RShields  MBKrupin  Teds.The Glaucomas, Glaucoma Therapy. 2nd ed. St Louis, Mo Mosby–Year Book Inc1996;1399- 1407
28.
Bárány  EH Dissociation of accommodation effects from outflow effects of pilocarpine. Paterson  GMiller  SJHPaterson  GDeds.Drug Mechanisms in Glaucoma The Gilston Glaucoma Symposium. Boston, Mass Little Brown & Co1966;275- 284
29.
Flügel  CBárány  EHLütjen-Drecoll  E Histochemical differences with the ciliary muscle and its function in accommodation. Exp Eye Res. 1990;50219- 226Article
30.
Citi  S Protein kinase inhibitors prevent junction dissociation induced by low extracellular calcium in MDCK epithelial cells. Journal Cell Biol. 1992;117169- 178Article
31.
Herbert  JMAugereau  JMGleye  JMaffrand  JP Chelerythrine is a potent and specific inhibitor of protein kinase C. Biochem Biophy Res Comm. 1990;172993- 999Article
32.
Birrell  GBHedberg  KKHabliston  DLGriffith  OH Protein kinase C inhibitor H-7 alters the actin cytoskeleton of cultured cells. J Cell Physiol. 1989;14174- 84Article
33.
Hall  A Ras-related proteins. Curr Opin Cell Biol. 1993;5265- 268Article
Laboratory Sciences
August 1998

Effects of H-7 on the Iris and Ciliary Muscle in Monkeys

Author Affiliations

From the Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison (Drs Tian, Millar, and Kaufman), the Wisconsin Regional Primate Research Center (Dr Kaufman), Madison; and the Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel (Drs Bershadsky, Becker, and Geiger). University of Wisconsin and Weizmann Institute of Science have filed a related patent application; accordingly, Drs Kaufman and Geiger have a proprietary interest. Alcon Laboratories, Inc, Fort Worth, Tex has paid a fee to University of Wisconsin and Weizmann Institute of Science for an option to license this technology and has provided some financial support in this general area. Dr Kaufman has served as a paid consultant on unrelated issues for Alcon Laboratories and several other pharmaceutical corporations.

Arch Ophthalmol. 1998;116(8):1070-1077. doi:10.1001/archopht.116.8.1070
Abstract

Objectives  To determine the effects of H-7 on (1) iris and ciliary muscles (CMs) in living monkeys; (2) isolated monkey CM strips; (3) actomyosin contractility in cultured Swiss 3T3 cells.

Methods  (1) Pupillary diameter (calipers) and accommodation (refractometer) in living monkeys were measured after topical, intracameral, or intravitreal administration of H-7 followed by systemic pilocarpine hydrochloride. (2) Pilocarpine-induced contraction of isolated monkey CM strips following administration of H-7 was measured in a perfusion chamber. (3) Actomyosin contractility in Swiss 3T3 cells cultured on thin silicone rubber film was determined by measuring cell-induced film wrinkles before and after administration of H-7.

Results  Topical H-7 prevented anesthesia-induced miosis but did not affect resting refraction. Intracameral or intravitreal H-7 dilated the pupil and inhibited miotic but not accommodative responses to pilocarpine. H-7 inhibited pilocarpine-induced contraction of isolated monkey CM strips and reduced Swiss 3T3 cell contraction.

Conclusions  H-7 inhibits actin-based contractility in non-muscle cells and in monkey iris sphincter and CM. Under our in vivo experimental conditions, the effect on the iris predominates over that on the CM.

SMOOTH MUSCLE contraction is associated with Ca++-dependent activation of myosin light chain kinase (MLCK) and consequent phosphorylation of myosin; MLCK activation is a primary factor increasing myosin light chain phosphorylation,1 leading from receptor activation to cellular contraction.24 H-7[1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine], a nonselective protein kinase inhibitor, reduces actomyosin-driven contractility in chick lens cells and fibroblasts, thus leading to deterioration of the actin microfilament system,2 and it inhibits endothelin 1–induced contraction of rabbit aortic 5 and airway smooth muscles.6 Since H-7 disrupts microfilament bundles with accompanying decreases in the incorporation of phosphorus 32 into phosphoproteins,7 and pretreatment with H-7 inhibits adenosine triphosphate–induced contraction of actin filaments,2 the H-7-induced reduction of actomyosin contractility is likely consequent to the decrease of phosphoproteins. However, the effects of the target kinase(s) for H-7 on the actin cytoskeleton, actomyosin contractility, and smooth muscle cell relaxation remain unclear.

