FIGURE 2.

Iris PMT mechanism. (A) Expression of OPN4 and CRY transcripts in the chick iris. PCR was conducted on cDNA templates obtained by reverse transcription of iris sphincter muscle RNA isolated at E17 (2 separate experiments) using primers based on chicken-specific sequences. OPN4x and OPN4m denote chick Xenopus- and mammalian-like melanopsin. CRY1 and CRY2 denote CRY subtypes. Reverse transcriptase presence or absence in the PCR reactions is indicated by + and – symbols, respectively. Reaction products were of predicted size (bp). (B) Involvement of CRY over OPN4 photopigments in PMT. (Left) Incubating irises with the small molecule OPN4 phototransduction inhibitor AA92593 (10–20 μM, 1–3 h) had no effect on iris PMT relative to time-matched controls (n = 4 test and control irises). (Right) Consistent with involvement of Flavin-containing, redox-signaling CRY proteins, inhibition of flavin-specific reduction following incubation with DPI (10 μM, 3 h) or increasing oxidation with H₂O₂ (0.4 mM, 1–2 h) significantly reduced PMT by 63 ± 6 or 76 ± 8%, respectively, relative to time-matched controls (n = 4 test and control irises in both cases). (C) Ca²⁺ dynamics underlying iris PMT. (Left) PMT requires external Ca²⁺ influx. PMTRs were reduced by 94 ± 1% in irises incubated for 10–20 min in Ca²⁺-free RS buffered with 2 mM EGTA (n = 3 test and 3 control irises) and reduced by 45 ± 7% in irises incubated for 30 min in RS (normal Ca²⁺) containing 2 mM Cd²⁺ to inhibit influx via Ca²⁺ channels (n = 8 test and 8 control irises). (Right) Iris PMT requires Ca²⁺ release from an intracellular store. Incubation in RS containing Thapsigargin (TG, 3 uM, 1.5h) to block the SERCA pump reduced the light response by 55 ± 6% (n = 5 test and 5 control irises). IP3-receptor inhibition with 2-APB (30–50 μM, 1.5 h) or with Xestospongin-C (XeC, 2 μM, 1.5 h) had little or no significant effect (n = 4–6 test and time- matched control irises). By contrast, RyR mediated Ca²⁺ release appeared critical since Ryanodine (100 μM, 1.5 h) significantly reduced the PMT by 42 ± 4% (n = 5 test and 5 time matched control irises). Results are expressed as mean maximal PMTR (±SEM) for irises in test RS conditions (blue/white check columns) relative to control irises (blue columns) from the same experiments assayed in normal RS.