Hugh R. Matthews

Measurement of cytoplasmic calcium concentration in the rods of wild-type and transducin knock-out mice

A 10 μm spot of argon laser light was focused onto the outer segments of intact mouse rods loaded with fluo‐3, fluo‐4 or fluo‐5F, to estimate dark, resting free Ca2+ concentration ([Ca2+]i) and changes in [Ca2+]i upon illumination. Dye concentration was adjusted to preserve the normal physiology of the rod, and the laser intensity was selected to minimise bleaching of the fluorescent dye. Wild‐type mouse rods illuminated continuously with laser light showed a progressive decrease in fluorescence well fitted by two exponentials with mean time constants of 154 and 540 ms. Rods from transducin α‐subunit knock‐out (Trα–/–) animals showed no light‐dependent decline in fluorescence but exhibited an initial rapid component of fluorescence increase which could be fitted with a single exponential (τ∼1–4 ms). This fluorescence increase was triggered by rhodopsin bleaching, since its amplitude was reduced by pre‐exposure to bright bleaching light and its time constant decreased with increasing laser intensity. The rapid component was however unaffected by incorporation of the calcium chelator BAPTA and seemed therefore not to reflect an actual increase in [Ca2+]i. A similar rapid increase in fluorescence was also seen in the rods of wild‐type mice just preceding the fall in fluorescence produced by the light‐dependent decrease in [Ca2+]i. Dissociation constants were measured in vitro for fluo‐3, fluo‐4 and fluo‐5F with and without 1 mm Mg2+ from 20 to 37 °C. All three dyes showed a strong temperature dependence, with the dissociation constant changing by a factor of 3–4 over this range. Values at 37 °C were used to estimate absolute levels of rod [Ca2+]i. All three dyes gave similar values for [Ca2+]i in wild‐type rods of 250 ± 20 nm in darkness and 23 ± 2 nm after exposure to saturating light. There was no significant difference in dark [Ca2+]i between wild‐type and Trα–/– animals.

Cytoplasmic calcium as the messenger for light adaptation in salamander rods.

1. In order to study the role of cytoplasmic calcium concentration (Ca2+i) in rod photoreceptor light adaptation, we have attempted to prevent light‐induced changes in Ca2+i by minimizing calcium fluxes across the outer segment plasma membrane. This was achieved by exposing the outer segment to a low‐Ca2+, 0‐Na+ solution, in which sodium was replaced with either guanidinium or lithium and the external calcium concentration (Ca2+o) was reduced to micromolar levels. 2. With guanidinium and 1‐3 microM‐Ca2+o, the circulating current in darkness was maintained for a period of at least 15 s, consistent with approximate stability of Ca2+i. With Li+ rather than guanidinium most of the initial current was suppressed, but the residual current was again relatively stable. 3. During prolonged exposures (greater than 30 s) to low‐Ca2+, 0‐Na+ solution followed by dim illumination, the circulating current did not remain constant but slowly increased. Incorporation of calcium buffer into the cytoplasm greatly reduced the rate of change of current, consistent with the idea that the increase arose from a gradual decrease in Ca2+i. 4. Light responses of rods exposed to low‐Ca2+, 0‐Na+ solution in darkness were altered in a characteristic manner. Although the initial rising phase of the light response was little changed, the peak amplitude of the response was larger and occurred later, and the response decayed more slowly than in control. The response‐intensity relation was steepened and was shifted towards lower intensities both for flashes and for steps of light. The normal sag in the response to steps disappeared, and the waveform of the step response could be predicted to a close approximation from the integral of the dim flash response. 5. Presentation of background illumination in Ringer solution produced a marked acceleration of the response to a subsequent bright flash. No such acceleration was observed if the background was given in low‐Ca2+, 0‐Na+ solution. 6. The results described in paragraphs 4 and 5 indicate that, under conditions expected to minimize changes in Ca2+i, all manifestations of light adaptation disappear, and the rod simply sums the effects of incident photons with an invariant integration time. 7. Exposure of a light‐adapted rod to low‐Ca2+, 0‐Na+ solution altered the responses to superimposed test flashes in much the same way as for rods in darkness. The initial rising phases in low‐Ca2+, 0‐Na+ solution were unchanged, but the responses were larger, reached peak later and decayed more slowly.(ABSTRACT TRUNCATED AT 400 WORDS)