H-7 increases aqueous humor outflow facility and reduces intraocular pressure in living monkeys, probably by affecting the trabecular meshwork cytoskeleton.8 Given potential therapeutic implications for glaucoma, and the effects of H-7 on cellular contractile systems, it seemed important to determine whether H-7 might interfere with contraction of the iris sphincter and ciliary muscles (CMs), which are responsible for miosis and accommodation respectively. We therefore determined the effects of H-7 on pupillary diameter and accommodation in the living monkey eye, and on contraction of isolated monkey CM strips in vitro. To gain insights at the cell biological level, we also studied the effects of H-7 on the contractility of cultured Swiss 3T3 cells.

MATERIALS AND METHODS
ANIMALS AND ANESTHESIA

Normal cynomolgus monkeys (Macaca fascicularis) weighing 2.1 to 4.3 kg were studied in accordance with University of Wisconsin-Madison, National Institutes of Health, and Association for Research in Vision and Ophthalmology guidelines. Anesthesia was induced by intramuscular ketamine (10 mg/kg), and maintained by intramuscular pentobarbital sodium (35 mg/kg).

DRUGS AND CHEMICALS

H-7, Medium 199 (containing glutamine), penicillin G-streptomycin solution, fetal bovine serum, and pilocarpine hydrochloride were obtained from Sigma Chemical Co, St Louis, Mo; dimethyl sulfoxide, from Research Industries Corporation, Salt Lake City, Utah; 2.5% phenylephrine hydrochloride (Mydfrin), from Alcon Laboratories, Inc, Fort Worth, Tex; and cyanoacrylate adhesive from Tri-Point Medical L.P., Raleigh, NC. H-7 solution for transcorneal anterior chamber (AC) injection (3 mmol/L) was freshly prepared in Bárány mock aqueous humor.9 For intravitreal injection, H-7 was formulated as 0.27 mg in 10 µL of 50% dimethyl sulfoxide (75 mmol/L) or 0.55 mg in 10 µL of 100% dimethyl sulfoxide (150 mmol/L). For topical application, H-7 was formulated as 1.1 mg in 20 µL of 25% dimethyl sulfoxide (150 mmol/L). Pilocarpine was dissolved in 0.1% citric acid buffer just before the experiments, so that intramuscular infusion of 3 mL delivered from 3 to 6 mg of pilocarpine hydrochloride (1.5 mg/kg of body weight).

PUPILLARY DIAMETER AND ACCOMMODATION MEASUREMENT IN LIVING MONKEYS

Accommodation (difference between baseline and postdrug refraction) was determined with a Hartinger (Zeiss-Jena, Jena, Germany) coincidence refractometer. Pupillary diameter was measured with vernier calipers under normal room light (350 lux).

Protocol 1

Baseline refraction and pupillary diameter were measured (average of 2-3 readings). Four 5-µL drops of 150-mmol/L H-7 or 25% dimethyl sulfoxide was administered to the central cornea of opposite eyes of supine monkeys at 30-second intervals, with blinking prevented between and for 1 minute after the last drops. Beginning 45 minutes later, refraction and pupillary diameter were measured every 15 minutes for 75 minutes (ie, until 2 hours after H-7 treatment). The H-7 dosage was chosen to give a 300-µmol/L H-7 concentration in the approximately 100-µL monkey AC,10 assuming 1% intracameral penetration and no drug loss from the AC.1113 A concentration of 300-µmol/L H-7 reduces actomyosin contractility and cytoskeletal cell junctional effects in cultured chick lens cells and fibroblasts,2 and it maximally increases outflow facility in living monkeys.8

Protocol 2

Baseline refraction and/or pupillary diameter were measured, followed by topical application of 2.5% phenylephrine (which stimulates the iris dilator muscle without influencing the iris sphincter and CM,14,15 thereby facilitating measurement of miosis and accommodation16). Refraction and/or pupillary diameter were measured again about 25 minutes later, after which 10 µL of 3-mmol/L H-7 was administered intracamerally (300 µmol/L in the approximately 100-µL AC), or 10 or 20 µL of 75- or 150-mmol/L (respectively) H-7 was administered intravitreally (300 µmol/L to 1.2 mmol/L in the approximately 2.5-mL vitreous17 ) to one eye and vehicle to the other. Refraction and pupillary diameter were determined 25 minutes after intracameral H-7 or 55 minutes after intravitreal H-7. Five minutes later, about 3 mL of pilocarpine hydrochloride solution was infused intramuscularly into the thigh (1.5 mg/kg) for from 10 to 15 minutes. Refraction was determined every 5 minutes after pilocarpine infusion until stable, and then final pupillary diameter was measured (Figure 1).

