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PubMed1993

Interactions between divalent cations and the gating machinery of cyclic GMP-activated channels in salamander retinal rods.

The effects of divalent cations on the gating of the cGMP-activated channel, and the effects of gating on the movement of divalent cations in and out of the channel's pore were studied by recording macroscopic currents in excised membrane patches from salamander retinal rods. The fractional block of cGMP-activated Na+ currents by internal and external Mg2+ as well as internal Ca2+ was nearly independent of cGMP concentration. This indicates that Mg2+ and Ca2+ bind with similar affinity to open and closed states of the channel. In contrast, the efficiency of block by internal Cd2+ or Zn2+ increased in proportion to the fraction of open channels, indicating that these ions preferentially occupy open channels. The kinetics of block by internal Ni2+, which competes with Mg2+ but blocks more slowly, were found to be unaffected by the fraction of channels open. External Ni2+, however, blocked and unblocked much more rapidly when channels were mostly open. This suggests that within the pore a gate is located between the binding site(s) for ions and the extracellular mouth of the channel. Micromolar concentrations of the transition metal divalent cations Ni2+, Cd2+, Zn2+, and Mn2+ applied to the cytoplasmic surface of a patch potentiated the response to subsaturating concentrations of cGMP without affecting the maximum current induced by saturating cGMP. The concentration of cGMP that opened half the channels was often lowered by a factor of three or more. Potentiation persisted after the experimental chamber was washed with divalent-free solution and fresh cGMP was applied, indicating that it does not result from an interaction between divalent cations and cGMP in solution; 1 mM EDTA or isotonic MgCl2 reversed potentiation. Voltage-jump experiments suggest that potentiation results from an increase in the rate of cGMP binding. Lowering the ionic strength of the bathing solution enhanced potentiation, suggesting that it involves electrostatic interactions. The strong electrostatic effect on cGMP binding and absence of effect on ion permeation through open channels implies that the cGMP binding sites on the channel are well separated from the permeation pathway.

Animals↗
PubMed1993

Visual pigment bleaching in isolated salamander retinal cones. Microspectrophotometry and light adaptation.

Visual pigment bleaching desensitizes rod photoreceptors greatly in excess of that due to loss of quantum catch. Whether this phenomenon also occurs in cone photoreceptors was investigated for isolated salamander red-sensitive cones. In parallel experiments, (a) visual pigment depletion by steps of bleaching light was measured by microspectrophotometry, and (b) flash sensitivity was measured by recording light-sensitive membrane current. In isolated cones, visual pigment bleaching permanently reduced flash sensitivity significantly below that due to the reduction in quantum catch, and there was little spontaneous recovery of visual pigment. The "extra" desensitization due to bleaching was most prominent up to bleaches of approximately 80% visual pigment and reached a level approximately 1 log unit beyond that due to loss of quantum catch. At higher bleaches, the effect of loss of quantum catch became more important. Bleaching did not greatly reduce the maximum light-suppressible membrane current. A 99% reduction of the visual pigment permanently reduced the circulating current by only 30%. Visual pigment bleaching speeded up the kinetics of dim flash responses. All electrical effects of bleaching were reversed on exposure to 11-cis retinal, which probably caused visual pigment regeneration. Light adaptation in photopic vision is known to involve significant visual pigment depletion. The present results indicate that cones operate with a maintained circulating current even after a large pigment depletion. It is shown how Weber/Fechner behavior may still be observed in photopic vision when the contributions of bleaching to adaptation are included.

Absorptiometry, Photon↗
PubMed1995

Bleached pigment activates transduction in salamander cones.

We have used suction electrode recording together with rapid steps into 0.5 mM IBMX solution to investigate changes in guanylyl cyclase velocity produced by pigment bleaching in isolated cones of the salamander Ambystoma tigrinum. Both backgrounds and bleaches accelerate the time course of current increase during steps into IBMX. We interpret this as evidence that the velocity of the guanylyl cyclase is increased in background light or after bleaching. Our results indicate that cyclase velocity increases nearly linearly with increasing percent pigment bleached but nonlinearly (and may saturate) with increasing back-ground intensity. In cones (as previously demonstrated for rods), light-activated pigment and bleached pigment appear to have somewhat different effects on the transduction cascade. The effect of bleaching on cyclase rate is maintained for at least 15-20 min after the light is removed, much longer than is required after a bleach for circulating current and sensitivity to stabilize in an isolated cone. The effect on the cyclase rate can be completely reversed by treatment with liposomes containing 11-cis retinal. The effects of bleaching can also be partially reversed by beta-ionone, an analogue of the chromophore 11-cis-retinal which does not form a covalent attachment to opsin. Perfusion of a bleached cone with beta-ionone produces a rapid increase in circulating current and sensitivity, which rapidly reverses when the beta-ionone is removed. Perfusion with beta-ionone also causes a partial reversal of the bleach-induced acceleration of cyclase velocity. We conclude that bleaching produces an "equivalent background" excitation of the transduction cascade in cones, perhaps by a mechanism similar to that in rods.

