Investigative Ophthalmology & Visual Science, Vol. 30, No. 10, October 1989 Copyright Association for Research in Vision and Ophthalmology

Investigative Ophthalmology & Visual Science, Vol. 30, No. 10, October 1989 Copyright Association for Research in Vision and Ophthalmology Activation of Beta-Adrenergic Receptors Causes Stimulation of Cyclic AMP, Inhibition of Inositol Trisphosphate, and Relaxation of Bovine Iris Sphincter Smooth Muscle Biochemical and Functional Interactions between the Cyclic AMP and Calcium Signalling Systems Souvenir D. Tachado, Rashid A. Akhrar, and Ara A. Abdel-Larif We have investigated the interactions between the camp and inositol 1,4,5-trisphosphate (IP 3 )-Ca 2+ signalling systems in the bovine iris sphincter by measuring the effects of /3-adrenergic and cholinergic muscarinic agonists and antagonists on camp formation, IP 3 accumulation and muscle contractionrelaxation. (1) Addition of 5 nm isoproterenol (ISO) or forskolin (5 ym) consistently produced stimulation of camp (540%), inhibition of IP 3 (34%) and complete relaxation of the muscle. The ISO effects were dose-dependent, with ECso values for camp formation, IP 3 inhibition and muscle relaxation of 2.8 X 10~ 7 M, 3.4 X 10~ 7 M and 0.45 X 10" 7 M, respectively. (2) Timolol, a /?-adrenergic antagonist, inhibited the ISO effects in a dose-dependent manner, with IC50 values for camp formation, IP 3 accumulation and muscle relaxation of 1.8 X 10~ 5 M, 3.2 X 10" s M and 2.2 X 10" 5 M, respectively. (3) The effects of ISO (0.5 nm) were time-dependent, and they clearly indicate a temporal relationship between the agonist-induced stimulation of camp, inhibition of IP 3, and relaxation of the muscle. Within 15 sec following the addition of ISO, there was a marked increase in the level of camp, a decrease in IP 3, and this was accompanied by an equally rapid relaxation of the muscle. (4) Addition of carbachol (CCh) to iris sphincter pretreated with ISO decreased camp formation and reversed muscle relaxation to complete contraction. Thus, when the sphincter was first treated with ISO and then stimulated with different concentrations of CCh, there was a dose-dependent inhibition of camp formation, an increased production of IP 3, and a parallel development of muscle contraction. Taken together, the data presented suggest a reciprocal interaction between the camp and IP 3 -Ca 2+ signalling systems in the iris sphincter: activation of /?-adrenergic receptors results in elevation of camp and inhibition of IP 3, and this leads to muscle relaxation, whereas stimulation of cholinergic muscarinic receptors lowers camp formation and increases IP 3 production, and this leads to Ca 2+ mobilization and muscle contraction. We propose that in the iris sphincter camp may act as regulator of responses to neurotransmitters, hormones and pharmacological agents that exert their action through the IP 3 -Ca 2+ second messenger system. Invest Ophthalmol Vis Sci 30:2232-2239,1989 There is now compelling experimental evidence that suggests that most tissues use at least two major signalling pathways for controlling cellular functions From the Department of Cell and Molecular Biology, Medical College of Georgia, Augusta, Georgia. Supported by grants from the National Institutes of Health, EY-04171 andey-04387. This is contribution number 1163 from the Department of Cell and Molecular Biology, Medical College of Georgia. Submitted for publication: December 27, 1988; accepted April 21, 1989. Reprint requests: Dr. Ata A. Abdel-Latif, Department of Cell and Molecular Biology, Medical College of Georgia, Augusta, GA 30912-2100. and proliferation. The A-kinase pathway is coupled to camp, while the Ca 2+ messenger system [Ca 2+ - calmodulin and protein kinase C (PKC)] is coupled to the breakdown of polyphosphoinositides (for reviews, see refs. 1, 2). In the former, the extracellular signal through its receptor at the plasma membrane activates adenylate cyclase which generates intracellular camp. In the latter, the receptor-activated phospholipase C leads to the release, from phosphatidylinositol 4,5-bisphosphate (PIP 2 *), of two putative * Abbreviations used are: PIP2, phosphatidylinositol 4,5-bisphosphate; DG, 1,2-diacylglycerol; IP 3, inositol 1,4,5-trisphosphate; ISO, isoproterenol; CCh, carbachol; MLC, myosin light chain, PKC, protein kinase C. 2232

