Signaling Pathways Involved in aAdrenergic Receptor Modulation of IKCa

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In most cell types, arARs couple to PTX-insensitive Gq proteins linked to activation of PLC, the generation of IP3, and subsequent Ca2+ stores release

Fig. 5. Activation of IK(Ca) by AR activation. (A, C) Whole-cell currents elicited from voltage steps from -82 to +58 mV from a holding potential of -62 mV, recorded in the absence (control) and presence of 100 |M epinephrine (EPI) or phenylephrine (PHe). (B) Mean (SEM) increase in IK measured at +58 mV after exposure of 4 cells to 100 |M EPI in the absence (EPI) and presence of IbTX (EPI + 10 nM IbTX). (D) Histogram shows mean (SEM) current measured at +58 mV for the same cells before and after 40-s application of 100 |M PHe in the absence of (PHe) or presence of 100 |M prazosin (PHe + PZ). Data have been normalized for cell capacitance.

Fig. 5. Activation of IK(Ca) by AR activation. (A, C) Whole-cell currents elicited from voltage steps from -82 to +58 mV from a holding potential of -62 mV, recorded in the absence (control) and presence of 100 |M epinephrine (EPI) or phenylephrine (PHe). (B) Mean (SEM) increase in IK measured at +58 mV after exposure of 4 cells to 100 |M EPI in the absence (EPI) and presence of IbTX (EPI + 10 nM IbTX). (D) Histogram shows mean (SEM) current measured at +58 mV for the same cells before and after 40-s application of 100 |M PHe in the absence of (PHe) or presence of 100 |M prazosin (PHe + PZ). Data have been normalized for cell capacitance.

(21,22). To investigate the involvement of this signaling pathway in PHe-mediated increases in IK(Ca), we employ a number of drugs that mimic or inhibit signaling molecules in this pathway.

1. Activation of IK(Ca) by phenylephrine could be mimicked by dialyzing PCE cells with 10 |M of IP3. Dialysis of PCE cells with 10 |M IP3 for 10 min increases the whole-cell outward current measured at +58 mV by a mean of 30 pA/pF (Fig. 6A).

2. To test the involvement of IP3-stimulated Ca2+ stores release in PHe-mediated IK(Ca) activation, we pretreat the cells with 5 | M of the membrane-permeant Ca2+-ATPase blocker TG to empty sarcoplasmic reticular Ca2+ stores (23). Before recording, PCE cells are incubated for 20 min in the dark in low-Ca2+ external

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Fig. 6. Involvement of IP3, PLC, and intracellular Ca2+ release in IK(Ca) activation. (A) Histogram shows mean (±SEM) current amplitude measured at +58 mV and normalized for cell capacitance in: 5 PCE cells before (Con) and after (PHe) 40 s of pressure application of 100 |M PHe; in 5 PCE cells treated for 30 min with 5 |M thapsigargin before (Con + TG) and after 40 s of pressure application of 100 |M PHe (PHe +TG); in 11 cells immediately after break-in to the whole-cell configuration (Break-in), and 10 min after intracellular dialysis with 10 |M IP3 (10 min IP3). (B) Inset shows representative whole-cell currents from a single PCE cell in response to a 500-ms step depolarization to +58 mV from a holding potential of -62 mV. Current is solution containing 5 pM TG. Whole-cell current is then recorded in PCE cells, which are constantly superfused at a flow rate of 1 mL/min using the low Ca2+ extracellular solution containing 5 pM TG. The intracellular electrode solution in this experiment is the low-Ca2+ solution containing 10 mM BAPTA. Outward current is measured before and after pneumatic pressure application of 100 pM PHe dissolved in low-Ca2+ external solution. PCE cells from the same culture without TG pretreatment are used as control. In comparison to control PCE cells, TG pretreatment prevents PHe-mediated increases in IK(Ca) (Fig. 6A).

