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Cholinergic and Adrenergic Modulation of the Ca²⁺ Response to Endothelin-1 in Human Ciliary Muscle Cells

¹ From the Department of Pharmacology, University of North Texas Health Science Center, Fort Worth, Texas; and the ² Department of Immunology, St. Paul Medical Center, Mary Kay Ash Institute for Cancer Research, Dallas, Texas.

	Abstract

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PURPOSE. To determine the cholinergic (carbachol, CCH) and adrenergic (norepinephrine, NE) modulation of Ca²⁺ response to endothelin-1 in human ciliary smooth muscle (HCSM) cells.

METHODS. Intracellular calcium levels were measured using the Fura-2 calcium imaging system in HCSM cells treated either singly with endothelin-1 (ET-1; 2–200 nM), CCH (1–100 µM), NE (0.1–10 µM) or isoproterenol (ISO; 1 µM) or in combinations of CCH, NE, or ISO with ET-1. Intracellular cAMP levels after NE and ISO treatments were also measured using a radioimmunoassay.

RESULTS. Endothelin-1 dose-dependently increased [Ca²⁺]_i and was characteristically biphasic (peak [Ca²⁺]_i for ET-1: 2 nM, 517 ± 73 nM; 20 nM, 785 ± 65 nM; and 200 nM, 2564 ± 359 nM). Carbachol also dose-dependently increased [Ca²⁺]_i; however, subsequent additions of ET-1 (200 nM) resulted in lower [Ca²⁺]_i (100 µM CCH + ET-1; 300 ± 21 nM) compared with that observed with 200 nM ET-1 alone (2564 ± 359 nM). Norepinephrine pretreatment also decreased ET-1–induced [Ca²⁺]_i (10 µM NE + ET-1; 619 ± 64 nM) compared with ET-1 alone, and NE’s effect could be reversed by propranolol (ß-adrenergic antagonist) treatment. Neither CCH nor NE was able to completely abolish ET-1’s ability to mobilize calcium in HCSM cells. Isoproterenol (a ß-agonist) mimicked NE’s effect on ET-1–induced [Ca²⁺]_i (1 µM ISO + ET-1; 254 ± 56 nM). Both ISO and NE elevated [cAMP] in HCSM cells.

CONCLUSIONS. In HCSM cells, CCH and ET-1 can activate common as well as specific [Ca²⁺]_i pools. The reduction in ET-1–induced [Ca²⁺]_i after NE/ISO treatment appears to be due to elevated cAMP levels via ß-receptor activation, suggesting the existence of receptor cross talk. The ability of CCH and NE to modulate ET-1’s actions on HCSM may be relevant to the regulation of ciliary muscle contraction and aqueous humor outflow.

	Introduction

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Endothelins (ETs: ET-1, -2, and -3) are a family of 21 amino acid, most potent vasoactive peptides identified to date and were first isolated from supernatants of porcine vascular endothelial cells.¹ Endothelins have been implicated to play a role in many pathologic and disease conditions, including hypertension, pulmonary fibrosis, myocardial infarction, diabetes, and ocular diseases like normal tension glaucoma.^{2 3}

The human eye abundantly expresses ETs (specifically ET-1 and ET-3) as well as ET receptors (ET_A and ET_B) in various structures, including retina, choroid, iris, ciliary body, and ciliary epithelium; and ET-1 is also present in the aqueous humor (AH).^{4 5 6} Endothelinlike immunoreactivity in AH of human and bovine eyes is two to three times greater than that in plasma.⁶ We have recently shown ET-1 synthesis and release in human nonpigmented ciliary epithelium to be under the regulation of cytokines like tumor necrosis factor-, suggesting that ET-1 thus released into AH could exert paracrine effects on target tissues like ciliary muscle and trabecular meshwork.⁷ Recently, it has been reported that ET-1 levels in AH of primary open-angle glaucoma (POAG) patients are significantly greater than those observed in non-POAG patients.⁸

In mammals, intravitreal injections of low doses of ET-1 elicit a prolonged lowering of intraocular pressure observed over several days.^{9 10 11} A possible mechanism contributing to ET-1–induced ocular hypotension is an increase in the outflow facility wherein ET-1 may have a direct effect on the contraction of the ciliary muscle, thus promoting fluid flow via the Schlemm’s canal.^{10 11} Endothelin-1 has been shown to contract isolated human ciliary smooth muscle (HCSM) cells¹² as well as isolated ciliary muscle strips of human,⁶ rhesus monkey,¹¹ and bovine¹³ eyes.

