Biomedical subjects

T D Lamb

Publications and source records attributed to T D Lamb.

At least 37 records · Page 2Linked to original sources

PubMed1997

Rod plateaux during dark adaptation in Sorsby's fundus dystrophy and vitamin A deficiency.

PURPOSE: To investigate the transitory plateaux observed during dark adaptation after partial bleaches in Sorsby's fundus dystrophy (SFD) and in systemic vitamin A deficiency (VAD). METHODS: Psychophysical dark adaptation functions were measured after bleaching exposures isomerizing from 2% to 99% of the rhodopsin. Narrow-band stimuli of 1.7 degrees diameter and 200 msec duration were presented at an eccentricity of 30 degrees. RESULTS: After a full bleach, the patients showed typical dark adaptation abnormalities reported for these diseases. The cone recovery was slowed, and the time to the rod-cone break was delayed; the final phase of rod recovery was also slowed but led to a normal final rod threshold. After partial bleaches, short wavelength stimuli produced a biphasic recovery function, with an initial rapid component and plateau, followed by a subsequent break-off and eventual return to prebleach thresholds. Action spectra obtained during the plateaux were consistent with thresholds for shorter wavelength stimuli mediated by rods and thresholds for longer wavelength stimuli mediated by cones. In the patient with VAD, vitamin A supplementation led to accelerated recovery and disappearance of the transitory rod plateaux. CONCLUSIONS: The biphasic dark adaptation functions resulting from fractional bleaches in SFD and VAD appear superficially similar to the classic biphasic adaptation functions obtained with full bleaches. However, thresholds during the plateaux are lower than the cone threshold, and action spectra indicate rod mediation. These transitory rod plateaux may increase our understanding of the normal visual cycle and its perturbation in retinal disease.

Adult↗

PubMed1996

Kinetics of desensitization induced by saturating flashes in toad and salamander rods.

1. The suction pipette technique was used to examine the effect of a conditioning pre-flash on the saturation time (tsat) of a bright test flash (intensity 10,000-250,000 isomerizations) delivered to intact salamander or toad rod outer segments. The conditioning flash was delivered 0-60 s before the test flash; its intensity was typically between six and sixty times dimmer than the test flash, and it was sufficient by itself to fully saturate the photocurrent. 2. A saturating pre-flash delivered before a saturating test flash reduced the tsat of the test flash. This was equivalent to a reduction in phototransduction gain (psi). 3. The pre-flash had little effect on tau zero the time constant of decay of the rate-limiting species in photoresponse inactivation (activated rhodopsin or the activated G-protein-phosphodiesterase complex). 4. The tsat declined exponentially as the separation time between a fixed intensity pre-flash and test flash was increased. The time constant (tau p) of decline in tsat was approximately 2.4s. The maximum reduction in tsat corresponded to a reduction in the apparent gain of phototransduction to approximately 0.10 of its original level. This exponential decline is consistent with a [Ca2+]i-mediated effect. 5. We conclude that the rate-limiting step in response inactivation and the step responsible for light-induced gain reduction constitute separate and distinct steps of the phototransduction cascade.

Animals↗

PubMed1996

Gain and kinetics of activation in the G-protein cascade of phototransduction.

The guanine nucleotide binding protein (G protein) cascade underlying phototransduction is one of the best understood of all signaling pathways. The diffusional interactions of the proteins underlying the cascade have been analyzed, both at a macroscopic level and also in terms of the stochastic nature of the molecular contacts. In response to a single activated rhodopsin (R*) formed as a result of a single photon hit, it can be shown that molecules of the G-protein transducin will be activated approximately linearly with time. This, in turn, will cause the number of activated molecules of the effector protein (the phosphodiesterase) also to increase linearly with time. These kinetics of protein activation provide an accurate description of the time course of the rising phase of the photoreceptor's electrical response over a wide range of flash intensities. Recent estimates indicate that at room temperature each R* triggers activation of the phosphodiesterase at a rate of 1000-2000 subunits.s-1. Now that a quantitative description of the activation steps in transduction has been obtained, perhaps the greatest challenge for the future is to provide a comprehensive description of the shutoff reactions, so that a complete account of the photoreceptor's response to light can be achieved.

Amphibians↗

PubMed1996

Ida Mann Lecture. Transduction in human photoreceptors.