Anterior chamber injections were made under an operating microscope, using a 30-gauge needle connected via polyethylene tubing to a micrometer syringe (Gilmont Instruments, Barnant Co, Barrington, Ill). The needle was threaded through the corneal stroma for approximately 6 mm, then directed into the AC so that the wound was self-sealing. For intravitreal injections, the needle was inserted 4 mm through the temporal pars plana 4 mm posterior to the limbus.

CLINICAL EXAMINATION

Slitlamp examination and indirect ophthalmoscopy were conducted by an ophthalmologist (B.T. or P.L.K.). Corneal epithelial integrity, the presence or absence of AC flare or cells, and lens clarity were noted 2 hours after topical or intracameral administration of H-7 and of corresponding vehicle in the contralateral eye. Vitreous transparency, retinal color and structure, and the state of the anterior segment were noted 1 week and 1 month or longer after intravitreal H-7 injection.

CONTRACTION OF ISOLATED CM
CM Preparation

Globes were enucleated from 6- to 18-year-old rhesus monkeys (Macaca mulatta) that were under deep anesthesia with intravenous pentobarbital sodium (25 mg/kg) just before or within 5 minutes of death by pentobarbital overdose. The CM was dissected, strips mounted in a perfusion chamber, and contractile responses to drugs in the circular (coronal) and longitudinal (sagittal) vectors measured simultaneously.1820

H-7 Experiments

After equilibration, 10-µmol/L pilocarpine hydrochloride (just maximal for contraction) was perfused through the chamber for 15 minutes. H-7 was then added to the Krebs-pilocarpine mixture at successive concentrations (1 to 300 µmol/L), each perfusing for 15 minutes. After the final dose, the strip was perfused with plain Krebs solution for 90 minutes (to reestablish baseline tension), and then rechallenged with pilocarpine (10 µmol/L) for 15 minutes. The CM strip was then perfused with plain Krebs solution for another 60 minutes, and then again challenged with pilocarpine for 15 minutes. Responses to H-7 were expressed as absolute Δ force from resting tension and as percent average pilocarpine response for each CM strip, the latter normalizing the data for interstrip variations due to muscle dimensions, mass, and mounting.

CULTURED CELLS

To test the effect of H-7 on the mechanical forces exerted by substrate-attached cells, Swiss 3T3 cells were plated on a silicone rubber film.21

Silicone Rubber Substrates

Dimethyl polysiloxane (silicone rubber, 12000 centistokes) was aliquotted into Petri dishes, allowed to spread for several hours, and then coated with gold-palladium using an evaporator sputter coater (Edwards High Vacuum International, Wilmington, Mass)22 at settings of 3 kV and 15 mA for 3 minutes. The UV light from the sputter coater polymerized the top layer of the silicone rubber to form a thin film floating on the unpolymerized rubber. The gold-palladium reduced the film's hydrophobicity and facilitated cell adhesion.

Visualization and Analysis of Cell Contractility

Swiss 3T3 cells were grown for 48 hours on thin silicone rubber film in Dulbecco modified Eagle medium plus 10% newborn calf serum. Two days after plating, cell-induced wrinkles appeared on the film. The cells were then treated with 300-µmol/L H-7. Time-lapse video cinematography was performed using National Institutes of Health Image software, taking 1 frame every 30 seconds. Intensity vs x-coordinate profiles were taken for the cross section of each of 5 wrinkles at each time point. The amplitudes of the profiles were measured and normalized so that the amplitude at the zero time points of each wrinkle were equal to 1.0.