No. 10 INTERACTIONS BETWEEN THE camp AND Ca 2+ SYSTEMS / Tachado er ol 2233 second messengers, 1,2-diacylglycerol (DG) and inositol 1,4,5-trisphosphate (IP 3 ). DG activates PKC and IP 3 induces Ca 2+ release from the endoplasmic reticulum. Rather than serving as distinctly separate second messenger systems, the two pathways can participate in a synergistic manner to regulate cell function. Thus, in platelets camp has a marked inhibitory effect on phosphoinositide breakdown. 1 Reciprocal effects were also reported for some phorbol esters, which are considered DG analogs, on the activity of receptors linked to camp. 3 ' 4 The mammalian iris sphincter is an ideal model for studies on the biochemical and functional interactions between the two second messenger systems. It is innervated by cholinergic nerve fibers, activation of which leads to muscle contraction and miosis. 5 Histological investigations have revealed that the iris sphincter muscle is innervated by both adrenergic and cholinergic neurons in the monkey, 6 rabbit 7 and man. 8 Physiological investigations have shown that the iris sphincter is functionally innervated by adrenergic nerve fibers in the dog, 9 monkey, 10 cattle, 1112 rat 13 and rabbit. 1014 In the human iris sphincter muscle, exogenously applied epinephrine or norepinephrine relaxes the tissue. 1516 In addition, the human iris sphincter muscle possesses both a and 0 adrenoceptors. 17 Considerable evidence suggests that the adrenergic nervous system plays a significant but complex role in the regulation of intraocular pressure (IOP) in the eye. 1819 Both sympathetic stimulation and locally applied /3-adrenergic agonists such as epinephrine decrease IOP. Paradoxically, /3-adrenergic antagonists such as timolol (which is effective in treating glaucoma) also decrease IOP. In previous communications from this laboratory we have established that in the iris smooth muscle the stimulated-hydrolysis of PIP 2 into DG and IP 3 by cholinergic muscarinic and a 1-adrenergic agonists is an early event which couples activated receptors to smooth muscle contraction 20-21 (for review, see ref. 22). Activation of cholinergic muscarinic receptors in rabbit ciliary processes also leads to inhibition of camp formation. 23 More recently, comparative studies on the effects of prostaglandins on DG and IP 3 production, camp formation, myosin light chain (MLC) phosphorylation and contraction in the iris sphincter smooth muscle of rabbit, bovine and other species revealed biochemical and functional interactions between the IP 3 -Ca 2+ and camp second messenger systems. 24 The overall objective of the present work was to investigate whether an interaction exists between receptor-mediated camp production, IP 3 accumulation and contraction-relaxation response of the bovine iris sphincter. In the present study we report on the reciprocal effects of isoproterenol (ISO) and other /3-adrenergic agents and carbachol (CCh) on IP 3 accumulation, camp formation and relaxation-contraction responses in the bovine iris sphincter. Materials and Methods Materials The following compounds were purchased from Sigma Chemical Co. (St. Louis, MO): Carbamylcholine chloride (carbachol), indomethacin, L-isoproterenol-HCl, propranolol, timolol, and 3-isobutyl-lmethylxanthine (IBMX). Sources for other reagents were as follows: Antibody to cyclic AMP from ICN ImmunoBiologicals (Lisle, IL); reagents for radioimmunoassay (RIA) of cyclic AMP including succinyl (methyl ester- 125 I) cyclic AMP tyrosine (2200 Ci/ mmol) from New England Nuclear (Boston, MA); myo-[ 3 H]inositol (15 Ci/mmol) from American Radiolabeled Chemical Inc. (St. Louis, MO). Incubation of the Sphincter Muscle with Myo-[ 3 H]inositol Bovine eyes were obtained from a local slaughterhouse, usually within 30 min after the death of the animals. After removal of the cornea, two sphincter muscle strips were prepared from each eye by making a radial cut about 3 mm from the pupillary margin at 3 o'clock and 9 o'clock positions and then cutting completely around the pupil on a line parallel to the pupillary margin. The paired sphincter strips from each eye were incubated at 37 C for 90 min in 1 ml of Krebs-Ringer bicarbonate buffer that contained 10 j*ci of 3 H-inositol/ml. 21 The ph of the buffer was adjusted to 7.4 with 97% O 2-3% CO 2. At the end of incubation, the sphincters were washed four times with 3 ml nonradioactive buffer and then suspended singly (of the paired strips, one was used as a control and the other as experimental) in 1 ml fresh nonradioactive buffer. At this time LiCl (10 mm final concentration) and IBMX (0.1 mm) were added to each incubation and 10 min later ISO or other pharmacological agents were added and incubations were continued for appropriate time intervals as indicated. Antagonists, when used, were added 5 min prior to the addition of the agonists. Incubations were terminated by the addition of 1 ml 10% (w/v) trichloroacetic acid (TCA), then homogenized in the same medium and 3 H-IP 3 and camp in the TCA extract analyzed as given below. Extraction and Analysis of 3 H-Inositol Phosphates The method used to extract and analyze 3 H-inositol phosphates was the same as described previously. 21 Briefly, the tissues were homogenized in the 5% TCA-containing medium, the homogenate