Phototransduction (the process by which light triggers a neural response in retinal rod and cone photoreceptors) is now understood at a molecular level. Indeed, the G-protein cascade of phototransduction is one of the best understood of all biological signalling pathways. The diffusional interactions of the proteins underlying the cascade are described and are briefly analysed. In response to a single activated rhodopsin (R*), formed as a result of a single photon hit, it can be shown that molecules of the G-protein will be activated (to G*) at an approximately constant rate. This, in turn, will cause the number of activated molecules of the third protein (the effector protein, E*, a phosphodiesterase) also to rise linearly with time. These kinetics of protein activation lead to an accurate description of the time-course of the rising phase of the photoreceptor's electrical response, both in single-cell recordings and also in recordings of the human electroretinogram (ERG). By analysing the a-wave of the ERG it is possible to determine the 'amplification' of transduction within living photoreceptors, and to begin to localise the molecular site of dysfunction is cases of photoreceptor abnormality.

Electroretinography↗

PubMed1995

Photoreceptor spectral sensitivities: common shape in the long-wavelength region.

Previous measurements of mammalian photoreceptor spectral sensitivity have been analysed, with particular attention to the long-wavelength region. The measurements selected for study come from rod and cone systems, and from human, monkey, bovine and squirrel sources. For the spectra from photoreceptor electrophysiology and from psychophysical sensitivity, the frequency scaling applied by Mansfield (1985, The visual system, pp. 89-106. New York: Alan Liss) provides a common shape over a range of at least 7 log10 units of sensitivity, from low frequencies (long wavelengths) to frequencies beyond the peak. The same curve is applicable to the absorbance spectrum of bovine rhodopsin, although the absorbance can only be measured down to about 2 log10 units below the peak. At the longest wavelengths the results exhibit a common limiting slope of 70 loge units (or 30.4 log10 units) per unit of normalized frequency. A simple equation is presented as a generic description for the alpha-band of mammalian photoreceptor spectral sensitivity curves, and it seem likely that the equation may be equally applicable to retinal1-based pigments in other species. Despite the lack of a theoretical basis, the equation has the correct asymptotic behaviour at long wavelengths, and it provides an accurate description of the peak. It also accounts accurately for the experimentally observed "yellowing" of long-wavelength lights that occurs beyond 700 nm.

Animals↗

PubMed1995

Transduction and adaptation in sensory receptor cells.

Sensory transduction shares common features in widely different sensory modalities. The purpose of this article is to examine the similarities and differences in the underlying mechanisms of transduction in the sensory receptor cells for vision, olfaction, and hearing. One of the major differences between the systems relates to the nature of the stimulus. In both the visual and olfactory systems a quantal mechanism of detection is possible, because the absorption of a photon or the binding of an odorant molecule provides an energy change significantly greater than the thermal noise in the receptor molecule. In hearing, on the other hand, the energy of a phonon is far lower, and detection occurs by a "classical" mechanism. For vertebrate photoreceptors and olfactory receptor cells, sensory transduction employs a G protein cascade that is remarkably similar in the two cases, and that is closely homologous to other G protein signaling cascades. For auditory and vestibular hair cells, transduction operates via a mechanism of direct coupling of the stimulus to ion channels, in a manner reminiscent of the direct gating of post-synaptic ion channels in various synaptic mechanisms. The three classes of sensory receptor cell share similarities in their mechanisms of adaptation, and it appears in each case that cytoplasmic calcium concentration plays a major role in adaptation.

Adaptation, Physiological↗

PubMed1994

Gating, selectivity and blockage of single channels activated by cyclic GMP in retinal rods of the tiger salamander.