Response properties of isolated mouse olfactory receptor cells

1 Response properties of isolated mouse olfactory receptor cells were investigated using the suction pipette technique. Cells were exposed to the odour cineole or to solutions of modified ionic content by rapidly changing the solution superfusing the cilia. All experiments were performed at 37°C. 2 Mouse olfactory receptor cells displayed a steep dependence of action potential frequency on stimulus concentration, a 3‐fold increase in stimulus concentration often saturating the firing frequency at 200‐300 Hz. The receptor current increased more gradually with increasing cineole concentration and did not saturate within the 100‐fold range of cineole concentrations applied. 3 When stimulated for 30 s with a low odour concentration, cells responded with sporadic spike firing. Higher concentrations led to the generation of a large receptor current at the onset of stimulation which returned to baseline levels within a few seconds, accompanied during its rising phase by a short burst of action potentials. Thereafter an oscillating response pattern was observed during the remainder of the stimulus, consisting of repetitive increases in receptor current of around 1 s duration accompanied by short bursts of action potentials. 4 Olfactory adaptation was studied by comparing the responses to two closely spaced odour stimuli. The response to the second odour stimulus recovered to 80% of its original magnitude when the cell was superfused with Ringer solution during the 5 s interval between odour exposures. In contrast, exposure to a choline‐substituted low Na+ solution between odour stimuli had two effects. First, the receptor current response to the first odour stimulus did not terminate as quickly as in the presence of Na+, suggesting the presence of a Na+‐Ca2+ exchanger. Second, the response to the second stimulus only recovered to 55% of its original magnitude, demonstrating the involvement of Na+‐Ca2+ exchange in the recovery of sensitivity in mouse olfactory receptor cells following stimulation.

Calcium, the two-faced messenger of olfactory transduction and adaptation.

Exposure of olfactory receptor cells to odour stimulates the influx of Ca(2+) through cyclic nucleotide-gated channels into the small volume within the cilia, the site of olfactory transduction. The consequent rise in intraciliary Ca(2+) concentration has two opposing effects: activation of an unusual excitatory Cl(-) conductance, and negative feedback actions on various stages of the odour transduction mechanism. Recent studies are beginning to unravel how Ca(2+) performs this dual function, and how the spatial and temporal dynamics of Ca(2+) modulate the odour response. The feedback actions of Ca(2+) on different elements of the transduction cascade seem to occur on different timescales, and are therefore responsible for shaping different parts of the receptor current response to odour stimulation.

Calcium and the mechanism of light adaptation in vertebrate photoreceptors.

Vertebrate photoreceptors transduce the absorption of light into a hyperpolarizing change in membrane potential. The mechanism of transduction is becoming fairly well understood and has been shown to occur via a G protein-coupled decrease in cyclic GMP. Attention is now turning to the way the enzymatic machinery in the outer segment of the photoreceptor cell is modulated during light adaptation. Recent studies show that light adaptation cannot occur if changes in the concentration of cytoplasmic free calcium in the outer segment are prevented, suggesting that calcium functions as a second messenger in sensitivity regulation.

Light adaptation in cone photoreceptors of the salamander: a role for cytoplasmic calcium.

