Replacing the rod with the cone transducin subunit decreases sensitivity and accelerates response decay.

Cone vision is less sensitive than rod vision. Much of this difference can be attributed to the photoreceptors themselves, but the reason why the cones are less sensitive is still unknown. Recent recordings indicate that one important factor may be a difference in the rate of activation of cone transduction; that is, the rising phase of the cone response per bleached rhodopsin molecule (Rh*) has a smaller slope than the rising phase of the rod response per Rh*, perhaps because some step between Rh* and activation of the phosphodiesterase 6 (PDE6) effector molecule occurs with less gain. Since rods and cones have different G-protein alpha subunits, and since this subunit (Talpha) plays a key role both in the interaction of G-protein with Rh* and the activation of PDE6, we investigated the mechanism of the amplification difference by expressing cone Talpha in rod Talpha-knockout rods to produce so-called GNAT2C mice. We show that rods in GNAT2C mice have decreased sensitivity and a rate of activation half that of wild-type (WT) mouse rods. Furthermore, GNAT2C responses recover more rapidly than WT responses with kinetic parameters resembling those of native mouse cones. Our results show for the first time that part of the difference in sensitivity and response kinetics between rods and cones may be the result of a difference in the G-protein alpha subunit. They also indicate more generally that the molecular nature of G-protein alpha may play an important role in the kinetics of G-protein cascades for metabotropic receptors throughout the body.

Removal of phosphorylation sites of gamma subunit of phosphodiesterase 6 alters rod light response.

The phosphodiesterase 6 gamma (PDE6 gamma) inhibitory subunit of the rod PDE6 effector enzyme plays a central role in the turning on and off of the visual transduction cascade, since binding of PDE6 gamma to the transducin alpha subunit (T alpha) initiates the hydrolysis of the second messenger cGMP, and PDE6 gamma in association with RGS9-1 and the other GAP complex proteins (G beta 5, R9AP) accelerates the conversion of T alpha GTP to T alpha GDP, the rate-limiting step in the decay of the rod light response. Several studies have shown that PDE6 gamma can be phosphorylated at two threonines, T22 and T35, and have proposed that phosphorylation plays some role in the physiology of the rod. We have examined this possibility by constructing mice in which T22 and/or T35 were replaced with alanines. Our results show that T35A rod responses rise and decay more slowly and are less sensitive to light than wild-type (WT). T22A responses show no significant difference in initial time course with WT but decay more rapidly, especially at dimmer intensities. When the T22A mutation is added to T35A, double mutant rods no longer showed the prolonged decay of T35A rods but remained slower than WT in initial time course. Our experiments suggest that the polycationic domain of PDE6 gamma containing these two phosphorylation sites can influence the rate of PDE6 activation and deactivation and raise the possibility that phosphorylation or dephosphorylation of PDE6 gamma could modify the time course of transduction, thereby influencing the wave form of the light response.

Multiple receptor activation elicits synergistic IP formation in nonpigmented ciliary body epithelial cells.

We have examined the interaction between muscarinic and alpha(2)-adrenergic receptor activation on inositol phosphate (IP) formation in the nonpigmented cells of the ciliary body epithelium (NPE cells) of the rabbit. We have compared these changes with those previously observed in the intracellular free Ca(2+) concentration. Whereas muscarinic receptor activation causes an increase in intracellular Ca(2+) and IP formation, activation of alpha(2)-receptors does not significantly increase either intracellular Ca(2+) or IPs over basal levels. However, simultaneous activation of muscarinic and alpha(2)-adrenergic receptors with the specific agonists carbachol and UK-14304 produces massive Ca(2+) increases and results in a synergistic increase in IP formation. This synergistic IP formation is inhibited by both muscarinic and alpha(2)-adrenergic receptor antagonists as well as by pertussis toxin and an inhibitor of phospholipase C. IP formation is predominantly independent of intracellular Ca(2+), because it is decreased but not prevented by blocking the entry of Ca(2+) with LaCl(3) or chelating intracellular Ca(2+) with 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Thus synergistic IP formation underlies, at least in part, the synergistic increase in intracellular Ca(2+) resulting from simultaneous activation of muscarinic and alpha(2)-adrenergic receptors.