RESULTS
PUPILLARY MEASUREMENTS AND REFRACTION IN LIVING MONKEYS
Protocol 1

The slight time-dependent miosis that occurred under anesthesia with ketamine and pentobarbital was inhibited by 150-mmol/L (1.1 mg) topical H-7 (P<.05, Figure 2), but resting refraction was not affected (Figure 3).

Protocol 2
Pupillary Diameter

Twenty-five minutes after phenylephrine administration, both pupils dilated equally (to 7.17±0.28 mm vs 7.20±0.26 mm [mean±SEM], P =not significant (P<.6), Figure 4, B, Figure 5, A). Twenty-five minutes after AC injection of 10 µL of 3-mmol/L H-7 ([H-7]AC=300-µmol/L), the pupils in the H-7–treated eyes dilated further relative to the contralateral controls (to 7.88±0.19 mm vs 6.78±0.46 mm [mean±SEM], P <.05, Figure 4, C, Figure 5, A). When pilocarpine was infused intramuscularly into the thigh, the control pupils constricted but the H-7–treated pupils did not (to 4.80±0.20 mm in controls vs 7.25±0.13 mm [mean±SEM] in H-7–treated eyes, P<.001, Figure 4, D, Figure 5, A). H-7 also dilated the pupil and inhibited the miotic response to pilocarpine dependent on the dose after intravitreal injection of 10 or 20 µL of 75- or 150-mmol/L H-7, respectively (300-, 600- or 1200-µmol/L H-7 in the vitreous, Figure 5, B-D).

Accommodation

No significant differences between pilocarpine-induced accommodation in H-7–treated vs control eyes were observed at 300-µmol/L intracameral H-7 or at 300- or 600-µmol/L intravitreal H-7 (Figure 5, E-H). At 1.2-mmol/L intravitreal H-7, a physiologically minimal but statistically significant difference was observed only at 40 and 45 minutes after intramuscular administration of pilocarpine, with the H-7–treated eyes accommodating around 1 diopter (D) less than the controls (Figure 5, H).

CLINICAL EXAMINATION

Mild punctate corneal epithelial defects and slight epithelial cloudiness were seen in H-7–treated eyes compared with controls 2 hours after 150-mmol/L (1.1-mg) topical H-7 or vehicle. No AC, lens, or retinal abnormalities were seen at any time following topical, intracameral, or intravitreal administration of H-7 or vehicle.

CONTRACTION OF ISOLATED CM

Resting CM tension was 100 to 200 mg (data not shown). Pilocarpine hydrochloride at a 10-µmol/L concentration induced reproducible, stable contractions averaging 22 and 37 mg above baseline in the circular and longitudinal vectors, respectively (Figure 6, A, Figure 7, A). H-7 dependent on the dose relaxed the pilocarpine-precontracted CM strips, the maximum effect occurring at 100 to 300 µmol of H-7 per liter and averaging 15 and 25 mg (69% and 75% of the pilocarpine response, respectively) in the respective vectors (Figure 6, A and B; Figure 7, A). H-7 was equipotent in the 2 vectors, with the dose-response curves for percent inhibition of pilocarpine-induced contraction virtually superimposable. After restoring the CM strips to baseline tension for 90 minutes by perfusion with drug-free Krebs solution, perfusion with 10-µmol/L pilocarpine hydrochloride for 15 minutes produced no contraction (Figure 7, B). After additional perfusion with drug-free Krebs solution for 60 minutes, perfusion with 10-µmol/L pilocarpine hydrochloride then yielded a weak, sluggish contraction in both vectors (Figure 7,C).

CULTURED CELLS

Microscopic examination of Swiss 3T3 cells cultured on a silicone rubber film revealed extensive wrinkles in the silicone substrate prior to H-7 treatment (Figure 8, A). After incubation with 300-µmol/L H-7 for up to 5.5 minutes, wrinkle amplitude was decreased by approximately 90% (Figure 8, B and C), indicating reduced cellular contractility.

COMMENT

H-7 increases outflow facility and reduces intraocular pressure in monkeys and therefore may have potential as an anti-glaucoma medication.8 We determined the effects of H-7 on the contraction of intraocular smooth muscles in vivo and in vitro.

Topical H-7 prevented time-dependent miosis under anesthesia with ketamine and pentobarbital and further dilated the phenylephrine–treated pupil in living monkeys, indicating that H-7 interferes with contraction of the iris sphincter muscle. H-7 theoretically should inhibit contraction of both the iris sphincter and dilator muscles. Therefore, our data also suggest that the iris sphincter predominates in determining pupillary diameter under our experimental conditions. However, an in vitro dose-response comparison between the iris sphincter and dilator would be helpful to identify potentially different sensitivities to H-7.