1. Patches in the inside-out configuration were excised from the membrane of outer and inner segments of the larval tiger salamander, Ambystoma tigrinum. The current flowing through single channels opened by cyclic GMP was studied with the voltage clamp technique. 2. Amplitude histograms of current recordings from patches containing only one flickering channel, excised from the inner segment and in the presence of 100 microM cyclic GMP, could be fitted by a theoretical scheme in which the single channel conductance was at least 55 pS at +40 mV and at least 45 pS at -40 mV. The mean open time was no longer than the time constant of our recording system, about 35 microseconds. Similar results were obtained by analysis of the amplitude histograms of patches from the outer segment containing many channels, and in the presence of 1-5 microM cyclic GMP. 3. In membrane patches excised from the outer segment, reducing the temperature from 24 to 8 degrees C did not reduce the flickering, but changed the amplitude histograms of current fluctuations activated by 1 microM cyclic GMP in a way consistent with a decrease of 50% in the single channel conductance and a decrease of 50% in the open probability. 4. In the presence of 1 microM cyclic GMP at +60 mV, when Na+ was replaced by NH4+ or K+, brief outward current transients flowing through single channels were observed. When Na+ was replaced with Li+, Rb+ or Cs+, current transients were very small. 5. The shape of the power spectrum of current fluctuations induced by 1 microM cyclic GMP at +60 mV did not change when the permeating ion was Na+, K+ or NH4+. Analysis of the amplitude histogram did not show any effect of the tested monovalent cations on the open probability or on channel gating. At +60 mV, the estimated single channel currents were at least 4, 2.8 and 2 pA for NH4+, Na+ and K+ respectively. 6. The addition of 0.5 or 1 mM Ca2+ to the medium bathing the cytoplasmic side of the membrane greatly reduced the frequency of openings, but single channel activity could still be observed. The blocking effect of 1 mM Ca2+ on the channel activity induced by 2 microM cyclic GMP could be counterbalanced by increasing the cyclic GMP concentration. The addition of 0.5 or 1 mM Ca2+ did not change the shape of power spectra obtained at membrane voltages between -100 and +100 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

Ambystoma↗

PubMed1994

Dark adaptation of toad rod photoreceptors following small bleaches.

The recovery of toad rod photoreceptors, following exposure to intense lights that bleached 0.02-3% of the rhodopsin, has been investigated using the suction pipette technique. The post-bleach period was accompanied by reduced flash sensitivity, accelerated kinetics, and spontaneous fluctuations (noise). The power spectrum of the fluctuations had substantially the form expected for the random occurrence of single-photon events, and the noise could therefore be expressed as a "photon-noise equivalent intensity". From the level of desensitization at any time, the after-effect of the bleach could also be expressed in terms of a "desensitization-equivalent intensity", and this was found to be at least a factor of 20 times higher than the noise-equivalent intensity at the corresponding time. Our results indicate that a bleach induces two closely-related phenomena: (a) a process indistinguishable from the effect of real light, and (b) another process which desensitizes and accelerates the response in the same way that light does, but without causing photon-like noise. We propose a mechanism underlying these processes.

Animals↗

PubMed1994

Stochastic simulation of activation in the G-protein cascade of phototransduction.

Activation of the G-protein cascade underlying phototransduction has been modeled by simulating the two-dimensional diffusional interactions that occur at the rod disc membrane between the three reacting protein species, which are the activated rhodopsin (R*), the G-protein (G), and the effector protein (E, the phosphodiesterase, PDE). The stochastic simulations confirm the main predictions of a simplified analytical model (Lamb, T. D., and E. N. Pugh, 1992, Journal of Physiology 449:719-758), and extend that treatment to more complicated cases, where there is a finite probability of reaction or a finite time for reaction. The simulations also provide quantitative estimates of the efficiency of coupling from activated G-protein (G*) to activated effector (E*) in terms of the concentrations, lateral diffusion coefficients, and binding rate constants of the participating molecules; the efficiency of coupling from G* to E* is found to be not as high as in the previous simplified analytical theory. The findings can be extended to other G-protein cascades, provided that the physical parameters of those cascades are specified.

Animals↗

PubMed1994

Analysis of ERG a-wave amplification and kinetics in terms of the G-protein cascade of phototransduction.

