A novel SERPINA12 variant and first European patients with diffuse palmoplantar keratoderma.

BACKGROUND: Hereditary palmoplantar keratodermas (hPPKs) comprise a heterogeneous group of skin disorders characterized by persistent palmoplantar hyperkeratosis. Loss-of-function variants in a serine peptidase inhibitor, SERPINA12, have recently been implicated in autosomal recessive diffuse hPPK. The disorder appears to share similarities with another hPPK associated with protease overactivity, namely Nagashima-type PPK (NPPK) caused by biallelic variants in SERPINB7. OBJECTIVES: The aim of this study was to enhance the understanding of the clinical and genetic characteristics of serine protease-related hPPKs caused by variants in SERPINA12 and SERPINB7. METHODS: Whole-exome sequencing (WES) was performed for hPPK patients. Haplotype analysis was completed for the patients with identified recessive SERPINA12 variants and their available family members. In addition, the current literature of SERPINA12- and SERPINB7-related hPPKs was summarized. RESULTS: The phenotype of SERPINA12-related hPPK was confirmed by reporting three new SERPINA12 patients, the first of European origin. A novel SERPINA12 c.1100G>A p.(Gly367Glu) missense variant was identified confirming that the variant spectrum of SERPINA12 include both truncating and missense variants. The previously reported SERPINA12 c.631C>T p.(Arg211*) was indicated enriched in the Finnish population due to a plausible founder effect. In addition, SERPINA12 hPPK patients were shown to share a similar phenotype to patients with recessive variants in SERPINB7. The shared phenotype included diffuse transgradient PPK since birth or early childhood and frequent palmoplantar hyperhidrosis, aquagenic whitening and additional hyperkeratotic lesions in non-palmoplantar areas. SERPINA12 and SERPINB7 hPPK patients cannot be distinguished without genetic analysis. CONCLUSIONS: Recessive variants in SERPINA12 and SERPINB7 leading to protease overactivity and hPPK produce a similar phenotype, indistinguishable without genetic analysis. SERPINA12 variants should be assessed also in non-Asian patients with diffuse transgradient PPK. Understanding the role of serine protease inhibitors will provide insights into the complex proteolytic network in epidermal homeostasis.

Mutations in the skeletal muscle alpha-actin gene in patients with actin myopathy and nemaline myopathy.

Muscle contraction results from the force generated between the thin filament protein actin and the thick filament protein myosin, which causes the thick and thin muscle filaments to slide past each other. There are skeletal muscle, cardiac muscle, smooth muscle and non-muscle isoforms of both actin and myosin. Inherited diseases in humans have been associated with defects in cardiac actin (dilated cardiomyopathy and hypertrophic cardiomyopathy), cardiac myosin (hypertrophic cardiomyopathy) and non-muscle myosin (deafness). Here we report that mutations in the human skeletal muscle alpha-actin gene (ACTA1) are associated with two different muscle diseases, 'congenital myopathy with excess of thin myofilaments' (actin myopathy) and nemaline myopathy. Both diseases are characterized by structural abnormalities of the muscle fibres and variable degrees of muscle weakness. We have identified 15 different missense mutations resulting in 14 different amino acid changes. The missense mutations in ACTA1 are distributed throughout all six coding exons, and some involve known functional domains of actin. Approximately half of the patients died within their first year, but two female patients have survived into their thirties and have children. We identified dominant mutations in all but 1 of 14 families, with the missense mutations being single and heterozygous. The only family showing dominant inheritance comprised a 33-year-old affected mother and her two affected and two unaffected children. In another family, the clinically unaffected father is a somatic mosaic for the mutation seen in both of his affected children. We identified recessive mutations in one family in which the two affected siblings had heterozygous mutations in two different exons, one paternally and the other maternally inherited. We also identified de novo mutations in seven sporadic probands for which it was possible to analyse parental DNA.