Synergistic receptor-activated calcium increases in single nonpigmented epithelial cells.

PURPOSE: To determine whether single nonpigmented ciliary body cells contain the signaling mechanism to produce synergistic drug-activated increases in Ca2+, or whether these responses are produced cooperatively by interaction among groups of cells. METHODS: Suspensions of single nonpigmented cells were plated onto soft collagen gels. Fura-2 fluorescence ratio imaging was used to examine receptor-evoked changes in intracellular Ca2+ concentration. RESULTS: Nonpigmented cells plated on soft collagen gels retained a rounded shape with membrane evaginations visible on their surface. Application of acetylcholine (10 microM) or epinephrine (1 microM) each produced small increases in intracellular Ca2+, but in combination they produced a Ca2+ increase of more than 10-fold. This synergistic Ca2+increase was a result of activation of muscarinic and alpha2-adrenergic receptors because a specific alpha2-adrenergic agonist could substitute for epinephrine in producing the response. The response could be blocked by a specific alpha2-antagonist and a muscarinic antagonist. An alpha1-agonist could not substitute for epinephrine in producing a synergistic increase nor could the synergism be blocked by alpha1- or beta-antagonists. The Ca2+ increase was largely produced by release from internal stores, because the peak amplitude of the response was nearly the same in the external solution containing a low Ca2+ concentration; however, the influx of Ca2+ into the cell was responsible for maintenance of a steady component of the Ca2+ increase during maintained drug stimulation and for refilling the internal stores. CONCLUSIONS: Single nonpigmented cells can produce synergistic increases in Ca2+ on multiple receptor activation, indicating that the mechanism of synergism does not require the interaction of multiple cells. The Ca2+ increase is a result of release from internal stores and Ca2+ entry through an as yet undefined conductance or transport system in the plasma membrane.

Functional and morphological differentiation of nonpigmented ciliary body epithelial cells grown on collagen rafts.

We have examined the effect of alteration in cell shape on promoting differentiated morphology and physiology in cultured nonpigmented epithelial cells from the ciliary body. We have grown pure populations of nonpigmented cells on collagen gels released from the culture dish to create collagen rafts. Shortly after the gels were detached, the cells shrank in diameter and increased in height while they contracted the gel. Concurrently, the actin cytoskeleton reorganized to the cell cortex as found in vivo. After this differentiated morphology developed, large changes in intracellular Ca2+ could be elicited by simultaneous activation of acetylcholine and epinephrine or acetylcholine and somatostatin receptors as seen in intact tissue. Explant cultures of isolated nonpigmented cell layers maintained their actin distribution and also showed synergistic Ca2+ increases. Spread cells, grown on rigid substrates, had a disorganized cytoskeleton and rarely showed synergism. These data suggest that the mechanism underlying synergistic Ca2+ responses in the ciliary body is functional in nonpigmented cells grown on collagen rafts. In addition, this pathway appears to be sensitive to the disposition of the cell's cytoarchitecture.

Synergistic increase in Ca2+ produced by A1 adenosine and muscarinic receptor activation via a pertussis-toxin-sensitive pathway in epithelial cells of the rabbit ciliary body.

The combined effects of adenosine and acetylcholine on the intracellular free-Ca2+ concentration in nonpigmented epithelial cells of the rabbit ciliary body were investigated using fura-2 fluorescence-ratio imaging. Acetylcholine (10 microM) by itself produced a modest increase in [Ca2+]i. Acetylcholine in combination with adenosine, or with the A1-specific agonists N6-cyclohexyl-adenosine, N6-cyclopentyl-adenosine and (R)-N6-(2-phenyl-1-methylethyl)-adenosine (0.1-1 microM), induced a massive increase in [Ca2+]i, which could be blocked by the A1-specific antagonist 8-cyclopentyl-1,3-dipropylxanthine. However, the A2-specific agonist 2-[(p-2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamide-ade nos ine and the antagonist 3,7-dimethyl-1-(2-propynyl)xanthine were without effect. Incubation of the tissue with pertussis toxin did not alter the response to ACh alone but eliminated the synergistic effect of adenosine (or of epinephrine). It was concluded that in the epithelial cells of the rabbit ciliary body, adenosine and epinephrine synergistically potentiate the rise in [Ca2+]i produced by ACh. This potentiation appears to occur via a pertussis-toxin-sensitive pathway, perhaps through G(i).