Resting refraction in monkeys under pentobarbital anesthesia is −1 to −3 D,23,24 The mild myopia is sensitive to atropine sulfate, and probably represents tonic accommodation.24 Eyes receiving topical H-7 and their contralateral controls both fell within this range (−2.26 to −2.65 D in H-7–treated eyes vs −2.17 to −2.99 D in controls) and did not differ significantly, indicating that topical H-7 did not affect tonic CM contraction. Possible reasons may be (1) weakness of tonic contraction, making small changes difficult to detect; (2) inadequate drug concentration at the posteriorly situated CM after topical administration25 (see paragraph below); or (3) that H-7 does not penetrate or is ineffective in CM cells.

Intracameral and intravitreal H-7 inhibited pupillary but not accommodative responses to intramuscular pilocarpine, excluding reason 1 and militating against reason 2 in the preceding paragraph. The experiments on isolated CM strips were designed to determine whether the pupillary-accommodative dissociation had a biological or a pharmacokinetic basis. Dependent on the dose, H-7 both prevented and reversed pilocarpine-induced contraction of isolated CM strips. Furthermore, the same dose of H-7 that prevented the pupillary response to pilocarpine in vivo prevented and reversed contraction of CM strips induced by a maximal pilocarpine dose in vitro, the effect lasting for several hours after removal of H-7 from the medium. Thus, H-7 inhibits contraction of both the iris sphincter and CM, but the effect on the latter is negligible under our in vivo experimental conditions. This has potential therapeutic implications for preserving pilocarpine's effect on outflow facility (which requires CM contraction)26 while inhibiting its vision-reducing miotic effect, especially in elderly patients with incipient cataract.27

Pilocarpine administered topically or intracamerally in monkeys induced miosis at a 100-fold lower dose than accommodation, but no difference in sensitivity occurred with systemically administered pilocarpine, suggesting that the pilocarpine concentration at the CM was 100 times less than at the iris following topical or intracameral dosing.25 Such differing drug concentrations could account for the dissociation of pupillary-accommodative responses to pilocarpine following intracameral H-7 in our in vivo experiments. H-7 was administered intravitreally to afford possibly better access to the circular, putatively more accommodation-relevant, portion of the CM.28,29 However, the circular CM is separated from the vitreous by the ciliary epithelium, the highly vascular ciliary process stroma, and the dense connective tissue ground plate between the stroma and the muscle. The first and third of these might constitute barriers and the second an efflux route, all preventing the drug from reaching the circular CM. Aqueous humor flow might carry some drug away from the ciliary body and into the AC. This may explain why even 1.2-mmol/L intravitreal H-7 only minimally inhibited the accommodative response to pilocarpine. Drug diffusion toward the target tissues was probably not a limiting factor, since the pupillary response was inhibited by intravitreal H-7. Further studies, such as an in vitro dose-response comparison between the iris sphincter and CM to identify potentially different sensitivities to H-7, are needed to unequivocally exclude a biological basis for the pupillary-accommodative dissociation, but a pharmacokinetic explanation seems plausible.

Smooth muscle contraction is an actin-myosin-based mechanical process. Activation of protein kinase C (PKC) and MLCK elicit muscle contraction by increasing myosin light chain phosphorylation. However, the inositol triphosphate -Ca++-MLCK signaling system, rather than the diacylglycerol–PKC system plays the major role in smooth muscle contraction.1 H-7 is a broad-spectrum, serine-threonine kinase inhibitor, inhibiting PKA, PKG, and PKC and MLCK.30,31. Since (1) H-7 has a specific and rapid effect on the actin cytoskeleton of cultured Swiss 3T3 and PTK2 cells, but a potent PKC inhibitor (sangivamycin) and a specific PKA/PKG inhibitor (HA1004) do not32; and (2) H-7 and a selective MLCK inhibitor (KT5926) inhibit actomyosin-driven contractility in chick lens cells and fibroblasts that leads to deterioration of the actin microfilament system, but highly potent PKC inhibitors (Ro31-8220 and GF109203X) do not,2 H-7 may inhibit actomyosin-driven contraction primarily by inhibiting MLCK or other enzymes involved in cellular contractility such as rho-kinase.22,33 Furthermore, cultured Swiss 3T3 cells grown on a silicone rubber film substrate induce wrinkling in the film by their normal contractility, and this wrinkling was reduced dramatically following the addition of 300-µmol/L H-7. A similar effect on smooth muscle cell contractility could explain the inhibition by H-7 of pilocarpine-induced contraction of the iris sphincter and CM in our monkeys.