Weber and noise adaptation in the retina of the toad Bufo marinus.

Responses to flashes and steps of light were recorded intracellularly from rods and horizontal cells, and extracellularly from ganglion cells, in toad eyecups which were either dark adapted or exposed to various levels of background light. The average background intensities needed to depress the dark-adapted flash sensitivity by half in the three cell types, determined under identical conditions, were 0.9 Rh*s-1 (rods), 0.8 Rh*s-1 (horizontal cells), and 0.17 Rh*s-1 (ganglion cells), where Rh* denotes one isomerization per rod. Thus, there is a range (approximately 0.7 log units) of weak backgrounds where the sensitivity (response amplitude/Rh*) of rods is not significantly affected, but where that of ganglion cells (1/threshold) is substantially reduced, which implies that the gain of the transmission from rods to the ganglion cell output is decreased. In this range, the ganglion cell threshold rises approximately as the square root of background intensity (i.e. in proportion to the quantal noise from the background), while the maintained rate of discharge stays constant. The threshold response of the cell will then signal light deviations (from a mean level) of constant statistical significance. We propose that this type of ganglion cell desensitization under dim backgrounds is due to a post-receptoral gain control driven by quantal fluctuations, and term it noise adaptation in contrast to the Weber adaptation (desensitization proportional to the mean background intensity) of rods, horizontal cells, and ganglion cells at higher background intensities.

Refined localisation of the genes for nebulin and titin on chromosome 2q allows the assignment of nebulin as a candidate gene for autosomal recessive nemaline myopathy.

A locus for autosomal recessive nemaline myopathy (NEM2) has been assigned by linkage analysis to a 13-cM region between the markers D2S150 and D2S142 on 2q21.2-q22. The genes for the giant muscle proteins nebulin and titin have previously been assigned by FISH to 2q24.1-q24.2 and 2q31, respectively. By using radiation hybrid mapping, we have reassigned the nebulin gene close to the microsatellite marker D2S2236 on 2q22 and the titin gene to the vicinity of the markers D2S384 and D2S364 on 2q24.3. The genomic orientation of the nebulin gene was determined as 5'-3' and of TTN as 3'-5' from the centromere. We conclude that the nebulin gene resides within the candidate region for NEM2 on the long arm of chromosome 2, while the titin gene is located outside this region.

Nebulin expression in patients with nemaline myopathy.

Nemaline myopathy is a structural congenital myopathy which may show both autosomal dominant and autosomal recessive inheritance patterns. Mutations in three different genes have been identified as the cause of nemaline myopathy: the gene for slow alpha-tropomyosin 3 (TPM3) at 1q22-23, the nebulin gene (NEB) at 2q21.1-q22, and the actin gene (ACTA1) at 1q42. The typical autosomal recessive form appears to be the most common one and is caused by mutations in the nebulin gene. We have studied the pattern of nebulin labeling, in patients with the typical congenital form (ten patients), the severe congenital form (two patients) or the mild, childhood-onset form (one patient), using antibodies against three different domains of nebulin. A qualitative and quantitative nebulin analysis in muscle tissue showed the presence of nebulin in myofibers from all patients. Some differences relating to the rod structure were observed. The majority of the largest subsarcolemmal rods were not labeled with the N2 nebulin antibody (I-band epitope) and showed an indistinct pattern with the two antibodies directed to the Z-band portion of nebulin (epitopes M176-181 and serine-rich domain). Diffuse rods were not revealed using the three antibodies. A discordant pattern of nebulin N2 epitope labeling was found in two affected sisters with a mutation in the nebulin gene, suggesting that modifications in nebulin distribution inside the rods might occur with the progression of the disease. Western blot analysis showed no direct correlation with immunofluorescence data. In nine patients, the band had a molecular weight comparable to the normal control, while in one patient, it was detected with a higher molecular weight. Our results suggest that presence/absence of specific nebulin Z-band epitopes in rod structures is variable and could depend on the degree of rod organization.

