We used whole‐cell patch clamp and fluorescent calcium imaging techniques to investigate the effects of adenosine 5′‐triphosphate (ATP) on membrane currents and intracellular calcium concentration ([Ca2+]i)in rat retinal pigment epithelial (RPE) cells. In 62 % of RPE cells, application of 100 μM ATP elicited a fast inward current at negative membrane potentials. In 38 % of RPE cells recorded, a biphasic response to ATP was observed in which activation of the fast inward current was followed by activation of a delayed outward current. The ATP‐activated inward current was a non‐selective cation (NSC) current that showed inward rectification, reversed at −1.5 ± 1 mV and was permeable to monovalent cations. The NSC current was insensitive to the P2 purinoceptor antagonists, suramin or PPADS but was activated by the purinoceptor agonists UTP, ADP and 2MeSATP. The outward current activated by ATP reversed at −68 ± 3 mV (equilibrium potential for potassium (EK) =−84 mV) and was blocked by Ba2+ ions, consistent with the activation of a K+ conductance. The outward K+ conductance was also reduced by the maxi‐KCa channel blocker iberiotoxin (IbTX; 10 nM), suggesting that ATP activated an outward Ca2+‐activated K+ channel in rat RPE cells. The Ca2+‐activated K+ current (IK(Ca)) was also activated by the purinoceptor agonists UTP, ADP and 2MeSATP. In fluo‐3 or fluo‐4 loaded RPE cells, ATP and the pyrimidine agonist UTP elevated [Ca2+]i. The increase in Ca2+ was not dependent on extracellular Ca2+ influx, but was sensitive to the Ca2+‐ATPase inhibitor thapsigargin, confirming the involvement of intracellular Ca2+ stores release. These results suggest that rat RPE cells express both P2X purinoceptors that gate activation of a non‐selective cation conductance and G protein‐coupled P2Y purinoceptors that mediate Ca2+ release from intracellular stores and activation of a calcium‐activated K+ current.
Neuropeptides are known to modulate the excitability of mammalian sympathetic neurons by their actions on various types of K+ and Ca2+ channels. We used whole cell patch-clamp recording methods to study the actions of substance P (SP) on dissociated adult guinea pig stellate ganglion (SG) neurons. Under current-clamp conditions, SG neurons exhibited overshooting action potentials followed by afterhyperpolarizations (AHP). The K+ channel blocker tetraethylammonium (1 mM), the Ca2+ channel blocker Cd2+ (0.1–0.2 mM), and SP (500 nM) depolarized SG neurons, decreased the AHP amplitude, and increased the action potential duration. In the presence of Cd2+, the effect of SP on membrane potential and AHP was reduced. Under voltage-clamp conditions, several different K+ currents were observed, including a transient outward K+ conductance and a delayed rectifier outward K+ current ( I K) consisting of Ca2+-sensitive [ I K(Ca)] and Ca2+-insensitive components. SP (500 nM) inhibited I K. Pretreatment with Cd2+ (20–200 μM) or the high-voltage-activated Ca2+ channel blocker ω-conotoxin (10 μM) blocked SP’s inhibitory effects on I K. This suggests that SP reduces I K primarily through the inhibition of I K(Ca) and that this may occur, in part, via a reduction of Ca2+ influx through voltage-dependent Ca2+ channels. SP’s actions on I K were mediated by a pertussis toxin-insensitive G protein(s) coupled to NK1 tachykinin receptors. Furthermore, we have confirmed that 500 nM SP reduced an inward Cd2+- and ω-conotoxin-sensitive Ba2+ current in SG neurons. Thus the actions of SP on I K(Ca) may be due in part to a reduction in Ca2+influx occurring via N-type Ca2+channels. This study presents the first description of ionic currents in mammalian SG neurons and demonstrates that SP may modulate excitability in SG neurons via inhibitory actions on K+ and Ca2+ currents.
The ciliary body epithelium (CBE) forms the inner surface covering of the ciliary processes of the eye and is composed of two different epithelial cell layers: a non-pigmented ciliary epithelial (NPCE) cell layer and a pigmented ciliary epithelial (PCE) cell layer (Caprioli, 1992). Both PCE and NPCE cells are involved in the production of aqueous humour, an isotonic solution composed primarily of water, Na¤, Cl¦ and HCOצ. The balance between the rate and quantity of aqueous humour produced and aqueous humour escape from the eye, via drainage pathways, is the primary determinant of intraocular pressure (IOP), and is subject to autonomic modulation (Caprioli, 1992). Transport data from intact and dispersed ciliary epithelial tissue suggest that PCE cells have solute uptake properties and are functionally coupled to the NPCE cells which have solute efflux properties (Wiederholt et al. 1991;Edelman et al. 1994). In this cell coupled model, ions and water from the stroma are taken up by PCE cells and passed to the NPCE cells via apical gap junctions (Raviola & Raviola, 1978), where they are secreted at the basolateral membrane into the posterior chamber as aqueous humour. Despite our understanding of this functional coupling between CE cells, the exact cellular transport mechanisms involved in fluid and ion secretion remain unresolved. However, it has now been shown that Na¤, K¤ and Cl¦ enter PCE cells via a furosemide-(frusemide-) and bumetamidesensitive Na¤-K¤-2Cl¦ symport and diffuse from PCE to NPCE cells via the apical gap junctions. Na¤, K¤ and Cl¦ ions are then secreted from NPCE cells through Na¤-K¤ exchange pumps and via basolateral K¤ and Cl¦ channels, and this is accompanied by paracellular Na¤ movement. A HCOצ-dependent transepithelial potential of approximately
1. The effects of adrenergic agonists on K¤ currents were studied in cultured rabbit pigmented ciliary epithelial (PCE) cells.
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