1. Light adaptation has been studied in isolated red‐sensitive cone photoreceptors of the salamander, using suction pipette recordings of circulating current. 2. In the presence of background illumination, the response to incremental dim flashes became desensitized according to the Weber‐Fechner law. The recovery phase of the flash response was accelerated significantly, although the time‐to‐peak was reduced only slightly, and for dim backgrounds the rising phase was unaltered. 3. The role of cytoplasmic calcium concentration, Cai2+, in mediating cone adaptation was investigated by minimizing light‐induced changes in Cai2+, either by incorporating calcium buffer into the cytoplasm or by exposing the outer segment to low‐Ca2+, 0‐Na+ solution. Both treatments appeared to slow dramatically or even to eliminate the onset of light adaptation in the cone. 4. When the low‐Ca2+, 0‐Na+ solution was presented in darkness, responses to subsequent illumination were affected in a characteristic manner: (i) the response‐intensity relation was steepened and shifted to lower intensities, (ii) the response to a step of light could be predicted by integration and compression of the flash response, and (iii) the flash sensitivity declined steeply as a function of background intensity. 5. After extended exposure of the cone to bright backgrounds, the sensitivity in darkness failed to return to its original level. The flash response kinetics were faster and more biphasic than for dark‐adapted responses or for responses desensitized to a comparable degree by exposure to steady background illumination. 6. The results indicate that, in cones isolated from the pigment epithelium, the primary factor influencing the adaptational state of the cell is the cytoplasmic concentration of free calcium, but that at high intensities the effects of pigment bleaching are likely to be significant.

Adaptation of the odour‐induced response in frog olfactory receptor cells

1 Receptor current and spiking responses were recorded simultaneously from isolated frog olfactory receptor cells using the suction pipette technique. Cells were stimulated with the odour cineole by rapid exchange of the solution bathing the olfactory cilia. 2 The receptor current response to a 1 s odour stimulus increased in a graded manner over a 300‐fold range of odour concentration without clear saturation, and was accompanied by a train of action potentials. As the concentration of the odour stimulus increased, the frequency of firing increased also, until it saturated at the highest concentrations. The number of spikes evoked by the stimulus first increased and then decreased with increasing concentration, reaching a maximum at intermediate odour concentrations. The dose‐response relation for spike firing rose at lower odour concentrations than the dose‐response relation for the receptor current response. 3 Adaptation to steady odour stimuli was investigated by exposing the cilia to a 4 s odour pre‐pulse and then to a 1 s odour test pulse. As the pre‐pulse concentration was increased the dose‐response relations derived from the receptor current and spiking responses shifted to higher absolute test pulse concentrations. However the number of spikes fired in response to a given test pulse was little affected by the pre‐pulse until, at the highest pre‐pulse concentrations spike firing was abolished despite the continued presence of a receptor current response. 4 The sensitivity of the receptor‐current response to incremental stimuli fell with increasing pre‐pulse concentration, declining with a limiting slope of 2.4 in double logarithmic co‐ordinates. The sensitivity determined from the spiking responses declined to zero at a lower pre‐pulse concentration, reflecting the abolition of spike firing at pre‐pulse concentrations which still evoked a graded receptor‐current response.

Dark adaptation in vertebrate photoreceptors

Exposure of the eye to bright light bleaches a significant fraction of the photopigment in rods and cones and produces a prolonged decrease in the sensitivity of vision, which recovers slowly as the photopigment is regenerated. This sensitivity decrease is larger than would be expected merely from the decrease in the concentration of the pigment. Recent experiments have shown that the decrease in sensitivity is produced largely by an excitation of the phototransduction cascade by bleached pigment; even in darkness, it produces an equivalent background similar to that produced by real steady background illumination. Thus, excitation produced by a form of rhodopsin thought previously to be inactive has a profound effect on the physiology of the photoreceptor. This raises the possibility that forms of other G protein-coupled receptors thought to be inactive might also play an important role in signal transduction and disease.