Clinical and genetic heterogeneity in autosomal recessive nemaline myopathy.

Autosomal recessive nemaline (rod) myopathy is clinically and genetically heterogeneous. A clinically distinct, typical form, with onset in infancy and a non-progressive or slowly progressive course, has been assigned to a region on chromosome 2q22 harbouring the nebulin gene Mutations have now been found in this gene, confirming its causative role. The gene for slow tropomyosin TPM3 on chromosome 1q21, previously found to cause a dominantly inherited form, has recently been found to be homozygously mutated in one severe consanguineous case. Here we wished to determine the degree of genetic homogeneity or heterogeneity of autosomal recessive nemaline myopathy by linkage analysis of 45 families from 10 countries. Forty-one of the families showed linkage results compatible with linkage to markers in the nebulin region, the highest combined lod scores at zero recombination being 14.13 for the marker D2S2236. We found no indication of genetic heterogeneity for the typical form of nemaline myopathy. In four families with more severe forms of nemaline myopathy, however, linkage to both the nebulin and the TPM3 locus was excluded. Our results indicate that at least three genetic loci exist for autosomal recessive nemaline myopathy. Studies of additional families are needed to localise the as yet unknown causative genes, and to fully elucidate genotype-phenotype correlations.

Gender and risk of autoimmune diseases: possible role of estrogenic compounds.

A striking common feature of many autoimmune diseases in humans and experimental animals, despite differences in pathology, is that females are highly susceptible to autoimmune conditions compared to males. In several animal models, estrogens promote, whereas androgens abrogate, B-cell-mediated autoimmune diseases. To understand mechanisms by which estrogens regulate autoimmunity, it is first necessary to decipher estrogen effects on the normal immune system. Estrogen treatment of nonautoimmune mice diminished lymphocyte numbers in both developmental and mature lymphoid organs. Estrogen dysregulated T- and B-cell balance by inducing selective T-cell hypoactivity and B-cell hyperactivity. Even though estrogen did not alter the relative percentages of splenic T-cell subsets, splenic lymphocytes had a reduced proliferative response to T-cell stimulants and were refractory to rescue from activation-induced apoptosis compared to cells from placebo-treated mice. In contrast, estrogen induced B-cell hyperactivity (promoted autoantibodies to double-stranded DNA and phospholipids, increased numbers of plasma cells, and increased autoantibody yield per B cell). Note that treatment of normal mice with estrogen can alter T- and B-cell regulation and overcome B-cell tolerance to result in autoimmunity in normal individuals. Could environmental estrogens promote some human autoimmune disorders? Is there a link between environmental estrogens and autoimmune disorders, especially since these disorders are reported possibly more frequently? These provocative questions warrant investigation. Our findings on immunomodulatory effects may serve as a benchmark to examine whether endocrine-disrupting chemicals will have similar immunologic effects.

Surround control of center adaptation in the receptive fields of frog retinal ganglion cells.

The sensitivity and intensity-response [R(log I)] functions of the receptive field center were determined by extracellular recording from frog retinal ganglion cells. The object was to study the steady-state adapting effects of peripheral background patterns: steady annuli and spinning "windmills" of light. Steady annular backgrounds could not be shown to directly effect any change of center responsiveness, only an enhancement of late response components attributable to depression of surround sensitivity. Movement of a windmill pattern shifted R(log I) functions to higher log intensities and decreased the maximal number of spikes in the response, but did not depress the saturation level of the impulse frequency. Its action thus resembled direct light-adaptation of the center.

Noise and the absolute thresholds of cone and rod vision.