Further studies of this interesting compound, especially with regard to ocular safety, seem warranted.

Back to top
Article Information

Accepted for publication April 17, 1998.

This study was supported by grants EY 02698 (Dr Kaufman) and RR00167 (Wisconsin Regional Primate Research Center [WPRC publication 37-044] [Madison]) from the National Institutes of Health, Bethesda, Md; by Catalyst Program Award (Drs Kaufman and Geiger) from the Glaucoma Research Foundation, San Francisco, Calif; by Senior Scientific Investigator Award (Dr Kaufman) from Research to Prevent Blindness Inc, New York, NY; by fellowship awards (Drs Tian and Millar) from the Ocular Physiology Research & Education Foundation, Madison; and by Yeda Ltd, the Neter Professorship for Cell and Tumor Biology, the Belilos Foundation, and the Rita Markus Foundation, Weizmann Institute of Science, Rehovot, Israel (Dr Geiger).

We thank William Hubbard, BS, for technical assistance.

Reprints: Paul L. Kaufman, MD, Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, F4/328 CSC, 600 Highland Ave, Madison, WI 53792-3220 (e-mail: kaufmanp@macc.wisc.edu).

References
1.
Yousufzai  SYKChen  ALAbdel-Latif  AA Species differences in the effects of prostaglandins on inositol triphosphate accumulation, phosphatidic acid formation, myosin light chain phosphorylation and contraction in iris sphincter of the mammalian eye: interaction with the cyclic AMP system. J Pharmacol Exp Ther. 1988;2471064- 1072
2.
Volberg  TGeiger  BCiti  SBershadsky  AD Effect of protein kinase inhibitor H-7 on the contractility, integrity, and membrane anchorage of the microfilament system. Cell Motil Cytoskeleton. 1994;29321- 338Article
3.
Kamm  KEStull  JT The function of myosin and myosin light chain kinase phosphorylation in smooth muscle. Annu Rev Pharmacol Toxicol. 1985;25593- 620Article
4.
Sommerville  LEHartshorne  DJ Intracellular calcium and smooth muscle contraction. Cell Calcium. 1986;7353- 364Article
5.
Sugiura  MInagami  THare  GMTJohns  JA Endothelin action: inhibition by a protein kinase C inhibitor and involvement of phosphoinositols. Biochem Biophys Res Commun. 1989;158170- 176Article
6.
Grunstein  MMChuang  STSchramm  CMPawlowski  NA Role of endothelin 1 in regulating rabbit airway contractility. AM J Physiol. 1991;260 ((4)) L75- L82
7.
Mobley  PLHedberg  KBonin  LChen  BGriffith  OH Decreased phosphorylation of four 20-kDa proteins precedes staurosporine-induced disruption of the actin/myosin cytoskeleton in rat astrocytes. Exp Cell Res. 1994;21455- 66Article
8.
Tian  BKaufman  PLVolberg  TGabelt  BTGeiger  B H-7 disrupts the actin cytoskeleton and increases outflow facility. Arch Ophthalmol. 1998;16633- 643Article
9.
Bárány  EH Simultaneous measurement of changing intraocular pressure and outflow facility in the vervet monkey by constant pressure infusion. Invest Ophthalmol. 1964;3135- 43
10.
Erickson-Lamy  KAKaufman  PLMcDermott  MLFrance  NK Comparative anesthetic effects of aqueous humor dynamics in the cynomolgus monkeys. Arch Ophthalmol. 1984;1021815- 1820Article
11.
Asseff  CFWeisman  RLPodos  SMBecker  B Ocular penetration of pilocarpine in primates. Am J Ophthalmol. 1973;75212- 215
12.
Harris  JE Problems in drug penetration. Leopoid  IHed.Symposium on Ocular Therapy. Vol 3. St Louis, Mo CV Mosby1968;96- 105
13.