2234 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / October 1989 Vol. 30 centrifuged at 3000g for 15 min, and the supernatant transferred to new tubes. The supernatant was extracted five times with 4 ml of anhydrous diethyl ether. At this time suitable aliquots of the extract were taken for camp determination (see below), the remainder was first neutralized with NaOH and then analyzed for 3 H-inositol phosphates by means of anion-exchange chromatography employing Biorad 1 X 8 resin (formate form, 200-400 mesh) as previously described. 21 The TCA-insoluble pellet was solubilized in 1 ml 0.1 N NaOH and suitable aliquots used for determination of protein by the method of Lowery et al. 25 Measurement of Cyclic AMP Measurement of camp was routinely performed in the same incubations used for the determination of 3 H-inositol phosphates. To protect camp from hydrolysis 0.1 mm IBMX, a camp phosphodiesterase inhibitor, was routinely included in the incubation mixture. This concentration of IBMX had no significant effect on 3 H-inositol phosphates formation. After appropriate dilution of the supernatant from the previous step, camp in the sample was succinylated and then assayed by RIA as described by Frandsen and Krishna. 26 Measurement of Agonist-Induced Tension Response in the Iris Sphincter For measurement of tension response, the two sphincter strips from the same iris were mounted in two separate 30 ml jacketed tissue baths that contained Krebs-Ringer bicarbonate buffer at 37 C. A mixture of O 2 (97%) and CO 2 (3%) was continuously bubbled through the solution. The tissue was allowed to equilibrate for 90 min under a resting tension of 300 mg. Shortly after mounting, the tissue started developing force which continued until about 1 hr, and thereafter the tissue did not develop any further tension. During the equilibration period the physiologic solution was changed every 20 min. At the end of equilibration, ISO or other drugs were added and the mechanical responses were monitored continuously using a Grass FT-03 force transducer connected to a Grass d.c. preamplifier. Dose-response curves for mechanical responses were constructed by cumulative addition of the agonist in the tissue bath. The concentration of the agonist was increased only after the effect of the previous concentration had stabilized. EC 5 o value is defined as that concentration of the agonist that produces 50% of the maximum response. In experiments where the effect of CCh on muscle contraction was investigated, the muscles were equilibrated as described above except that 1 ^M indomethacin was included in the equilibration buffer and the resting tension during equilibration period was 50 mg. The bovine sphincter, unlike the rabbit sphincter, develops inherent tone probably due to endogenous prostaglandin synthesis. Because of this the bovine sphincter does not respond maximally to cholinergic stimulation unless the tissue is pretreated with indomethacin. To inhibit the endogenous formation of prostaglandins, 1 ixm indomethacin was routinely added to the tissue baths. Calculations and Statistical Analysis of the Data To correct for variation in tissue size the data for 3 H-inositol phosphates, camp and tension responses were normalized to tissue protein content. Data are expressed as the mean ± SE. Statistical differences between the two means were determined by a paired student's t-test. When P was <0.05, the values were considered to be significantly different. Results Effects of /?-Adrenergic and Cholinergic Muscarinic Agonists on camp Formation, IP 3 Accumulation and Muscle Tension in the Bovine Iris Sphincter In the following studies we used IP 3 accumulation as an index of stimulated PIP 2 hydrolysis by phospholipase C, and camp formation as an index of adenylate cyclase activity. As shown in Table 1, addition of ISO or forskolin to iris sphincter resulted in a 5- to 6-fold increase in camp formation. These effects were blocked by the 0-adrenergic antagonists timolol and propranolol. Addition of the antagonists Table 1. Effects of 0-adrenergic agonists and antagonists on camp formation, IP 3 accumulation and muscle tension in the bovine iris sphincter* Addition None ISO (5 nm) Forskolin (5 j^m) Timolol (50 pm) ISO (5 MM) + Timolol (50 fim) Propranolol (50 ^M) ISO (5 MM) + Propranolol (50 nm) CCh (50 MM) camp (pmol/mg protein) 14 ± 1 90 ±8 98 ±4 13± 1 16±2 12± 1 17±3 17±2 IPi (dpm/mg protein) 3922 ± 254 2686 ± 168 2714 ± 108 3020 ± 179 3961 ±234 2980 ± 147 3804 ± 151 9471 ±803 Tension} (mg/mg wet tissue wt) 24.8 ± 0.4 3.0 ±0.1 1.6 ±0.1 28.5 ± 0.05 19.9 ± 1.3 27.2 ± 0.7 15.6 ± 1.0 26.5 ± 0.8* * The data are means ± SE of three experiments, each conducted in triplicate (N = 9). (For details of experiment, see Materials and Methods.) t The concentrations of ISO and CCh used in measurement of the pharmacological response were 1 fim. $ The basal tension in the absence of carbachol was 4.4 mg/mg wet weight.