Literature data on light detection by cone and rod vision at absolute threshold are analysed in order (1) to decide whether the threshold performance of dark-adapted cone vision can, like that of rod vision, be consistently explained as limited by noise from a "dark light"; (2) to obtain comparable estimates of the dark noise and dark light of (foveal) cones and (peripheral) rods. The dark noise was estimated by a maximum-likelihood procedure from frequency-of-seeing data and compared with the dark light derived from increment-threshold functions. In both cone and rod vision, the estimated dark noise coincides with Poisson fluctuations of the estimated dark light if 17% (best estimate) of lambda max-quanta incident at the cornea produce excitations. At that fraction of quanta exciting, dark lights are equivalent to 112 isomerisations per sec in each foveal cone and 0.011 isomerisations per sec in each rod. It is concluded that (1) the threshold performance of dark-adapted cone as well as rod vision can be consistently described as noise-limited, but not by postulating a multi-quantum coincidence requirement for single receptors; (2) the underlying intrinsic activity in both the cone and the rod system is light-like as regards correspondence between noise effect and background adaptation effect. One possibility is that this activity is largely composed of events identical to the single-photon response, originating in the visual pigment, in cones as well as in rods.

On the relation between ERG waves and retinal function: inverted rod photoresponses from the frog retina.

In rod mass receptor photoresponses recorded across the isolated frog retina, a paradoxical cornea-positive wave may precede the response of normal polarity. We present a model which shows that the light-induced decrease in rod current can give rise to inverted or biphasic ERG signals if the distal part (tip) of the rod outer segment responds more slowly and/or less sensitively than the proximal part (base). The condition is that current entering at the tip is represented with greater weight in the ERG. The model reproduces recorded ERG waveforms well. It further predicts that if there is a light-insensitive conductance in the tip membrane, ERG photoresponses may be non-recordable although current photoresponses are only slightly reduced. The model reveals a type of complexity in the relation between mass potentials and underlying physiological processes which has not previously received attention.

The effects of temporal noise and retinal illuminance on foveal flicker sensitivity.

We measured foveal flicker sensitivity with and without external added temporal noise at various levels of retinal illuminance and described the data with our model of flicker sensitivity comprising: (i) low-pass filtering of the flickering signal plus external temporal and/or quantal noise by the modulation transfer function (MTF) of the retina (R): (ii) high-pass filtering in proportion to temporal frequency by the MTF of the postreceptoral neural pathways (P): (iii) addition of internal white neural noise; and (iv) detection by a temporal matched filter. Without temporal noise flicker sensitivity had a band-pass frequency-dependence at high and medium illuminances but changed towards a low-pass shape above 0.5 Hz at low luminances, in agreement with earlier studies. In strong external temporal noise, however, the flicker sensitivity function had a low-pass shape even at high and medium illuminances and flicker sensitivity was consistently lower with noise than without. At low luminances flicker sensitivity was similar with and without noise. An excellent fit of the model was obtained under the assumption that the only luminance-dependent changes were increases in the cut-off frequency (fc) and maximum contrast transfer of R with increasing luminance. The results imply the following: (i) performance is consistent with detection by a temporal matched filter, but not with a thresholding process based on signal amplitude; (ii) quantal fluctuations do not at any luminance level become a source of dominant noise present at the detector; (iii) the changes in the maximum contrast transfer reflect changes in retinal gain, which at low to moderate luminances implement less-than-Weber adaptation, with a 'square-root' law at the lowest levels; (iv) the changes of fc as function of mean luminance closely parallels time scale changes in cones, but the absolute values of fc are lower than expected from the kinetics of monkey cones at all luminances; (v) the constancy of the high-pass filtering function P indicates that surround antagonism does not weaken significantly with decreasing light level.

Center and surround excitation in the receptive fields of frog retinal ganglion cells.

We have reexamined the receptive fields of frog retinal ganglion cells focussing on their surround properties. Carefully excluding artifacts due to stimulation of the (Gaussian) RF center, we found that spiking responses can be elicited by step stimulation of any receptor type in the surrounds of all the classes 1-4 Maturana et al. (1960) (J. gen. Physiol. 43, 129-175). The surround responses are antagonized by the responsive center and suppressed by the inhibitory surround, but are seen because of their slower dynamics. The responsive surround differs spectrally from the center: in the latter, cones and green rods compete, in the former, their signals sum.

