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1. Changes in cytosolic free calcium concentration ([Ca2+]i) in response to cholinergic and adrenergic agents alone and in combination were investigated using fura-2 fluorescence imaging in intact non-pigmented epithelial cells of rabbit ciliary body.2. Resting ('baseline') [Ca2+]i was 147 + 6 nm (mean + S.E.M.). Acetylcholine (ACh, 10/uM) Ciliary body epithelium is considered responsible for the secretion of aqueous humour in the eye, but the regulatory mechanisms of aqueous humour formation are not yet fully understood (Brubaker, 1991). Though there is no evidence for direct neural innervation of ciliary body epithelial cells, both sympathetic and parasympathetic nerves have been observed within the ciliary processes in the vicinity of the epithelial cells (Stone, Kuwayama & Laties, 1987). Furthermore, agents synthesized in the eye and circulating hormones such as adrenaline may reach these cells by diffusion (Elayan, Kennedy & Ziegler, 1990).Since the ciliary body epithelium appears not to receive direct synaptic input, and since there is such a multitude of receptors present on these cells (see Wax, 1992), it seemed to us possible that the epithelial cells may be regulated by the combined effect of multiple receptor activation, as has been suggested for platelet aggregation (Ruffolo, Nichols, Stadel & Hieble, 1993). In this report, we show such an effect for combined application of muscarinic and adrenergic agonists. Whereas ACh and adrenaline applied by themselves each produce small increases in [Ca2+]i, simultaneous application produces an over 7-fold increase, in part as a result of Ca2+ influx across the plasma membrane. This influx appears to occur through a La3+-sensitive entry pathway. METHODS Tissue isolationIntact ciliary body epithelial processes were isolated from pigmented rabbits by the following procedure. Rabbits weighing 2-3 kg were killed with a lethal dose of sodium pentobarbitone (200 mg). The eyes were then rapidly enucleated, collected and rinsed in Hepes-buffered Ringer solution (for formulation, see below). The anterior segments were isolated, pinned (cornea down) in a dissecting dish filled with Ringer solution, and the lens and lens capsule carefully removed. Single processes were sectioned from the ciliary body by cutting along the base of the process from the iridial margin to pars plana. Individual processes were laid on their sides in Ringer solution in 35 mm Petri dishes that had been modified by cementing a glass coverslip over a 13 mm hole drilled into the bottom of the dish. The processes were barely covered and held down with a 2-3 mm square piece of glass coverslip, to improve mechanical stability.A plexiglass insert was placed in the chamber to reduce the chamber volume. The process was continuously superfused 215 MS 3083, pp. 215-221

When amphibian papillar hair cells (APHCs) of the leopard frog, Rana pipiens pipiens, are osmotically challenged, they exhibit a characteristically asymmetric (rectifying) response: Small decreases (5%, or less) in the extracellular solution’s osmolarity do not significantly affect the cells’ volume; larger decreases produce a relatively slow volume increase in APHCs, while exposure to a hyperosmotic medium leads to rapid shrinking of these cells. Furthermore, the rate of volume change appears to be a function of the rate of extracellular osmotic change. These characteristics make the application of methods devised for the estimation of the osmotic permeability coefficient (Pf) for semipermeable membranes – i.e. those with significant permeability only to water – to APHC membrane rather futile. We have therefore devised a method that takes both the permeability to solutes as well as the kinetics of the osmolarity change into consideration, in order to obtain estimates of Pf that are to a large degree independent of these factors. We have compared the new and earlier methods. Using the new method, we have estimated the Pf of APHCs’ plasma membrane to be in the 10−2-cm/s range, and thus significantly larger than those reported for lipid bilayers. APHC’s membrane Pf appears to be cell-size independent and insensitive to extracellular mercury. These results suggest that APHCs express water-permeable channels in their plasma membrane. Furthermore, we suggest that asymmetric and rate dependent shape changes produced by osmolarity changes in APHCs imply the presence of significant permeability to solutes. The significance of transmembrane solute transport and water channel expression in amphibian auditory hair cells is discussed.

1. The whole‐cell recording mode of the patch‐clamp technique was used to investigate the presence of voltage‐activated currents in the isolated pigmented cells from the rabbit ciliary body epithelium grown in culture. 2. In Ringer solution with composition similar to that of the rabbit aqueous humour, depolarizing voltage steps activated a transient inward current and a delayed outward current, while hyperpolarization elicited an inwardly rectified current. 3. The depolarization‐activated inward current was mainly carried by Na+ and was blocked by submicromolar concentrations of tetrodotoxin. This current in many cells was sufficiently large to produce a regenerative Na+ spike. 4. The depolarization‐activated outward current was carried by K+ and blocked by external TEA and Ba2+. Its activation appeared to be Ca2(+)‐independent. 5. The hyperpolarization‐activated inward current was almost exclusively carried by K+ and was blocked by Ba2+ and Cs+. For large hyperpolarizations below ‐120 mV, this current exhibited a biphasic activation with a fast transient peak followed by a slower sag, that appeared to be due to K+ depletion. 6. The voltage‐dependent K+ conductances probably act to stabilize the cell membrane resting potential and may also play a role in ion transport. The function of the Na(+)‐dependent inward current is unclear, but it may permit the electrically coupled epithelial cells of the ciliary body to conduct propagated action potentials.

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