No. 10 INTERACTIONS BETWEEN THE camp AND Co 2+ SYSTEMS / Tochodo er ol 2235 alone had no effect on the basal formation of camp. In contrast to their effects on camp, both ISO and forskolin inhibited IP 3 accumulation by about 31% of that of the control, and these effects were reversed by the 0-adrenergic antagonists. Interestingly, both timolol and propranolol significantly depressed (P < 0.01) IP 3 formation by about 23% of that of the control. Parallel studies on the effect of ISO and forskolin on iris sphincter, preequilibrated for 90 min under the same experimental conditions employed for the biochemical studies resulted in a complete relaxation of the muscle (Table 1, Fig. 1). As with the biochemical findings, the /3-adrenergic antagonists inhibited the agonist-induced muscle relaxation, timolol being more potent than propranolol. In contrast to the effects of the 0-adrenergic agonists, CCh, a rnuscarinic agonist, elicited a 140% increase in IP 3 accumulation and this was accompanied by a large increase in the contractile response (Fig. 1). The contractile response consisted of a rapid (30 sec) phasic component and this was followed by a slightly lower tonic response which lasted for several minutes. CCh had no effect on the basal level of tissue camp. Dose-Response Effect of ISO on camp Formation, IP 3 Accumulation and Muscle Relaxation in Bovine Iris Sphincter 75 mg 1 min 75 mg 1 min Phasic (30 s) CCh (1 MM) ISO (1 (60() Fig. 1. Representative recordings of mechanical responses of bovine iris sphincter muscle to CCh (1 MM) and ISO (1 nm). The experimental details were the same as described in the legend to Table 1 and in Methods. Muscle Relaxation Dose-response studies on the effects of ISO on the biochemical and physiological responses showed that the /3-adrenergic agonist dose-dependently increased the formation of camp with a maximal effect obtained at 1 /um of the agonist (Fig. 2). Under the same experimental conditions ISO inhibited IP 3 accumulation in a dose-dependent manner. Increasing concamp Accumulation Log (Isoproterenol), M Fig. 2. Dose-response effect of ISO on camp formation, IP 3 accumulation and muscle relaxation in bovine iris sphincter. All experimental conditions were the same as described in Table 1, except that the agonist concentration was varied as indicated. The results are means of two experiments each done in triplicate. centrations of ISO resulted in increased relaxation of the muscle and a maximal relaxant effect of the agonist was observed at 0.2 fxm. The finding that the dose-relaxation curve for ISO lies to the left of those for camp and IP 3 could suggest the presence of spare receptors for eliciting the pharmacological response. The EC 5{) values were: 2.8 X 10" 7 M for camp formation, 3.4 X 10~ 7 M for IP 3 inhibition and 4.5 X 10" 8 M for muscle relaxation. Effect of Timolol on ISO-Induced camp Formation, IP 3 Accumulation and Muscle Relaxation in Bovine Iris Sphincter Timolol is a potent /3-adrenergic antagonist and is widely used as an antiglaucoma agent. However, the mechanism of its hypotensive effect is still unclear. Thus, it was of interest to investigate its effect on ISO-induced camp formation, IP 3 accumulation and muscle relaxation in the iris sphincter. As shown in Figure 3, the biochemical and pharmacological responses induced by ISO (0.5 nm) were dose-dependently inhibited by timolol. The IC 50 values for camp formation, IP 3 accumulation and muscle relaxation were 1.8 X 10" 5 M, 3.2 X 10" 5 M, and 2.2 X 10~ 5 M, respectively. Time Course of the Effects of ISO on camp Formation, IP 3 Accumulation and Muscle Relaxation in Bovine Iris Sphincter In these experiments we have investigated the temporal relationships between ISO-induced camp formation, inhibition of IP 3 accumulation and muscle relaxation. Upon addition of ISO (0.5 nm) the level of intracellular camp was increased by 40% within 15 sec, and the maximal effect was observed at about 5 min (Fig. 4). In contrast to its stimulatory effect on camp formation, ISO inhibited IP 3 accumulation in