Light adaptation of cone photoresponses studied at the photoreceptor and ganglion cell levels in the frog retina.

The sensitivity and time scale of the dominant (562 nm) cone system of the frog, Rana temporaria, were studied as functions of steady adapting illuminance (IB). Photoreceptor responses to brief flashes of light were recorded as aspartate-isolated ERG mass potentials from the isolated retina. The characteristics of the cone signal after transmission through the retina were derived from response thresholds and stimulus--intensity-response--latency functions for extracellularly recorded spike discharges of single ganglion cells in the eyecup. At 14 degrees C, the single-photon response of dark-adapted cones, extrapolated from ERG intensity-response functions, had an amplitude of 0.5% of the saturated response (Umax) and peaked at tp approximately 0.4 sec. Steady background illumination decreased both tp and flash sensitivity (SF), starting from apparent "dark lights" of, respectively, less than 10 (for time scale) and about 100 (for sensitivity) photoisomerisations per cone per second [P*sec-1]. From there upwards, two distinct ranges of background adaptation were apparent. Under moderate backgrounds (up to IB approximately 10(4) - 10(5) P*sec-1), sensitivity fell according to the relation SF alpha IB-0.64 and time scale shortened according to tp alpha IB-0.16. Under brighter backgrounds, from approx. 10(5) P*sec-1 up to the limit of our light source at 10(7) P*sec-1, the decrease in SF was significantly stronger than predicted by the Weber relation (SF alpha IB-1), while the decrease in tp levelled out and even tended to reverse. All these changes were virtually identical at the photoreceptor and ganglion cell levels, although the absolute time scale of cone signals apparent at the latter level was 2-fold longer. Our general conclusion is that photoreceptors have several distinct regimes for light adaptation, and traditional descriptions of functional changes (in sensitivity and kinetics) relevant to vision need to be restated with higher resolution, in view also of recent insights into the diversity of underlying mechanisms.

Flicker sensitivity as a function of target area with and without temporal noise.

Flicker sensitivities (1-30 Hz) in foveal, photopic vision were measured as functions of stimulus area with and without strong external white temporal noise. Stimuli were circular, sinusoidally flickering sharp-edged spots of variable diameters (0.25-4 degrees ) but constant duration (2 s), surrounded by a uniform equiluminant field. The data was described with a model comprising (i) low-pass filtering in the retina (R), with a modulation transfer function (MTF) of a form derived from responses of cones; (ii) normalisation of the temporal luminance distribution by the average luminance; (iii) high-pass filtering by postreceptoral neural pathways (P), with an MTF proportional to temporal frequency; (iv) addition of internal white neural noise (N(i)); (v) integration over a spatial window; and (vi) detection by a suboptimal temporal matched filter of efficiency eta. In strong external noise, flicker sensitivity was independent of spot area. Without external noise, sensitivity increased with the square root of stimulus area (Piper's law) up to a critical area (A(c)), where it reaches a maximum level (S(max)). Both A(c) and eta were monotonic functions of temporal frequency (f), such that log A(c) increased and log eta decreased linearly with log f. Remarkably, the increase in spatial integration area and the decrease in efficiency were just balanced, so A(c)(f)eta(f) was invariant against f. Thus the bandpass characteristics of S(max)(f) directly reflected the composite effect of the distal filters R(f) and P(f). The temporal equivalent (N(it)) of internal neural noise (N(i)) decreased in inverse proportion to spot area up to A(c) and then stayed constant indicating that spatially homogeneous signals and noise are integrated over the same area.

pH regulation in frog cones studied by mass receptor photoresponses from the isolated retina.