Janes  RGStiles  JF The penetration of C14-labeled atropine into the eye: a comparison of methods of application. Arch Ophthalmol. 1959;6269- 74Article
14.
Kaufman  PL Accommodation and presbyopia: neuromuscular and biophysical aspects. Hart  WM  Jred.Adler's Physiology of the Eye. 9th ed. St Louis, Mo Mosby–Year Book Inc1992;391- 411
15.
Thompson  HS The pupil. Hart  WM  Jred.Adler's Physiology of the Eye. 9th ed. St Louis, Mo Mosby–Year Book Inc1992;412- 441
16.
Bito  LZDeRousseau  CJKaufman  PLBito  JW Age-dependent loss of accommodative amplitude in rhesus monkeys: an animal model for presbyopia. Invest Ophthalmol Vis Sci. 1982;2323- 31
17.
Kaufman  PLCalkins  BTErickson  KA Ocular biometry of the cynomolgus monkey. Curr Eye Res. 1981;1307- 309Article
18.
Poyer  JFKaufman  PLFlügel  C Age does not affect contractile responses of the isolated rhesus monkey ciliary muscle to muscarinic agonists. Curr Eye Res. 1993;12413- 422Article
19.
Poyer  JFMillar  CKaufman  PL Prostaglandin F2α effects on isolated rhesus monkey ciliary muscle. Invest Ophthalmol Vis Sci. 1995;362461- 2465
20.
Millar  CPoyer  JFGabelt  BTKaufman  PL Endothelin subtypes: effect on isolated rhesus monkey ciliary muscle. J Pharmacol Exp Ther. 1995;2751143- 1147
21.
Harris  AKWild  PStopak  D Silicone rubber substrata: a new wrinkle in the study of cell locomotion. Science. 1980;208177- 179Article
22.
Chrzanowska-Wodnicka  MBurridge  K Rho-stimulated contractility drives the formation of stress fibers and focal adhesions. J Cell Biol. 1996;1331403- 1415Article
23.
Westheimer  GBlair  SM Accommodation of the eye during sleep and anesthesia. Vision Res. 1973;131035- 1040Article
24.
Crawford  KGabelt  BTKaufman  PLBito  LZ Effects of various anesthetic and autonomic drugs on refraction in monkeys. Curr Eye Res. 1990;9525- 532Article
25.
Törnqvist  G Comparative studies of the effect of pilocarpine on the pupil and on the refraction in two species of monkey (Cercopithecus ethiops and Macaca irus). Invest Ophthalmol Vis Sci. 1964;3388- 398
26.
Kaufman  PLBárány  EH Loss of acute pilocarpine effect on outflow facility following surgical disinsertion and retrodisplacement of the ciliary muscle from the scleral spur in the cynomolgus monkey. Invest Ophthalmol. 1976;15793- 807
27.
Nardin  GFZimmerman  TJ Ocular cholinergic agents. Ritch  RShields  MBKrupin  Teds.The Glaucomas, Glaucoma Therapy. 2nd ed. St Louis, Mo Mosby–Year Book Inc1996;1399- 1407
28.
Bárány  EH Dissociation of accommodation effects from outflow effects of pilocarpine. Paterson  GMiller  SJHPaterson  GDeds.Drug Mechanisms in Glaucoma The Gilston Glaucoma Symposium. Boston, Mass Little Brown & Co1966;275- 284
29.
Flügel  CBárány  EHLütjen-Drecoll  E Histochemical differences with the ciliary muscle and its function in accommodation. Exp Eye Res. 1990;50219- 226Article
30.
Citi  S Protein kinase inhibitors prevent junction dissociation induced by low extracellular calcium in MDCK epithelial cells. Journal Cell Biol. 1992;117169- 178Article
31.
Herbert  JMAugereau  JMGleye  JMaffrand  JP Chelerythrine is a potent and specific inhibitor of protein kinase C. Biochem Biophy Res Comm. 1990;172993- 999Article
32.
Birrell  GBHedberg  KKHabliston  DLGriffith  OH Protein kinase C inhibitor H-7 alters the actin cytoskeleton of cultured cells. J Cell Physiol. 1989;14174- 84Article
33.
Hall  A Ras-related proteins. Curr Opin Cell Biol. 1993;5265- 268Article
×