2236 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / October 1989 Vol. 30 100-1 40 60 Timolol 100 1.0 1.5 Time (Minutes) Fig. 3. Effects of timolol on ISO-induced camp formation, IP 3 accumulation and muscle relaxation in bovine iris sphincter. All experimental details were the same as described in Table 1, except that different concentrations of timolol were added 5 min prior to the addition of ISO (0.5 /xm). The incubation time with the agonist was 10 min. The data are means of three separate experiments. Fig. 4. Time course of the effects of ISO (0.5 i*m) on camp formation, IP 3 accumulation and muscle relaxation in bovine iris sphincter. Experimental details were the same as described in Table 1, except that incubations were carried out at various time intervals as indicated. The results are means of three to six separate experiments. a time-dependent manner. Inhibition of IP 3 accumulation by ISO was evident after 30 sec of incubation and reached maximal (30% inhibition) at about 5 min (Fig. 4). In general the time course profile for ISO-induced muscle relaxation parallels that of camp formation. About 50% of muscle relaxation by the agonist is achieved within 15 sec and it is complete within 2 min. These data clearly demonstrate a temporal relationship between the ISO-induced stimulation of camp, inhibition of IP 3 and relaxation of the bovine iris sphincter. Discussion The data presented suggest a reciprocal relationship between the two second messenger systems in the bovine iris sphincter: activation of muscarinic re- Reversal by CCh of ISO-Induced camp Formation, IP 3 Inhibition and Muscle Relaxation in the Bovine Iris Sphincter Further support for interaction between the camp and IP 3 -Ca 2+ signalling systems came from studies on the effects of CCh on ISO-induced stimulation of camp, inhibition of IP 3 and relaxation of the iris sphincter (Fig. 5). In this experiment the sphincter strips were pretreated with ISO (0.5 \im) prior to the addition of various concentrations of CCh. Under these experimental conditions the muscle: (1) is relaxed; (2) has an elevated level of camp (540% increase); and (3) has a reduced level of IP 3 (30% decrease). Addition of CCh reversed the three responses which resulted in: (1) a decrease in camp formation, with maximal effect (40% inhibition) obtained at 20 nm CCh; (2) a dose-dependent increase in IP 3 accumulation; and (3) a dose-dependent reversal of muscle relaxation to maximal contraction obtained at 50 CCh. Fig. 5. Reversal of ISO-induced camp formation, IP 3 inhibition and muscle relaxation by various concentrations of CCh in bovine iris sphincter. The sphincter muscles were prelabeled with myo- [ 3 H]inositol and suspended in 1 ml of Krebs-Ringer bicarbonate buffer (ph 7.4) containing 10 mm LiCl and 0.1 mm IBMX as described in Table 1. After 10 min of incubation, 0.5 nm ISO was added to the tissues followed by the addition of different concentrations of CCh and the incubations were continued for 10 min. The reactions were terminated with 10% (w/v) TCA and inositol phosphates and camp were analyzed as described in Methods. For the relaxation studies, the sphincter muscle was equilibrated for 90 min under a resting tension of 300 mg. At this time 0.5 /im ISO was added and muscle relaxation recorded. After the muscle had relaxed, different concentrations of CCh were added to the organ bath and changes in isometric tension recorded. The results are means of two separate experiments each conducted in triplicate.

