Role of the non-neuronal cholinergic system in the eye: a review.

Pharmacologically active preparations directed towards modulating muscarinic receptor activity in the eye have been used for over 2000 years when extracts from Atropa belladonna were first applied to enhance eye appearance through pupillary dilation. The first clinically active drugs targeting a specific eye disease were anticholinesterases (e.g. ecothiophate) applied as eye drops to treat glaucoma in the 1960's. However, cataract was soon detected as a relatively frequent side effect and such drugs are now only used to treat glaucoma as a last resort. As muscarinic agonists have been found to reduce intraocular pressure both by decreasing aqueous humour production (through Na,K-ATPase pump inhibition) and increasing outflow (by muscle contraction), it is likely that treatments will be developed that target specific muscarinic subtypes. Recently, it has been shown that the M1 receptor subtype predominates in the lens. It is therefore important that this subtype is not targeted in future ocular therapies so that the side-effect of cataract is avoided. Form-deprived myopia resulting from an increased axial length in the affected eye can be reduced by the application of atropine. This effect has been achieved both in a chick model system and in human clinical trials, and in the former system atropine has been shown to reduce the production of scleral extracellular proteins. Carbachol stimulates tear fluid production through the activation of muscarinic receptors. Interestingly, at least part of the stimulation occurs via epidermal growth factor (EGF) receptors and although the precise signalling mechanisms are not completely understood, it has been shown that calcium mobilisation plays a critical role in both muscarinic and EGF receptor activity. It should be noted that in the four examples described above, the cell types responsible for producing the physiological output are non-neuronal in origin. Therefore cholinergic receptor activation plays diverse roles in the eye and pharmacological intervention based on specific receptor sub-types has potential benefit in a number of ocular problems. However, potential side effects have also recently been identified.

Regional differences in functional receptor distribution and calcium mobilization in the intact human lens.

PURPOSE: To investigate regional differences in Ca(2+) mobilization kinetics in the intact human lens produced by exposure to agonists of tyrosine-kinase and G-protein-coupled receptors and to characterize the major receptor subtypes involved in Ca(2+) signaling in the different regions. METHODS: Whole human lenses were placed anterior side down in a plastic chamber and perifused with artificial aqueous humor (AAH) at 30 degrees C. After fura-2 incorporation, cytosolic Ca(2+) levels were monitored by using epifluorescence techniques in either the equatorial or central anterior epithelial cells of the intact lens. Agonists dissolved in AAH were applied to the lens in successive short pulses. RESULTS: Central anterior lens epithelial cells produced a large response to 10 microM acetylcholine (ACh) and histamine; only a small response to adenosine triphosphate (ATP); and no response to 10 microM adrenalin, 10 ng/ml epithelial growth factor (EGF) or TGF alpha, or 50 ng/ml platelet-derived growth factor (PDGF)-AB. Conversely, the equatorial cells produced a strong response to 10 microM ATP and histamine, 10 ng/ml EGF (or TGF alpha), and 50 ng/ml PDGF-AB, but failed to respond to 10 microM ACh or 10 microM adrenalin. The EGF-induced response in the equatorial cells was blocked completely by tyrphostin (AG1478), a specific inhibitor of the EGF receptor tyrosine kinase. Carbachol, a nonhydrolyzable analogue of ACh, and pilocarpine, the M1 muscarinic receptor-specific agonist, both produced the same trend of response amplitude elicited by ACh in each region of the lens. The potency order of purinergic agonist-induced Ca(2+) mobilization at the equator was consistent with the P2Y(2) receptor subtype. The histamine-induced response was abolished by 10 microM triprolidine, a specific H(1) receptor antagonist, but remained unaffected by the specific H(2) and H(3) antagonists, ranitidine and thioperamide, respectively. CONCLUSIONS. There is a spatial heterogeneity in functional receptor activity in different regions of the whole lens. The important growth factor receptors for EGF and PDGF are functionally active only in the equatorial cells of the mature human lens. This study further shows that the ACh, histamine, and ATP-induced responses arise from the activation of M1 muscarinic, H(1) histamine, and P2Y(2) purinergic receptors, respectively.

Role of the endoplasmic reticulum in shaping calcium dynamics in human lens cells.