1-Methyl-3-isobutylxanthine↗
PubMed1996

Equivalence of background and bleaching desensitization in isolated rod photoreceptors of the larval tiger salamander.

Psychophysical experiments have shown an equivalence between sensitivity reduction by background light and by bleaches for the human scotopic system. We have compared the effects of backgrounds and bleaches on the light-sensitive membrane-current responses of isolated rod photoreceptors from the salamander Ambystoma tigrinum. The quantum catch loss was factored out from the desensitization due to bleaching to give the fraction of "extra" desensitization due to adaptation. For backgrounds, desensitization is well described by the Weber/Fechner equation. The extra desensitization after bleaches can also be described by the Weber/Fechner equation, if an "equivalent" background produced by bleaching is made linearly proportional to the fraction of pigment bleached. A background which produces an extra desensitization of a factor of two is equivalent to a fractional bleach of approximately 6%. Equivalent background and bleaching desensitizations were associated with similar reductions in circulating current. There is a linear relation between log flash sensitivity and decrease in circulating current. Equivalent background and bleaching desensitizations were associated with similar increases in cGMP phosphodiesterase and guanylate cyclase activity. These were inferred from membrane current changes after steps into lithium or IBMX solutions. There were also similar reductions in the integration times of dim flash responses for equivalent desensitizations produced by backgrounds and bleaches. These results suggest that the equivalence between background and bleaching found psychophysically may arise at the very earliest stages of visual processing and that these two processes of desensitization have similar underlying mechanisms.

Adaptation, Physiological↗
PubMed1996

Persistent activation of transducin by bleached rhodopsin in salamander rods.

The hydrolysis-resistant GTP analogue GTP-gamma-S was introduced into rods isolated from the retina of the salamander Ambystoma tigrinum to study the origin of the persistent excitation induced by intense bleaching illumination. Dialysis of a dark-adapted rod with a whole-cell patch pipette containing 2 mM GTP-gamma-S resulted in a gradual decrease in circulating current. If the rod was first bleached and its sensitivity allowed to stabilize for at least 30 min, then dialysis with GTP-gamma-S produced a much faster current decay. The circulating current could be restored by superfusion with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, suggesting that the decay in current originated from persistent excitation of the phosphodiesterase by transducin bound to GTP-gamma-S. We conclude that the persistent excitation which follows bleaching is likely to involve the GTP-binding protein transducin, which mediates the normal photoresponse. This observation suggests that a form of rhodopsin which persists long after bleaching can activate transducin much as does photoisomerized rhodopsin, although with considerably lower gain.

1-Methyl-3-isobutylxanthine↗
PubMed1998

Kinetics of recovery of the dark-adapted salamander rod photoresponse.

The kinetics of the dark-adapted salamander rod photocurrent response to flashes producing from 10 to 10(5) photoisomerizations (Phi) were investigated in normal Ringer's solution, and in a choline solution that clamps calcium near its resting level. For saturating intensities ranging from approximately 10(2) to 10(4) Phi, the recovery phases of the responses in choline were nearly invariant in form. Responses in Ringer's were similarly invariant for saturating intensities from approximately 10(3) to 10(4) Phi. In both solutions, recoveries to flashes in these intensity ranges translated on the time axis a constant amount (tauc) per e-fold increment in flash intensity, and exhibited exponentially decaying "tail phases" with time constant tauc. The difference in recovery half-times for responses in choline and Ringer's to the same saturating flash was 5-7 s. Above approximately 10(4) Phi, recoveries in both solutions were systematically slower, and translation invariance broke down. Theoretical analysis of the translation-invariant responses established that tauc must represent the time constant of inactivation of the disc-associated cascade intermediate (R*, G*, or PDE*) having the longest lifetime, and that the cGMP hydrolysis and cGMP-channel activation reactions are such as to conserve this time constant. Theoretical analysis also demonstrated that the 5-7-s shift in recovery half-times between responses in Ringer's and in choline is largely (4-6 s) accounted for by the calcium-dependent activation of guanylyl cyclase, with the residual (1-2 s) likely caused by an effect of calcium on an intermediate with a nondominant time constant. Analytical expressions for the dim-flash response in calcium clamp and Ringer's are derived, and it is shown that the difference in the responses under the two conditions can be accounted for quantitatively by cyclase activation. Application of these expressions yields an estimate of the calcium buffering capacity of the rod at rest of approximately 20, much lower than previous estimates.

Animals↗
PubMed1999

Light-dependent changes in outer segment free-Ca2+ concentration in salamander cone photoreceptors.

