Abstract
Purpose: :
The rod phototransduction cascade contains two steps of amplification, rhodopsin (R*) and phosphodiesterase (E*) activation. The R* shut-off is known to be accelerated with lowered intracellular [Ca2+], while the E* inactivation is thought to be calcium-independent. Recently, compelling evidence has been given by Krispel et al. (Neuron 51(4): 391-3, 2006) that the rate-limiting step of rod inactivation is the shut-off of E*. Therefore it is expected that removal of calcium should not affect the dominant time constant of photoresponse recovery (TD). The effect of lowered external [Ca2+] on TD was tested.
Methods: :
Saturated ERG photoresponses to flashes of increasing stimulus intensities were recorded at 25 oC across isolated mouse (Mus musculus) retinas perfused at the photoreceptor side. The b-wave and higher-order neuron components were blocked with 2 mM aspartate and the glial component was removed by adding barium (10 mM BaCl2) into the electrode space in contact with the proximal side of the retina. The normal Ringer contained 1 mM Ca2+ while the free [Ca2+] in the low-Ca2+ solution (containing 0,1 mM Ca2+ + 2 mM Mg2+ + 0,4 mM EGTA) was ~ 12 nM. The dominant recovery time constants (TD) were determined from the semilog "Pepperberg plots" (Pepperberg et al., Vis Neurosci. 8(1):9-18, 1992).
Results: :
The average value of TD at 25 oC was 340 ms in normal Ringer and 650 ms in low Ca2+ (11 retinas). The increase in TD becomes apparent only in very low [Ca2+]free (~10-8 M) but is completely reversible. Two possible sources for the extended TD in low [Ca2+] were hypothesized: 1) Increased outer segment [cGMP], or 2) decreased [ATP]/[GTP] due to increased ATPase activity (Winkler and Riley, IOVS 16(12): 1151-54, 1977). Hypothesis 1) was tested by application of moderate background lights in low Ca2+ (up to ~500 Rh*/s/rod) in order to decrease [cGMP]. Background light did not shorten TD. Also, application of IBMX (30 and 100 µM) to increase [cGMP] in normal Ringer did not affect TD. Hypothesis 2) was tested by replacing 128 mM of Na+ by choline in the low-Ca2+ solution in order to decrease Na-K ATPase activity and thereby to raise ATP/GTP levels. This did not shorten TD in low [Ca2+].
Conclusions: :
Our results suggest that either a) the inactivation mechanism of PDE* contains a calcium-dependent component or b) in low Ca2+ some other mechanism, e.g. the inactivation of R*, comes over as the rate-limiting step of photoresponse termination.
Keywords: photoreceptors • calcium • electrophysiology: non-clinical