PURPOSE: To test rigorously the hypothesis that the a-wave of the electroretinogram (ERG) is proportional to the rod photocurrent by examining the applicability to a-waves of a recent model of the activation steps in the G-protein cascade of phototransduction. METHODS: ERGs were recorded in response to flashes of graded intensity, from six dark-adapted normal subjects and from two patients, one with retinitis pigmentosa (RP) and one with cone retinal dystrophy with rod involvement (CRD). The a-wave portions of the responses were analyzed with a model of the activation steps of the G-protein cascade. The model is characterized by a parameter, A, the amplification constant, with units of s-2 (per photoisomerization), which may be expressed as the product of physical and biochemical parameters of the transduction cascade. RESULTS: Each a-wave family was well described by the model. For the six normal subjects, we obtained A approximately 7 s-2, about 100-fold greater than in isolated amphibian rods at 22 degrees C, but close to the value for isolated primate rods. For the patient with RP, the maximum a-wave amplitude (amax) was considerably reduced, but the amplification constant was normal (A = 7.5 s-2). In contrast, the patient with CRD had a nearly normal amax but had an amplification constant about sixfold lower than normal (A = 1.1 s-2). CONCLUSIONS: The authors conclude that the a-wave is a direct reflection of the rod photo-current and that the rising phase kinetics are accurately described by a simple model of the G-protein cascade. They show that the small volume of the human rod outer segment is crucial to the achievement of high amplification, and they show how their observations constrain the possible pathologies of phototransduction in patients with retinal disease.

Adolescent↗

PubMed1993

Amplification and kinetics of the activation steps in phototransduction.

We can summarize our investigation of amplification in the activation steps of vertebrate phototransduction as follows. (1) A theoretical analysis of the activation steps of the cGMP cascade shows that after a brief flash of phi photoisomerizations the number of activated PDE molecules should rise as a delayed ramp with slope proportional to phi, and that, as a consequence, the cGMP-activated current should decay as a delayed Gaussian function of time (Eqn. 20). (i) Early in the response to a flash, the normalized response R(t) can be approximated as rising as 1/2 phi At2 (after a short delay), where A is the amplification constant characteristic of the individual photoreceptor. (ii) The delayed ramp behavior of PDE activation and the consequent decline of current in the form of the delayed Gaussian are confirmed by experiments in a variety of photoreceptors; the analysis thus yields estimates of the amplification constant from these diverse photoreceptors. (iii) Eqn. 20 further predicts that the response-intensity relation at any fixed time should saturate exponentially, as has been found experimentally. (2) The amplification constant A can be expressed as the product of amplification factors contributed by the individual activation steps of phototransduction, i.e., A = nu RG cGP beta sub n (Eqns. 9 and 21), where (i) nu RG is the rate of G* production per Rh*; (ii) cGP is the efficiency of the coupling between G* production and PDE* production; (iii) beta sub is the increment in hydrolytic rate constant produced by one PDE*, i.e., a single activated catalytic subunit of PDE; and (iv) n is the Hill coefficient of opening of the cGMP-activated channels. (3) The amplification factor beta sub includes the ratio kcat/Km, which characterizes the hydrolytic activity of the PDE in vivo where cG << Km. Two different analyses based upon photocurrents were developed which provide lower bounds for kcat/Km in vivo; these analyses establish that kcat/Km probably exceeds 10(7) M-1 s-1 (and is likely to be higher) in both amphibian and mammalian rods. Few biochemical studies (other than those using trypsin activation) have yielded such high values. A likely explanation of many of the relatively low biochemical estimates of kcat/Km is that Km may have been overestimated by a factor of about 4 in preparations in which stacks of disks are left intact, due to diffusion with hydrolysis in the stacks.(ABSTRACT TRUNCATED AT 400 WORDS)

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed1992

Different channel-gating properties of two classes of cyclic GMP-activated channel in vertebrate photoreceptors.

We report that two types of cGMP-activated channel coexist in the photoreceptor plasma membrane, with the most commonly encountered class appearing broadly similar to the channel reported in previous patch-pipette experiments. However, we find that flickering of this channel between the open and closed states is so rapid that a discrete single-channel conductance cannot unequivocally be resolved; the occurrence of flickering is largely independent of membrane voltage and of the presence of cytoplasmic Ca2+ or Mg2+. In recordings from the inner segment we occasionally find a second class of cGMP-gated channel, with activity resembling that reported for cloned channels. This channel does not flicker, but instead exhibits distinct open-close transitions. Our results suggest that the predominant form of channel in vivo differs significantly from cloned channels, and that its gating properties are not as simple as reported previously.

Ambystoma↗

PubMed1992

G-protein cascades: gain and kinetics.

G-protein cascades provide amplification in a wide variety of biological signal transducers--from hormonal and synaptic systems to the receptor cells of vision and olfaction. Through recent understanding of the molecular mechanisms involved, it is possible to construct a quantitative description of the amplification and speed of response of the cascade. The gain and kinetics can now be described in terms of physical parameters, such as enzyme activities and the densities and lateral diffusion coefficients of the proteins involved.