Mass cone photoresponses were recorded across the aspartate-treated frog retina under treatments chosen to affect putative pH-regulating mechanisms. The saturated response amplitude (Umax) was found to be a monotonically increasing function of perfusion pH in the range 7-8, and thus presumably of intracellular pH (pHi). Accepting that Umax can be used as an index of pHi changes, two results indicate the importance of bicarbonate transport for preventing intracellular acidification: (1) bicarbonate-buffered (6 mM HCO3- + 6 mM HEPES) perfusate increased Umax compared with nominally bicarbonate-free perfusate (12 mM HEPES); (2) the anion transport blocker DIDS (0.1 mM) caused a strong decrease in the amplitude of photoresponses. Substitution of 95 mM chloride by gluconate in the perfusing fluid boosted photoresponses indicating that at least part of the bicarbonate transport involves HCO3-/Cl- exchange. Amiloride (2 mM) also caused a decrease of photoresponse amplitude, which suggests that Na+/H+ exchange contributes to pHi regulation. In all these respects, cones behaved similarly to rods. Cones differed from rods (in the intact retina) in that addition of 0.5 mM of the carbonic anhydrase inhibitor acetazolamide reduced (never augmented) photoresponses. The difference is considered in relation to the presence of carbonic anhydrase in cone, as opposed to rod, outer segments.

Changes in retinal time scale under background light: observations on rods and ganglion cells in the frog retina.

The kinetics of rod responses to flashes and steps of light was studied as a function of background intensity (IB) at the photoreceptor and ganglion cell levels in the frog retina. Responses of the rod photoreceptors were recorded intracellularly in the eyecup and as ERG mass potentials across the isolated, aspartate-superfused retina. The kinetics of the retinally transmitted signal was derived from the latencies of ganglion cell spike discharges recorded extracellularly in the eyecup. In all states of adaptation the linear-range rod response to dim flashes could be modelled as the impulse response of a chain of low-pass filters with the same number of stages: 4 (ERG) or 4-6 (intracellular). Dark-adapted time-to-peak (tp, mean +/- SD) at 12 degrees C was 2.4 +/- 0.6 sec (ERG) or 1.7 +/- 0.4 sec (intracellular). Under background light, the time scale shortened as a power function of background intensity, I-bB with b = 0.19 +/- 0.03 (ERG) or 0.14 +/- 0.04 (intracellular). The latency-derived time scale of the rod-driven signal at the ganglion cell agreed well with that of the photoreceptor responses. The apparent underlying impulse response had tp = 2.0 +/- 0.7 sec in darkness and accelerated as I-bB with b = 0.17 +/- 0.03. The photoreceptor-to-ganglion-cell transmission delay shortened by 30% between darkness and a background delivering ca 10(4) photoisomerizations per rod per second. Data from the literature suggest that all vertebrate photoreceptors may accelerate according to similar power functions of adapting intensity, with exponents in the range 0.1-0.2. It is noteworthy that the time scale of human (foveal) vision in experiments on flicker sensitivity and temporal summation shortens as a power function of mean luminance with b approximately 0.15.

The effect of background luminance on visual responses to strong flashes: perceived brightness and the early rise of photoreceptor responses.

The threshold intensity for large-long incremental stimuli rises proportionally to adapting background luminance IB (Weber adaptation), but the intensity required to evoke a criterion high-brightness sensation rises much less steeply. We propose that this difference originates in the very first stage of visual processing, in the phototransduction and adaptation properties of the retinal photoreceptor cells. A physiological model previously found to account for visual latency and brightness as functions of stimulus intensity in the dark-adapted state [Donner, K. (1989). Visual Neuroscience, 3, 39-51] is extended to cover different states of adaptation. It is assumed that the neural coding of high intensities is based on the rate of rise (quasi-derivative) of the photoreceptor response just after it reaches a small threshold amplitude. The shallow background adaptation functions for high-brightness criteria emerge as a consequence of the relative constancy of the leading edge of large responses under backgrounds, a phenomenon that can be formally described by compensating changes in photoreceptor sensitivity and time scale. We first test the model on supra-threshold responses in the frog retina, where the discharge rate of ganglion cells (a possible neural code for brightness) and the primary rod hyperpolarizations can be recorded under identical conditions. The two are related as predicted over at least 3 log units of background intensity. We then show that published data on the background adaptation of human foveal high brightness judgments conform to the same model, assuming that human cones accelerate as IB-b with b = 0.14-0.15.