No. 10 INTERACTIONS BETWEEN THE camp AND Co 2+ SYSTEMS / Tochodo er ol 2237 ceptors induces IP 3 accumulation, Ca 2+ mobilization and smooth muscle contraction, whereas stimulation of 0-adrenergic receptors results in elevation of camp production, inhibition of IP 3 accumulation and muscle relaxation. This conclusion is supported by the following findings: (1) Addition of ISO or forskolin to the sphincter muscle increased camp formation, inhibited IP 3 accumulation, and caused muscle relaxation. These effects of the 0-agonist were dose-dependent (Figs. 2, 4) and were blocked by the /^-antagonists, propranolol and timolol (Table 1). The observation that the dose-response curve for relaxation (EC 50 = 0.045 MM) was shifted to the left of those for IP 3 inhibition (EC 50 = 0.34 /xm) and camp production (EC 50 = 0.28 nm) could be attributed to the presence of spare receptors in this tissue. Lack of receptor reserve for agonist-mediated increase in inositol phosphate accumulation has been reported in several types of smooth muscle including rabbit aorta, 27 ' 28 guinea pig visceral longitudinal smooth muscle 29 and rabbit iris dilator. 30 A possible receptor reserve for a,-adrenoreceptor-mediated [ 3 H]inositol phosphate accumulation has been reported in rat vas deferens. 31 In the present study it is also possible that other events may be responsible for relaxation at lower concentrations of ISO and that camp and IP 3 effects are more important at higher concentrations of the j8-agonist (0.5-5 jim). (2) The effects of ISO on the biochemical and physiological responses were time dependent, and they clearly demonstrated a temporal relationship between the ISO-induced stimulation of camp, inhibition of IP 3, and relaxation of the bovine iris sphincter (Fig. 4). (3) Addition of CCh to iris sphincter pretreated with ISO decreased camp formation, with maximal effect (40% inhibition) obtained at 20 /xm of the muscarinic agonist, induced a dose-dependent increase in IP 3 accumulation, and caused a dose-dependent reversal of muscle relaxation (Fig. 5). These data show that in the iris sphincter, as with other ocular tissues, 23 muscarinic receptors are negatively coupled to adenylate cyclase, and that this inhibition is significant only when adenylate cyclase is activated by js-adrenergic agonists. Further, as with other tissues, 32 CCh alone produced little if any significant effect on basal camp metabolism, although it did inhibit adrenergic stimulation (Fig. 5). In light of these and previous findings on the iris sphincter, 2-2224 we propose the following scheme on possible interactions between the IP 3 -Ca 2+ -DG and cyclic AMP signalling systems in the iris sphincter (Fig. 6). Over the past 2 years, the importance of the interaction between the two signalling Fig. 6. Scheme showing the possible roles of the two second messengers, IP 3 - Ca 2+ -DG and camp, in mediating the action of 0- agonists and Ca 2+ -mobilizing agonists on the tension response in the iris sphincter smooth muscle. + and - indicate stimulation and inhibition, respectively. Abbreviations used: PG, prostaglandin; CCh, carbachol; NE, norepinephrine; subst. P, substance P; CaM, calmodulin; PS, phosphatidylserine; PIP2, phosphatidylinositol 4,5- bisphosphate; IP 3, inositol 1,4,5-trisphosphate; DG, 1,2-diacylglycerol; PKC, protein kinase C; G-protein, GTP-binding protein; MLC, myosin light chain. Plasma _ Membrane" PKC /3-Adrenergic Agonist or PG I Receptor i G-Protein 4 Adenylatecyclase ATP CAMP + PPj I ^ camp-dependent Protein Kinase Relaxation Ca 2 + -Mobilizing Agonist (e.g. CCh, NE, Subst. P, PG) 1 Receptor 4 G-Protein I Phospholipase C PIP 2 DG (Ca 2+, PS) PKC P + Actin-»Contraction MLC Phosphatase

2238 INVESTIGATIVE OPHTHALMOLOGY G VISUAL SCIENCE / Ocrober 1989 Vol. 30 systems in the regulation of Ca 2+ -dependent functions, such as smooth muscle contraction and secretion, has been well documented. In smooth muscle (Fig. 6), activation of Ca 2+ -mobilizing receptors leads to the generation of IP 3 and DG. The increase in intracellular Ca 2+ leads to activation of MLC kinase, phosphorylation of MLC and consequently phasic contraction. This lasts for about 30 sec in the iris sphincter (Fig. 1). DG activates PKC to: (1) Stimulate MLC phosphorylation and consequently tonic contraction; (2) feedback-inhibit phospholipase C activity; and (3) stimulate adenylate cyclase to raise camp production and consequently inhibit phospholipase C and MLC kinase activities. The tonic response lasts for several