Animals↗

PubMed1992

A quantitative account of the activation steps involved in phototransduction in amphibian photoreceptors.

1. We have undertaken a theoretical analysis of the steps contributing to the phototransduction cascade in vertebrate photoreceptors. We have explicitly considered only the activation steps, i.e. we have not dealt with the inactivation reactions. 2. From the theoretical analysis we conclude that a single photoisomerization leads to activation of the phosphodiesterase (PDE) with a time course which approximates a delayed ramp; the delay is contributed by several short first-order delay stages. 3. We derive a method for extracting the time course of PDE activation from the measured electrical response, and we apply this method to recordings of the photoresponse from salamander rods. The results confirm the prediction that the time course of PDE activation is a delayed ramp, with slope proportional to light intensity; the initial delay is about 10-20 ms. 4. We derive approximate analytical solutions for the electrical response of the photoreceptor to light, both for bright flashes (isotropic conditions) and for single photons (involving longitudinal diffusion of cyclic GMP in the outer segment). The response to a brief flash is predicted to follow a delayed Gaussian function of time, i.e. after an initial short delay the response should begin rising in proportion to t2. Further, the response-intensity relation is predicted to obey an exponential saturation. 5. These predictions are compared with experiment, and it is shown that the rising phase of the flash response is accurately described over a very wide range of intensities. We conclude that the model provides a comprehensive description of the activation steps of phototransduction at a molecular level.

Animals↗

PubMed1990

Cyclic GMP and calcium: the internal messengers of excitation and adaptation in vertebrate photoreceptors.

The roles of cyclic GMP (cGMP) and calcium (Ca2+) in vertebrate rod phototransduction are reviewed, with the emphasis on developments since the discovery of the cGMP-activated conductance of the rod outer segment. The first hypothesis subjected to critical examination is that cGMP acts as the sole internal messenger of excitation. This hypothesis is evaluated with a formal, quantitative model of the biochemical actions of cGMP. Application of the model shows a remarkable agreement between independent electrophysiological and biochemical measurements of the resting dark amounts of (1) total cGMP (2) free cGMP (3) fraction of open cGMP-activated channels and (4) the rate of cGMP hydrolysis. The second hypothesis examined is that Ca2+ acts as an internal messenger in rod light adaptation. Recent electrophysiological evidence has shown minimization of the normal light-induced reduction of free Ca2+ prevents rods from exhibiting the change in sensitivity and speed characteristic of light adaptation. Physiological effects, formerly attributed to a role of calcium as an excitational messenger are shown to be consistent with a biochemical model in which Ca2+ serves as the cytoplasmic signal in a powerful feedback loop that acts to restore the concentration of cGMP both during and after exposure to light. Residual problems facing the "cGMP cascade theory of phototransduction" are reviewed. Issues are itemized that will have to be resolved quantitatively before it will be possible to develop a fully comprehensive theory of photoreceptor excitation, restoration and adaptation combining the roles of Ca2+ and cGMP.

Adaptation, Physiological↗

PubMed1990

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.

Action Potentials↗

PubMed1989

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)

Adaptation, Ocular↗

PubMed1988

Photoreceptor light adaptation is mediated by cytoplasmic calcium concentration.

The vertebrate visual system can operate over a large range of light intensities. This is possible in part because the sensitivity of photoreceptors decreases approximately in inverse proportion to the background light intensity. This process, called photoreceptor light adaptation, is known to be mediated by a diffusible intracellular messenger, but the identity of the messenger is still unclear. There has been considerable speculation that decreased cytoplasmic Ca2+ concentration (Cai2+) may play a role in light adaptation, and recent experiments in which Ca2+ buffer was incorporated into rod-cells have supported this notion. The extent of the contribution of calcium, however, remains unresolved. We now show that light-dependent changes in sensitivity in amphibian photoreceptors can be abolished by preventing movements of Ca2+ across the outer-segment plasma membrane. These experiments demonstrate that light adaptation in photoreceptors is mediated in cones primarily, and in rods perhaps exclusively, by changes in Cai2+.

Adaptation, Ocular↗