Responses to prolonged odour stimulation in frog olfactory receptor cells

1 The suction pipette technique was used to record receptor current and spiking responses from isolated frog olfactory receptor cells during prolonged odour stimuli. 2 The majority (70 %) of cells displayed ‘oscillatory’ responses, consisting of repeated bursts of spikes accompanied by regular increases in receptor current. The period of this oscillation varied from 3.5 to 12 s in different cells. The remaining cells responded either with a ‘transient’ burst of spikes at the onset of stimulation (10 %), or by ‘sustained’ firing throughout the odour stimulus (20 %). 3 In cells with oscillatory responses, the Ca2+‐activated Cl− channel blocker niflumic acid prolonged the period of oscillation only slightly, despite a 3.8‐fold decrease in the receptor current. A 3‐fold reduction in the external Cl− concentration nearly doubled the receptor current, but had little effect on the oscillation period. These results imply that the majority of the receptor current underlying these oscillatory responses is carried by the Ca2+‐activated Cl− conductance, suggesting that the intracellular Ca2+ concentration oscillates also. 4 In cells with oscillatory responses, the period of oscillation was prolonged 1.5‐fold when stimulated in a low‐Na+ solution designed to incapacitate Na+‐Ca2+ exchange, irrespective of whether Na+ was replaced by permeant Li+ or impermeant choline. The dependence of the oscillation period upon external Na+ suggests that it may be governed by the dynamics of Ca2+ extrusion via Na+‐Ca2+ exchange. 5 Exposure to the membrane‐permeable cyclic nucleotide analogue CPT‐cAMP evoked a sustained rather than an oscillatory response even in cells with oscillatory responses to odour. The inability of CPT‐cAMP to evoke an oscillatory response suggests that the cAMP concentration is likely to oscillate also. 6 Perforated‐patch recordings revealed that oscillatory responses could only be evoked when the membrane potential was free to change, but not when it was clamped near the resting potential. Since substantial changes in Ca2+‐activated Cl− current, and hence odour‐induced depolarisation, had little effect upon the period of oscillation, changes in membrane potential are suggested to play only a permissive role in these oscillatory responses. 7 These results are interpreted in terms of the coupled oscillation of Ca2+ and cyclic nucleotide concentrations within the olfactory cilia during prolonged odour stimulation.

Effects of lowered cytoplasmic calcium concentration and light on the responses of salamander rod photoreceptors.

1. In order to study the interactions between cytoplasmic calcium concentration ([Ca2+]i) and light in modulating the responses of rod photoreceptors, [Ca2+]i was held at different levels by manipulating Ca2+ fluxes across the outer segment membrane. 2. If [Ca2+]i was reduced by the removal of external Ca2+ in the continued presence of Na+, and then held near this reduced level by exposure to 0 Ca(2+)‐0 Na+ solution, the onset of the recovery phase of the response to a bright flash delivered just before the return to Ringer solution was accelerated, much as is the case during light adaptation, provided that precautions were taken to minimize Na+ influx. 3. If the rod was first allowed to adapt to steady light, [Ca2+]i held near the appropriate light‐adapted level by superfusion with 0 Ca(2+)‐0 Mg(2+)‐0 Na+ solution and the light extinguished, the onset of the recovery phase of the bright flash response varied with the original background intensity in the same way as in the continued presence of steady light. These results indicate that reduction of [Ca2+]i is sufficient to induce this manifestation of light adaptation in darkness. 4. When [Ca2+]i was held at a reduced level in darkness, not only was the sensitivity to dim flashes reduced, but the response rising phase was also delayed and its amplitude increased supralinearly with flash intensity, neither of which changes is seen during light adaptation. However, similar changes in response kinetics resulted when [Ca2+]i was held near its normal dark level and the phosphodiesterase was partially inhibited by 3‐isobuty‐1‐methylxanthine (IBMX), suggesting that they arose indirectly from an elevated cyclic GMP concentration rather than from a direct effect of Ca2+. 5. If [Ca2+]i was held near the normal dark level and bright steady light presented, the circulating current was completely suppressed. Partial inhibition of the phosphodiesterase by superfusion with 0 Ca(2+)‐0 Na+ solution including IBMX resulted in restoration of the circulating current. Dim flash responses recorded under these conditions exhibited kinetics similar to those recorded in 0 Ca(2+)‐0 Na+ solution in darkness, in contrast to the response acceleration seen when [Ca2+]i was held near the appropriate light‐adapted level. These results indicate that the kinetics of the flash response depend on [Ca2+]i rather than on the steady light intensity.