Flicker sensitivity as a function of spectral density of external white temporal noise.

Foveal flicker sensitivity at 0.5-30 Hz was measured as a function of the spectral density of external, white, purely temporal noise for a sharp-edged 2.5 deg circular spot (mean luminance 3.4 log phot td). Sensitivity at any given temporal frequency was constant at low powers of external noise, but then decreased in inverse proportion to the square root of noise spectral density. Without external noise, sensitivity as function of temporal frequency had the well-known band-pass characteristics peaking at about 10 Hz, as previously documented in a large number of studies. In the presence of strong external noise, however, sensitivity was a monotonically decreasing function of temporal frequency. Our data are well described (goodness of fit 90%) by a model comprising (i) low-pass filtering by retinal cones, (ii) high-pass filtering in the subsequent neural pathways, (iii) adding of the temporal equivalent of internal white spatiotemporal noise, and (iv) detection by a temporal matched filter, the efficiency of which decreases approximately as the power -0.58 of temporal frequency.

Response univariance in bull-frog rods with two visual pigments.

Rods in the bull-frog retina contain varying proportions of rhodopsin (lambda max = 502 nm) and porphyropsin (lambda max = 527 nm) in a dorso-ventral gradient from the porphyropsin-rich dorsal rim to the virtually pure rhodopsin fields of the central and ventral retina. We investigated if quantal excitations in the same rod are different depending on whether they are initiated by isomerization of a rhodopsin or a porphyropsin molecule. Current photoresponses were recorded from dark-adapted rods by sucking the outer segment into a recording pipette. The relation between pigment composition and spectral sensitivity was established by comparison with microspectrophotometrically measured absorbance spectra of rods from the same neighbourhood. Rods with suitable porphyropsin: rhodopsin mixtures (ideally between 1:4 and 1:2) were stimulated with flashes of red (608 nm) and blue (465 nm) light, whereby the red light will isomerize porphyropsin much more often than rhodopsin, and the reverse will be true of the blue light. The amplitude and shape of the single-photon response were found to be identical for the "red" and "blue" flash series to within measurement error (ca 10%). This indicates that the quantal responses initiated by the two pigments are identical.

Changes in the light-sensitive current of salamander rods upon manipulation of putative pH-regulating mechanisms in the inner and outer segment.

The light-sensitive current of dark-adapted rods isolated from the Ambystoma retina was recorded while either the inner or the outer segment (IS or OS) protruding from the suction pipette was exposed to treatments intended to reveal the physiological roles of pH-regulating transport mechanisms. Applied to the IS, both amiloride (presumed to block Na+/H+ exchange, 2 mM) and 4-4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) (presumed to block bicarbonate transport, 0.1 mM) generally abolished light sensitivity completely but reversibly, consistent with acidification of the IS. Yet, the circulating ("dark") current often persisted, implying that the OS was not acidified. Applied to the OS, amiloride depressed but DIDS increased the dark current and photoresponses. Given the fact that the current increases with rising OS-pHi, this suggests alkalinization, which could be due to DIDS inhibiting bicarbonate extrusion by HCO3-/Cl- exchangers in the OS. Consistent with this idea, replacing external Cl- by other anions increased the current as would be expected if HCO3-/Cl- exchange is reversed. We propose that the IS and OS manage their acid balances independently and with different sets of transport mechanisms. Acidosis in either compartment suppresses the photosensitivity of the rod, but by differing mechanisms.