We have previously shown ET-1’s actions on HCSM cells to be mediated via an ET_A receptor, resulting in the mobilization of intracellular calcium ([Ca²⁺]_i) and cAMP, via the production of the prostanoid prostaglandin E₂.¹⁴

It is known that the ciliary muscle tone is under the regulation of both, cholinergic (acetylcholine) innervation, mediated by muscarinic receptors, and adrenergic innervation, mediated via ₂- and ß₂-receptor subtypes.^{15 16 17} However, the interactions between ETs and cholinergic and adrenergic agents have not been well established in the ciliary muscle. This is important because ETs in the AH can either singly or in combination with the autonomic innervation modulate the tone of ciliary muscle and enhance AH outflow and/or accommodation. Because calcium is an important mediator of smooth muscle contraction, its intracellular mobilization dynamics in response to various agonist treatments were monitored using the Fura-2 intracellular calcium imaging technique. Presently, we report on the interactions that exist between ET-1, carbachol (CCH; a cholinergic agonist), and norepinephrine (NE; an - and ß-adrenergic agonist) in isolated cultured HCSM cells.

	Methods

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Isolation of Cells and Culture Conditions
HCSM cells were a generous gift from Iok–Hou Pang (Alcon Laboratories, Ft. Worth, TX) and were harvested from the tissue 12 hours after death, using the procedure described by Tamm et al.¹⁸ The cells were maintained at 37°C in Dulbecco’s modified Eagle medium (DMEM) (GIBCO, Grand Island, NY) supplemented with 44 mM NaHCO₃, 10% fetal bovine serum (Hyclone Laboratories, Logan, UT), and antibiotics (GIBCO). HCSM cells used in the present study were from passages 12 through 14.

[Ca²⁺]_i Measurement
[Ca²⁺]_i measurements were recorded as previously described.¹⁹ Briefly, primary HCSM cells (passages 12–14) were seeded on coverslips and incubated with 3 µM of Fura-2 dye (Molecular Probes, Eugene, OR) in a modified Krebs-Ringer buffer solution (KRB, in millimoles: 115 NaCl, 2.5 CaCl₂, 1.2 MgCl₂, 24 NaHCO₃, 5 KCl, 5 glucose, and 25 HEPES, pH 7.4) for 30 minutes at 37°C. Fura-2 fluorescence from these cells was monitored at 37°C by the ratio technique (excitation at 340 and 380 nm, emission at 500 nm) under a Nikon Diaphot microscope using Metafluor software (Universal Imaging, West Chester, PA). [Ca²⁺]_i was calculated according to the formula by Grynkiewicz et al.²⁰ Calibrations were performed in vivo, and conditions of high [Ca²⁺]_i were achieved by adding the Ca²⁺ ionophore 4-Bromo-A23187 (1–3 µM; Calbiochem, San Diego, CA), whereas conditions of low [Ca²⁺]_i were obtained by adding EGTA (4–5 mM). [Ca²⁺]_i for each treatment was measured at least in two coverslips, such that 5 to 10 cells were monitored each time. Statistical significance of [Ca²⁺]_i between control and treatments was determined either parametrically by Student’s t-test at P < 0.05 or by one-way ANOVA with Student–Newman–Keuls multiple comparison test at P < 0.05 and nonparametrically by Mann–Whitney ranked sum test or Kruskal–Wallis one-way ANOVA with Dunn’s multiple comparison test.