PURPOSE: Localized cortical cataracts in the human lens have been shown to involve a selective increase in calcium with no change in sodium content. Recent studies in the rat lens in vitro have shown that the store-operated channel is highly selective for calcium over sodium, and therefore this channel was characterized further in human lens cells. METHODS: Human primary cultures were initiated from epithelial explants and passaged onto coverslips. After incorporating Fura-2, agonist- or thapsigargin-induced changes in cytosolic calcium were monitored and calibrated using fluorometric digital imaging techniques. RESULTS: Histamine and adenosine triphosphate (ATP; 10 microM) induced a large transient increase in cytosolic calcium followed by a maintained lower plateau phase in the continued presence of the calcium-signaling agonist. The second phase was abolished by removing external calcium and represented the contribution from the store-operated influx. The store-operated pathway was blocked by inorganic agents such as zinc and nickel (100 microM) but was insensitive to the voltage-sensitive calcium channel blocker, nifedipine (1 mM). Depolarizing the membrane voltage by raising the external potassium (75 mM) also blocked the influx. Similar results were obtained if the store was first emptied directly using thapsigargin (1 microM), and with this agent it was also possible to observe the very slow activation and inactivation kinetics (>10 seconds) of the channel. Addition of manganese to the bathing medium initiated a quench of Fura-2 isobestic fluorescence that was enhanced 2.9 +/- 0.3-fold after 10 microM ATP addition. There was a delay of 82 +/- 16 seconds between initiation of the calcium spike and the Mn2+ quench rate, indicating the presence of a delayed entry pathway. In the resting state, removal of, or increasing extracellular calcium concentration 10-fold did not perturb the level of cytosolic Ca2+. Similar maneuvers performed after agonist- or thapsigargin-induced store depletion of intracellular stores brought about dramatic changes in cytosolic Ca2+ consistent with the activation of a Ca2+ entry pathway. Lower concentrations of agonist induced oscillations of Ca2+ that continued for a short time in Ca-free solution. No increase in Mn2+ quench rate was associated with oscillations. A 100-microM zinc- and KCl-induced blockade of Ca2+ entry had no effect on the form of agonist-induced oscillations. Inhibition of Ca2+ influx by zinc (100 microM) converted a sustained Ca2+ response to a train of repetitive Ca2+ spikes. CONCLUSIONS: Human lens cells normally have very low Ca2+ permeability. Depletion of intracellular stores by agonists or thapsigargin initiates a Ca2+ entry pathway that is not required for the Ca2+ oscillations induced by low concentrations of agonist. This potentially provides a signal transduction mechanism with minimal risk of Ca2+ overload to the lens, whereas overactivation of the store-operated channel is a possible way of increasing calcium in the lens and could explain the distribution found in localized cataracts.

Characterization of muscarinic receptors in human lens cells by pharmacologic and molecular techniques.

PURPOSE: Activation of muscarinic receptors has been implicated in an increased risk of cataract after anticholinesterase treatment for glaucoma. The purpose of the present study was to determine the acetylcholine muscarinic receptor subtype(s) present in native human lens epithelial cells (NHLECs) and a human lens cell line, HLE-B3, and to compare the distribution in other ocular cells. METHODS: Human lens cells were perfused with artificial aqueous humor (35 degrees C) after fura-2 incorporation, and calcium levels were measured using a fluorometric single-cell digital imaging system. Acetylcholine was the primary muscarinic agonist, and the receptor subtypes were elucidated by determining the relative effectiveness of pirenzepine and AF-DX 384 in blocking the agonist-induced response. The levels of expression of mRNA for the receptor subtypes M1 through M5 were determined by quantitative reverse transcription-polymerase chain reaction (QRT-PCR) using a sequence detection system (ABI Prism 7700; Perkin-Elmer, Foster City, CA). This was performed using total RNA extracted from native lens, retina, iris, and sclera and also cultured lens cells. RESULTS: Acetylcholine induced a similar concentration-dependent increase in peak-amplitude cytosolic calcium in the range 100 nM to 100 microM in both native and HLE-B3 cells. However, the kinetics of the response waveforms to 30-second pulses of acetylcholine were different in the two cell types. At higher concentrations (> 1 microM), a second phase appeared in the HLE-B3 cells that was absent in the NHLEC response. The 50% inhibitory concentration (IC50) values for blockade of a 1 microM acetylcholine response by pirenzepine and AF-DX 384 were 30 nM and 230 nM, respectively, for NHLECs, and 300 nM and 92 nM, respectively, for HLE-B3 cells. The QRT-PCR data showed that more than 90% of the total muscarinic receptor mRNA from NHLEC was of M1 origin. In the HLE-B3 cells, however, more than 95% of the mRNA was of M3 origin. mRNA for M3 was also in greatest abundance in other eye tissues, although there was a significant contribution from M1 in iris and sclera. CONCLUSIONS: Both NHLECs and HLE-B3 cells express muscarinic receptors that produce significant changes in cytosolic calcium in response to acetylcholine. Both pharmacologic and QRT-PCR evidence shows that whereas the M1 subtype predominates in NHLECs, M3 is the major contributor in HLE-B3 cells. In all other eye tissues, M3 appears to be the major contributor. These data should be taken into account when choosing particular models to investigate cataract mechanisms and also when designing muscarinic agonists to treat glaucoma.