3. The PLC inhibitor U-73122 is employed to examine the effect of PLC inhibition on responses to PHe. Whole-cell currents are recorded before and after a 40-s pneumatic pressure application of 100 pM PHe in standard extracellular solution. The cell is then superfused at a flow rate of 1 mL/min for 5-10 min with standard extracellular solution containing 10 pM U-73122. The effect of PHe application on whole-cell outward current is then recorded in the presence of U-73122 and compared to control response in the absence of U-73122. PHe-mediated increases in IK(Ca) are blocked by U-73122 (Fig. 6B) confirming the involvement of PLC.

4. To examine the involvement of G-proteins in PHe-mediated increases in IK(Ca), whole-cell current is recorded in PCE cells dialyzed with 2 mM of the G-protein inhibitor GDPpS included in standard intracellular solution. The effect of GDPpS on the response of a cell to PHe is recorded 10 min after attaining the whole-cell configuration to ensure adequate exchange of the solution. The response of PCE cells from the same culture to PHe is also recorded following dialysis of the cells for 10 min with standard electrode solution containing 0.1 mM GTP. GDPpS significantly reduces the PHe-mediated increase in IK(Ca) compared to control cells from the same culture dialyzed with GTP (Fig. 7A).

5. The sensitivity of the PHe response to PTX is examined by treating PCE cells with PTX for 12-14 h (see Note 2). PTX blocks G-proteins of the Gi/o/z/t class by ADP ribosylating cysteine residues at the GTP binding site on the a-subunit (24).

Following PTX pretreatment, PCE cells are placed in the experimental chamber and superfused with standard external solution with K+ aspartate internal solution. The whole-cell current response of control and PTX-treated cells is then recorded after a test application of 100 mM phenylephrine (by pneumatic pressure injection for 40 s). PTX treatment has no effect on the PHe-mediated increase in IK(Ca) measured at +58 mV (Fig. 7B), confirming the involvement of a PTX-insensitive G protein(s) in arAR modulation of KCa channels.

Fig. 6. (continued from opposite page) shown after pressure application of 100 pM PHe in the same cell before (4) and after 20-30 min of incubation with 50 pM U-73122 (2). Histogram shows mean (±SEM) PHe-mediated increase in the current amplitude, normalized for cell capacitance, measured at +58 mV in 4 cells before (Con + U-73122) and after incubation with U-73122 (PHe + U73122).

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Fig. 7. G-proteins are involved in PHe-mediated IK(Ca) increases. (A) Histogram shows mean (±SEM) current amplitude measured at +58 mV with 0.1 mM GTP or 2 mM GDPPS in the pipet before (GTP; GDPpS) or after 40-s application of 100 pM PHe (PHe + GTP; PHe + GDPpS). (B) Mean (±SEM) current amplitude measured at +58 mV in 4 untreated cells before (Con) and after PHe application (+PHe), and in 4 cells pretreated with 500 ng/mL of PTX before (Con + PTX) and after PHe application (PHe + PTX). Data have been normalized for cell capacitance.

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Fig. 7. G-proteins are involved in PHe-mediated IK(Ca) increases. (A) Histogram shows mean (±SEM) current amplitude measured at +58 mV with 0.1 mM GTP or 2 mM GDPPS in the pipet before (GTP; GDPpS) or after 40-s application of 100 pM PHe (PHe + GTP; PHe + GDPpS). (B) Mean (±SEM) current amplitude measured at +58 mV in 4 untreated cells before (Con) and after PHe application (+PHe), and in 4 cells pretreated with 500 ng/mL of PTX before (Con + PTX) and after PHe application (PHe + PTX). Data have been normalized for cell capacitance.

3.6. Summary

Using patch-clamp recording techniques, we have demonstrated that IKCa in rabbit PCE cells can be activated by arARs coupled, via a PTX-insensitive G-protein to a PLC-dependent signaling pathway and intracellular IP3-sensitive Ca2+ stores. In vivo, released transmitters and hormones could modulate K current and provide for both paracrine and endocrine regulation of aqueous humor secretion by the ciliary epithelium.

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