minutes in the iris sphincter (Fig. 1). Furthermore, the iris muscle is highly enriched in the enzyme PKC. 33 Recently, Mittag et al 34 reported that intravitreal injection of 50 pmoles of phorbol ester into rabbit eyes produced approximately a 40% decrease of intraocular pressure relative to the control eye, lasting for more than 72-hr. Very recently, we demonstrated that ISO activates MLC phosphatase in the iris sphincter to release Pi (S. D. Tachado, R. A. Akhtar and A. A. Abdel-Latif, unpublished data). This could underlie the mechanism of relaxation in this smooth muscle (Fig. 6). Timolol depressed basal IP 3 formation by 23% (Table 1), and both biochemical and pharmacological responses induced by ISO were dose-dependently inhibited by the drug (Fig. 3). More recently, we found that timolol (10 nm) inhibited IP 3 formation in bovine ciliary processes by 47% after 1 min of incubation (S. D. Tachado, R. A. Akhtar and A. A. Abdel- Latif, unpublished data). The mechanism of the hypotensive effect of this antiglaucoma drug is still unknown. 35 " 38 The present study indicates, however, that in addition to acting as a /3-adrenergic antagonist timolol exerts a significant inhibitory effect on the IP 3 -Ca 2+ signalling system. The formation of aqueous humor appears to decrease after topical treatment with timolol, while the outflow facility seems to remain unchanged (for reviews, see refs. 37, 38). Since secretion and muscle contraction are Ca 2+ -dependent functions, it is possible that timolol by inhibiting IP 3 formation and consequently Ca 2+ mobilization could reduce the formation of aqueous humor in the ciliary processes. In conclusion, the data presented demonstrate biochemical and functional interactions between the Ca 2+ and camp systems in the bovine iris sphincter. In this tissue camp may act as regulator of responses to neurotransmitters, hormones and pharmacological agents which exert their action through the IP 3 - Ca 2+ -DG second messenger system. This conclusion is in accord with recent findings on the species differences in the effects of prostaglandins on the two second messenger systems and on muscle contraction in the iris sphincter. 24 Key words: bovine iris sphincter, adrenergic agonists and antagonists, cyclic AMP, inositol trisphosphate-ca 2+, muscle tension Acknowledgment We thank Mr. David Miller for technical assistance. References 1. Nishizuka Y: The role of protein kinase C in cell surface signal transduction and tumor promotion. Nature (Lond) 308:693, 1984. 2. Abdel-Latif AA: Calcium-mobilizing receptors, polyphosphoinositides, and the generation of second messengers. Pharmacol Rev 38:227, 1986. 3. Yoshimasa T, Sibley DR, Bouvier M, Lefkowitz RJ, and Caron MG: Cross-talk between cellular signalling pathways suggested by phorbol-ester induced adenylate cyclase phosphorylation. Nature (Lond) 327:67, 1987. 4. Phaneuf S, Berta P, Le Peuch C, Haiech J, and Cavadore JC: Phorbol ester modulation of cyclic AMP accumulation in a primary culture of rat aortic smooth muscle cells. J Pharmacol ExpTherap 245:1042, 1988. 5. Thompson HS: The pupil. In Adler's Physiology of the Eye: Clinical Application, Moses RA, editor. St. Louis, CV Mosby, 1981, pp.326-356. 6. Nomura T and Smelser GK: The identification of adrenergic and cholinergic nerve endings in the trabecular meshwork. Invest Ophthalmol 13:525, 1974. 7. Laties A and Jacobowitz D: A histochemical study of the adrenergic and cholinergic innervation of the anterior segment of the rabbit eye. Invest Ophthalmol 3:592, 1964. 8. Ehinger B: Adrenergic nerves to the eye and related structures in man and in the cynomolgus monkey (Macaca irus). Invest Ophthalmol 5:42, 1966. 9. Yoshitomi T and Ito Y: Double reciprocal innervations in dog iris sphincter and dilator muscles. Invest Ophthalmol Vis Sci 27:83, 1986. 10. van Alphen GWHM, Kern R, and Robinette S: Adrenergic receptors of the intraocular muscles: comparison to cat, rabbit and monkey. Arch Ophthalmol 74:253, 1965. 11. Schaeppi U and Koella WP: Adrenergic innervation of cat iris sphincter. Am J Physiol 207:273, 1964. 12. Kalsner S: The effect of yohimbine on presynaptic and postsynaptic events during sympathetic nerve activation in cattle iris: A critique of presynaptic receptor theory. Br J Pharmacol 78:247, 1983. 13. Narita S and Watanabe M: Response of the isolated rat iris sphincter to cholinergic and adrenergic agents and electrical field stimulation. Life Sci 29:285, 1981. 14. Wahlestedt C, Bynke G, Beding B, von Leithner P, and Hakanson R: Neurogenic mechanisms in contraction of the rabbit iris sphincter muscle. Eur J Pharmacol 117:303, 1985. 15. Kern R: Die adrenergischen Receptoren der intraocularen Muskeln des Meschen: Eine in vitro Studie. Graefes Arch Klin Exp Ophthalmol 180:231, 1970. 16. van Alphen GWHM: The adrenergic receptors of the muscles of the human eye. Invest Ophthalmol 15:502, 1976. 17. Yoshitomi T, Ito Y, and Inomata H: Functional innervation