Measurement of cAMP
The assay for measuring cAMP was performed as previously described.¹⁴ Briefly, HCSM cells were grown to confluence in a 24-well plate after supplementation with DMEM and 10% fetal bovine serum. On the day of the experiment, the cells were incubated with serum-free DMEM and pretreated with 1 mM 3-isobutyl-1-methylxanthine at room temperature for 10 minutes (IBMX: phosphodiesterase inhibitor). After various agonist/antagonist treatments (10 minutes for agonists and pretreatment with antagonists for 15 minutes), the reaction was stopped by replacing the medium with 0.2 ml ice-cold 0.1 M acetic acid and incubated at room temperature for 5 minutes, after which 0.3 ml of ice-cold 0.1 M sodium acetate was added. The concentration of cAMP was measured by radioimmunoassay using an aliquot of cell extract (100 µl) according to the instructions given by the manufacturer. Statistical significance of [cAMP] between control and treatments was determined either by a Student’s t-test at P < 0.05 or by one-way ANOVA with Student–Newman–Keuls multiple comparison test at P < 0.05.

Treatments
For [Ca²⁺]_i measurements, HCSM cells were treated individually with ET-1 (2, 20, and 200 nM; Peninsula Laboratories, Belmont, CA), NE (0.1, 1, and 10 µM), CCH (1, 10, 100 µM), isoproterenol (ISO; 1 µM; Sigma–Aldrich Chemical, St. Louis, MO). In HSCM cells, [Ca²⁺]_i was measured after the sequential addition of ET-1 followed by CCH. Also, NE, ISO, or dibutryl cAMP (a cell-permeable and nonhydrolyzable analogue of cAMP) treatments were followed by ET-1. The involvement of - and ß-adrenergic receptors after NE treatment was ascertained by preincubating HCSM cells (for 30 minutes) with benexatramine (irreversible antagonist of ₁-adrenergic receptor; 300 µM; Sigma) or propranolol (potent ß-adrenergic receptor antagonist; 100 µM; Sigma). HCSM cells were also pretreated for 30 minutes with U-73122 (a phospholipase C_ß (PLC_ß) inhibitor; 1 µM; Calbiochem) followed by NE (0.1 µM) or ET-1 (200 nM) treatments. Also, the involvement of prostaglandins after NE treatment was determined by preincubating the cells with INDO (a cyclooxygenase inhibitor; 10 µM; Sigma).

For cAMP measurements using a radioimmunoassay, HCSM cells were treated with NE (0.1, 1, and 10 µM) and ISO (1 µM) in triplicate wells for 10 minutes. Some cells were also preincubated with propranolol (100 µM) for 15 minutes and then treated with NE (10 µM) for an additional 10 minutes.

	Results

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Effects of ET-1, CCH, and NE on Mobilization of [Ca²⁺]_i in HCSM Cells
Endothelin-1 dose-dependently increased [Ca²⁺]_i compared with baseline and was highest at 200 nM ET-1, suggesting the existence of functional ET receptors in HCSM cells (Table 1 A ). Endothelin-mediated [Ca²⁺]_i elevation was typically biphasic with a transient spike (mainly due to release from intracellular calcium stores) followed by a sustained plateau phase (combination of intra- and extracellular calcium) that was higher than the baseline (Fig. 1A ). In our experiments, CCH, like ET-1, was able to biphasically mobilize [Ca²⁺]_i in HSCM cells with the highest [Ca²⁺]_i observed at 10 and 100 µM doses (Table 1 B ; Fig. 1B ). The [Ca²⁺]_i response to NE was different from that observed either with ET-1 or CCH in that no transient spikes were detected at any concentration. A gradual rise in [Ca²⁺]_i was observed at lower doses of NE (0.1 and 1 µM), compared with that observed with 10 µM NE (Table 1 C ; Fig. 1C ). To summarize, in our experiments the [Ca²⁺]_i mobilized by the 3 agonists in HCSM cells was highest for ET-1 (200 nM > 20 nM = 2 nM) followed by CCH (100 µM = 10 µM > 1 µM) and finally by NE (0.1 µM = 1 µM > 10 µM).

View larger version (22K): [in this window] [in a new window]   Figure 1. A representative graph showing the effects of ET-1, CCH, and NE on [Ca2+]i mobilization in HCSM cells as measured by Fura-2 calcium imaging. HCSM cells seeded on coverslips were treated with 200 nM ET-1 (A), 100 µM CCH (B), and 100 nM NE (C). [Ca2+]i was calculated as described in the Methods section.