Synergistic effect of adrenergic and muscarinic receptor activation on [Ca2+]i in rabbit ciliary body epithelium.

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 microM) doubled [Ca2+]i, and adrenaline (1 microM) increased it by about 36%. When ACh (10 microM) and adrenaline (1 microM) were applied together [Ca2+]i was transiently increased to 1160 +/- 160 nM, about 7 times the response induced by ACh alone. 3. Noradrenaline and 5-bromo-6-(2-imidazolin-2-yl-amino)-quinoxaline (UK 14304) had effects similar to adrenaline in enhancing the response to ACh. Phenylephrine (Phe) had a relatively smaller effect and none was observed for methoxamine and isoprenaline (Iso). 4. The response to ACh and adrenaline could be blocked by atropine (1 microM, 87 +/- 5%), yohimbine (1 microM, 73 +/- 8%), and to a lesser degree by prazosin (1 microM). Propranolol had no effect. 5. Lowering the extracellular calcium concentration to 3 nM dropped the baseline [Ca2+]i by half and reduced the response to ACh and adrenaline to a small and transient rise in [Ca2+]i. Addition of La3+ to Ca(2+)-containing solution also lowered [Ca2+]i and largely reduced the response. 6. We conclude that simultaneous activation of muscarinic and alpha 2-adrenergic receptors induces a large increase in [Ca2+]i, which is the result of both Ca2+ release and influx.

Dihydropyridine-sensitive Ca2+ spikes and Ca2+ currents in rabbit ciliary body epithelial cells.

Intracellular microelectrode and whole-cell patch-clamp recordings were used to investigate a Ba(2+)-induced regenerative depolarization and its underlying Ba2+ current in the ciliary body epithelial cells of the rabbit eye. Exposure of these epithelial cells to 4-10 mmol l-1 Ba2+ depolarized the membrane potential and caused the generation of one or more spikes, before the membrane potential reached a steady-state level. The spikes, but not the slow phase of depolarization, could be blocked with Co2+ (2 mmol l-1), Gd3+ (25 mumol l-1), La3+ (20 mumol l-1), Cd2+ (10 mumol l-1), verapamil (30 mumol l-1) and nifedipine (1 mumol l-1). Tetrodotoxin at 100 nmol l-1 had no effect. In the absence of Na+, but in the presence of external Ba2+, step depolarization of the membrane potential activated an inward current that could be blocked with Co2+ (2 mmol l-1), Cd2+ (10 mumol l-1) and nifedipine (1 mumol l-1), but not with Ni2+ (50 mumol l-1) or omega-conotoxin (1-10 mumol). This inward current could be enhanced with the dihydropyridine agonist (+/-)BAY K 8644 (1 mumol l-1). The inactivation characteristics of the inward current (v1/2 = -38.7 mV, k = 12.6 mV) is most like that seen in neurons. These findings indicate that the epithelial cells of the ciliary body possess dihydropyridine-sensitive, voltage-activated Ca2+ channels.

Tissue culture of rabbit ciliary body epithelial cells on permeable supports.