No. 10 INTERACTIONS BETWEEN THE camp AND Co 2+ SYSTEMS / Tochado er al 2239 and contractile properties of the human iris sphincter muscle. Exp Eye Res 46:979, 1988. 18. Krieglstein GK: The uses and side effects of adrenergic drugs in the management of intraocular pressure. In Glaucoma: Applied Pharmacology in Medical Treatment, Drange SM and Neufeld AH, editors. New York, Grune & Stratton, Inc., 1984, pp. 255-276. 19. Neufeld AH: The mechanism of action of adrenergic drugs in the eye. In Glaucoma: Applied Pharmacology in Medical Treatment Drange SM and Neufeld AH, editors. New York, Grune & Stratton, Inc., 1984, pp. 255-276. 20. Akhtar RA and Abdel-Latif AA: Carbachol causes rapid phosphodiesteratic cleavage of phosphatidylinositol 4,5-bisphosphate and accumulation of inositol phosphates in rabbit iris smooth muscle: Prazosin inhibits norepinephrine- and ionophore A23187-stimulated release of inositol phosphates. BiochemJ 224:291, 1984. 21. Howe PH, Akhtar RA, Naderi S, and Abdel-Latif A A: Correlative studies on the effect of carbachol on myo-inositol trisphosphate accumulation, myosin light chain phosphorylation, and contraction in sphincter smooth muscle of rabbit iris. J Pharmacol Exp Ther 239:574, 1986. 22. Abdel-Latif AA: Polyphosphoinositides, generation of second messengers, myosin light chain phosphorylation and contraction in rabbit sphincter smooth muscle. Molec Cell Biochem 82:125, 1988. 23. Jumblatt JE, North GT, and Hackmiller RC: Muscarinic cholinergic inhibition of adenylate cyclase in rabbit ciliary processes. ARVO Abstracts. Invest Ophthalmol Vis Sci (Suppl) 29:324, 1988. 24. Yousufzai SYK, Chen, AL, and Abdel-Latif AA: Species differences in the effects of prostaglandins on inositol trisphosphate 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 247:1064, 1988. 25. Lowery OH, Rosebrough JN, Fair AL, and Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 193:265, 1951. 26. Frandsen EK and Krishna G: A simple ultrasensitive method for the assay of cyclic AMP and cyclic GMP in tissues. Life Sci 18:529, 1977. 27. Villalobos-Molina R, Uc M, Hong E, and Garcia-Sainz JA: Correlation between phosphatidylinositol labeling and contraction in rabbit aorta: Effect of a-l adrenergic activation. J Pharmacol Exp Ther 222:258, 1982. 28. Campbell MD, Deth RC, Payne RA, and Honeyman TW: Phosphoinositide hydrolysis is correlated with agonist-induced calcium flux and contraction in the rabbit aorta. Eur J Pharmacol 116:129, 1985. 29. Best L, Brooks KJ, and Bolton TB: Relationship between stimulated inositol lipid hydrolysis and contractility in guinea-pig visceral longitudinal smooth muscle. Biochem Pharmacol 34:2297, 1985. 30. Akhtar RA and Abdel-Latif AA: Surgical sympathetic denervation increases a,-adrenoceptor-mediated accumulation of myo-inositol trisphosphate and muscle contraction in rabbit iris dilator smooth muscle. J Neurochem 46:96, 1986. 31. Fox AW, Abel PW, and Minneman K.P: Activation of a r adrenoceptors increased [ 3 H]inositol metabolism in rat vas deferens and caudal artery. Eur J Pharmacol 116:145, 1985. 32. Grammas P, Diglio CA, Giacomelli F, and Wiener J: Cholinergic-adrenergic receptor interactions in cerebral microvessels. J Neurochem 44:1732, 1985. 33. Howe PH and Abdel-Latif A A: Purification and characterization of protein kinase C from rabbit iris smooth muscle: Myosin light-chain phosphorylation in vitro and in intact muscle. Biochem J 255:423, 1988. 34. Mittag TW, Yoshimura N, and Podos SM: Phorbol ester: Effect on intraocular pressure, adenylate cyclase, and protein kinase in the rabbit eye. Invest Ophthalmol Vis Sci 28:2057, 1987. 35. Neufeld AH: Epinephrine and timolol: How do these drugs lower intraocular pressure. Ann Ophthalmol 13:1109, 1981. 36. Caprioli J and Sears M: The adenylate cyclase receptor complex and aqueous humor formation. Yale J Biol Med 57:283, 1984. 37. Yorio T: Review: Cellular mechanism in the actions of antiglaucoma drugs. J Ocular Pharmacol 1:397, 1985. 38. Leopold IH and Duzman E: Observations on the pharmacology of glaucoma. Ann Rev Pharmacol Toxicol 26:401, 1986.