Effect of ISO and Dibutryl cAMP Pretreatment on ET-1–Induced [Ca²⁺]_i in HCSM Cells
Previously, we have shown an elevation of cAMP after ISO (1 µM) treatment in HCSM cells.¹⁴ To determine whether the attenuation of ET-1–induced [Ca²⁺]_i after NE treatment could be due to ß-adrenergic receptor–induced elevation of cAMP levels, we pretreated HCSM cells with ISO (1 µM) followed by ET-1 (200 nM). As expected, ISO pretreatment decreased the ET-1–induced [Ca²⁺]_i response by nearly 10-fold, compared with 200 nM ET-1 alone (Table 5) .

Effect of INDO on NE and ET-1–Induced Changes in [Ca²⁺]_i in HCSM Cells
Because NE may also mediate its effects via the production of prostaglandins, we treated HCSM cells with INDO (10 µM) and then sequentially added NE (0.1 or 10 µM) followed by ET-1. Although INDO alone did not elevate [Ca²⁺]_i, compared with baseline, it was unable to attenuate the effect of NE-induced reduction in ET-1’s ability to mobilize [Ca²⁺]_i in HCSM cells (Table 6) .

	Discussion

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Our experiments showed that individually both CCH and ET-1 were able to mobilize [Ca²⁺]_i; however, in sequential treatments, the addition of the first agonist diminished the ability of the second agonist to mobilize [Ca²⁺]_i in a dose-dependent manner (Table 2) . Also, [Ca²⁺]_i in response to the second agonist was dependent on the dose of the first agonist. These data suggest that CCH and ET-1 activate similar [Ca²⁺]_i pools. Our observations are consistent with previous reports on HCSM cells treated with acetylcholine and ET-1.²² It is interesting that 200 nM ET-1 was able to mobilize [Ca²⁺]_i even after pretreatment with the highest dose of CCH (100 µM), suggesting that not all [Ca²⁺]_i stores are depleted after a high dose of any agonist treatment.

In the past, ß-adrenergic agonists like NE have been used in conjunction with cholinomimetics to test their effects on the ciliary smooth muscle tissue because both agonists appear to regulate muscle tone in vivo and thus regulate AH outflow.²¹ However, no previous work has focused on how ß-adrenergics, specifically NE, modulate ET-1’s effects on the ciliary muscle. Such interactions are possible because there is an abundance of ß₂-adrenergic receptors and ET_A receptors present on ciliary muscle and also because ET-1 causes ciliary muscle contraction. Although NE is primarily a ß₁-adrenergic agonist, it also has some affinity for ß₂ receptors and can elevate cAMP levels. Because ß₂ receptors account for 90% of total ß-adrenergic receptors on the ciliary muscle with less than 10% of ß₁ receptors present,²³ it is highly likely that NE at the doses tested in the present study was interacting with ß₂ receptors and elevating cAMP.

In HCSM cells, the gradual rise in [Ca²⁺]_i after 0.1 µM NE treatment was mediated via PLC_ß because U-73122 treatment attenuated this response. Similar observations were made in cells pretreated with U-73122 followed by ET-1 treatment. It is possible that the decrease in ET-1 response after low doses of NE was due to changes in releasable calcium pools activated by NE. The ability of BEX, a potent irreversible ₁-adrenergic antagonist,²⁴ to attenuate the NE-mediated elevation of [Ca²⁺]_i strongly suggests the involvement of ₁ receptor. However, [Ca²⁺]_i for ET-1 after BEX + NE (0.1 µM) treatments continued to be lower than that seen with ET-1 alone, suggesting that other adrenergic receptors and/or other second messenger pathways may be activated at the low dose of NE. Prostaglandins did not appear to mediate the 0.1 µM NE-induced reduction in ET-1’s ability to mobilize [Ca²⁺]_i as tested using INDO (an inhibitor of cyclooxygenases). Although it is presently unclear how 0.1 µM NE inhibits ET-1’s actions in HCSM cells, it is quite possible that NE pretreatment could desensitize the common [Ca²⁺]_i pools shared by these two agonists, thus decreasing ET-1’s ability to elevate [Ca²⁺]_i.