The aqueous humor is produced by the epithelium of the ciliary body, a complex structure encircling the anterior segment of the eye. Aqueous humor production occurs by active transport, but the mechanism of this process is not understood. To produce a preparation in which active transport can be investigated, we have attempted to prepare cultures suitable for measurements of ion and water flux. We have grown rabbit ciliary body epithelial cells on permeable supports, coated with several extracellular matrix proteins. We then examined the ability of these proteins to promote the growth of a differentiated layer of epithelial cells. Non-pigmented and pigmented cells formed sheets of contiguous cells when grown on a variety of support media. The most successful substrate was a permeable support produced by Falcon/Becton Dickinson coated with a mixture of collagen IV, laminin and heparan sulfate. Under these conditions, cultures could be maintained for several months, but pigmented cell cultures did not develop a measurable transepithelial resistance (TER), and the TER of non-pigmented cell cultures was typically only 20-30 omega cm2. Much higher TERs exceeding 200 omega cm2 could be measured from non-pigmented cell cultures 3-5 days after plating, but these high values were unstable. Examination of the cultures with electron microscopy revealed that the cells were partially differentiated with the formation of a basal lamina and intercellular junctions. Labelling with a specific monoclonal antibody marker for tight junction protein (ZO-1) suggested that non-pigmented cell cultures showed extensive tight junction formation. The low TER of the non-pigmented cell cultures appears therefore not to be due to the lack of tight junctions but rather to the presence of spaces between cells.

Nonpigmented cells of the rabbit ciliary body epithelium. Tissue culture and voltage-gated currents.

The aqueous humor of the eye is thought to be secreted by the epithelium of the ciliary body. This epithelium has been difficult to study, in part because of its complicated morphology. The authors attempted to circumvent this difficulty by growing the epithelial cells in tissue culture. A procedure is described for producing pure primary cultures of rabbit nonpigmented ciliary body epithelial cells. This procedure was used with whole-cell patch-clamp recording to characterize voltage-activated currents in the nonpigmented cells. These experiments show that most nonpigmented cells contain two kinds of currents: a rapidly activating and inactivating inward current, carried by Na+ and blocked by tetrodotoxin (TTX), and a more slowly activating and inactivating outward current, blocked by tetraethylammonium (TEA+), Ba2+, and 4-aminopyridine (4-AP) and presumably carried by K+. Both of these currents have been observed in freshly dissociated cells and in cultures up to 7 days old. The voltage-gated currents in ciliary body epithelial cells are remarkably similar to those of neurons and raise the possibility that these epithelial cells are capable of spike propagation.

Isolation of non-pigmented epithelial cells from rabbit ciliary body.

We describe a new technique for the separation and isolation of nonpigmented ciliary body epithelial cells from rabbit. Excised ciliary body is incubated in a medium buffered with EGTA to a free-Ca2+ concentration of 10(-8) M, and the nonpigmented cell layer is separated from the pigmented layer by microdissection under direct visualization. This technique yields intact cells which are greater than 99% nonpigmented. It can be used to produce preparations of nonpigmented cell plasma membrane and of viable whole cells, which may be useful for biochemistry, pharmacology, cell transport studies and tissue culture.

Monoclonal antibodies to the H+-K+ ATPase of gastric mucosa selectively stain the non-pigmented cells of the rabbit ciliary body epithelium.

Monoclonal antibodies developed against the transport enzyme H+-K+ ATPase of gastric mucosa selectively bind to the nonpigmented cell layer in both the pars plana and pars plicata of the rabbit ciliary body epithelium. The most specific of these antibodies (HK 12.18) reacts with the nonpigmented cells but with no other cell type in ciliary body, retina or iris. Antibody reactivity is evinced by a distinct particulate staining which is greater on the surfaces of the nonpigmented cells than within the cytoplasm and seems most intense on the apical membrane between the pigmented and nonpigmented cell layers. This pattern of staining was present in 2-3-day-old pups and in young adults, in both pigmented and albino animals. Similar staining was not observed in control sections treated with preimmune serum, nonspecific mouse monoclonal antibody, or HK 12.18 preabsorbed to gastric microsomes. In addition to staining intact ciliary body, the antibody HK 12.18 can be shown to react with a population of cells lacking pigment granules in suspensions of dissociated ciliary body epithelium. This antibody may therefore be useful as a marker for the identification of nonpigmented cells in tissue culture.