However, the addition of ISO (1 µM), a potent ß-agonist known to elevate [cAMP] in HCSM cells,¹⁴ also attenuated ET-1’s response, suggesting that cAMP generated by ISO or by higher doses of NE decreased ET-1–induced elevation of [Ca²⁺]_i by an unknown mechanism. Furthermore, propranolol reversed the NE-induced inhibition (for all 3 doses of NE) of ET-1’s ability to mobilize [Ca²⁺]_i, suggestive of ß-receptor involvement. It was also demonstrated that NE dose-dependently elevated cAMP levels via the ß receptor in these HCSM cells, which is consistent with the premise that cAMP regulated the ET-1 response.

In the rat myometrium, a low concentration of ISO (20 nM), has been shown to attenuate the formation of inositol trisphosphate and to lower [Ca²⁺]_i in a cAMP-independent manner by activating K⁺ channels, leading to the closure of voltage-gated Ca²⁺ channels.²⁵ Additional support for the possible involvement of cAMP (after NE or ISO treatment) comes from an observed reduction in ET-1–induced [Ca²⁺]_i in HCSM cells after pretreatment with dibutryl cAMP. It is important to note that ET-1 also indirectly produces cAMP in HCSM cells via prostaglandin E₂ production within 8 minutes of incubation; however, cAMP thus produced does not affect [Ca²⁺]_i caused by ET-1.¹⁴ The reasons for this are probably related to the time course of cAMP production relative to [Ca²⁺]_i mobilization by ET-1. Moreover, the amount of cAMP generated by this action may not be as high compared with that obtained after treatments with NE or ISO and therefore does not affect ET-1–induced [Ca²⁺]_i signaling. In a previous report,¹⁴ in HCSM cells, the amount of cAMP generated by 1 nM ET-1 was 3.5 pmol/well and 10 to 12 pmol/well for 1 µM ET-1, compared with 63 pmol/well for 10 µM NE and 92 pmol/well for 1 µM ISO seen in the present study. Moreover, cAMP production via ß-adrenergic receptor activation is a direct consequence of adenylyl cyclase activation, whereas ET-1’s effects on cAMP production are mediated through prostaglandin production.

Therefore, our results strongly suggest the existence of "cross talk" between second messengers cAMP and [Ca²⁺]_i in HCSM cells, ultimately resulting in the regulation of ET-1–induced [Ca²⁺]_i levels. There are a number of targets that cAMP could influence, relative to the ET-1–induced [Ca²⁺]_i signaling in HCSM cells, including modifying receptor states through cAMP-dependent phosphorylation, affecting the inositol trisphosphate–calcium signaling pathway, or inhibiting myosin light chain kinase activity, similar to what has been suggested for bovine ciliary muscle.²⁶ Further research is necessary to understand the exact mechanism involving cAMP and [Ca²⁺]_i cross talk signaling in HCSM cells.

In conclusion, we presently report that CCH and NE modulate ET-1–induced [Ca²⁺]_i signaling in HCSM cells. Although CCH and ET-1 share similar [Ca²⁺]_i pools, NE reduces ET-1–induced [Ca²⁺]_i signaling probably by elevating cAMP levels via the activation of ß receptors. The interactions of these autonomic neurotransmitters with ET-1 on ciliary muscle could result in the regulation of ciliary muscle contraction and ultimately affect AH dynamics and accommodation.

	Footnotes

 
Supported by a Texas Advanced Research Grant (ARP-009768) and a NEI/NIH Grant (EY11979).

Submitted for publication April 20, 1999; revised September 17 and November 17, 1999; accepted November 30, 1999.

Commercial relationships policy: N.

Presented at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May, 1999.

Corresponding author: Ganesh Prasanna, Department of Pharmacology, University of North Texas Health Science Center, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107. gprasann{at}molly.hsc.unt.edu

	References

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