Simultaneous measurements of photocurrent and outer segment Ca2+ were made from isolated salamander cone photoreceptors. While recording the photocurrent from the inner segment, which was drawn into a suction pipette, a laser spot confocal technique was employed to evoke fluorescence from the outer segment of a cone loaded with the Ca2+ indicator fluo-3. When a dark-adapted cone was exposed to the intense illumination of the laser, the circulating current was completely suppressed and fluo-3 fluorescence rapidly declined. In the more numerous red-sensitive cones this light-induced decay in fluo-3 fluorescence was best fitted as the sum of two decaying exponentials with time constants of 43 +/- 2.4 and 640 +/- 55 ms (mean +/- SEM, n = 25) and unequal amplitudes: the faster component was 1.7-fold larger than the slower. In blue-sensitive cones, the decay in fluorescence was slower, with time constants of 140 +/- 30 and 1,400 +/- 300 ms, and nearly equal amplitudes. Calibration of fluo-3 fluorescence in situ from red-sensitive cones allowed the calculation of the free-Ca2+ concentration, yielding values of 410 +/- 37 nM in the dark-adapted outer segment and 5.5 +/- 2.4 nM after saturating illumination (mean +/- SEM, n = 8). Photopigment bleaching by the laser resulted in a considerable reduction in light sensitivity and a maintained decrease in outer segment Ca2+ concentration. When the photopigment was regenerated by applying exogenous 11-cis-retinal, both the light sensitivity and fluo-3 fluorescence recovered rapidly to near dark-adapted levels. Regeneration of the photopigment allowed repeated measurements of fluo-3 fluorescence to be made from a single red-sensitive cone during adaptation to steady light over a range of intensities. These measurements demonstrated that the outer segment Ca2+ concentration declines in a graded manner during adaptation to background light, varying linearly with the magnitude of the circulating current.

Ambystoma↗
PubMed2000

Extracellular HCO(3)(-) dependence of electrogenic Na/HCO(3) cotransporters cloned from salamander and rat kidney.

We studied the extracellular [HCOabstract (3) (-)] dependence of two renal clones of the electrogenic Na/HCO(3) cotransporter (NBC) heterologously expressed in Xenopus oocytes. We used microelectrodes to measure the change in membrane potential (DeltaV(m)) elicited by the NBC cloned from the kidney of the salamander Ambystoma tigrinum (akNBC) and by the NBC cloned from the kidney of rat (rkNBC). We used a two-electrode voltage clamp to measure the change in current (DeltaI) elicited by rkNBC. Briefly exposing an NBC-expressing oocyte to HCOabstract (3 )(-)/CO(2) (0.33-99 mM HCOabstract (3)(-), pH(o) 7.5) elicited an immediate, DIDS (4, 4-diisothiocyanatostilbene-2,2-disulfonic acid)-sensitive and Na(+)-dependent hyperpolarization (or outward current). In DeltaV(m) experiments, the apparent K(m ) for HCOabstract (3)(-) of akNBC (10. 6 mM) and rkNBC (10.8 mM) were similar. However, under voltage-clamp conditions, the apparent K(m) for HCOabstract (3)(-) of rkNBC was less (6.5 mM). Because it has been reported that SOabstract (3)(=)/HSO abstract (3)(-) stimulates Na/HCO(3 ) cotransport in renal membrane vesicles (a result that supports the existence of a COabstract (3)(=) binding site with which SOabstract (3)(=) interacts), we examined the effect of SOabstract (3)(=)/HSO abstract (3)(-) on rkNBC. In voltage-clamp studies, we found that neither 33 mM SOabstract (4)(=) nor 33 mM SOabstract (3) (=)/HSOabstract (3)(-) substantially affects the apparent K(m) for HCO abstract (3)(-). We also used microelectrodes to monitor intracellular pH (pH(i)) while exposing rkNBC-expressing oocytes to 3.3 mM HCOabstract (3 )(-)/0.5% CO(2). We found that SO abstract (3)(=)/HSOabstract (3 )(-) did not significantly affect the DIDS-sensitive component of the pH(i) recovery from the initial CO(2 )-induced acidification. We also monitored the rkNBC current while simultaneously varying [CO(2)](o), pH(o), and [COabstract (3)(=)](o) at a fixed [HCOabstract (3)(-)](o) of 33 mM. A Michaelis-Menten equation poorly fitted the data expressed as current versus [COabstract (3)(=)](o ). However, a pH titration curve nicely fitted the data expressed as current versus pH(o). Thus, rkNBC expressed in Xenopus oocytes does not appear to interact with SOabstract (3 )(=), HSOabstract (3)(-), or COabstract (3)(=).

Ambystoma↗
PubMed2000

The role of steady phosphodiesterase activity in the kinetics and sensitivity of the light-adapted salamander rod photoresponse.

We investigated the kinetics and sensitivity of photocurrent responses of salamander rods, both in darkness and during adaptation to steady backgrounds producing 20-3,000 photoisomerizations per second, using suction pipet recordings. The most intense backgrounds suppressed 80% of the circulating dark current and decreased the flash sensitivity approximately 30-fold. To investigate the underlying transduction mechanism, we expressed the responses as a fraction of the steady level of cGMP-activated current recorded in the background. The fractional responses to flashes of any fixed intensity began rising along a common trajectory, regardless of background intensity. We interpret these invariant initial trajectories to indicate that, at these background intensities, light adaptation does not alter the gain of any of the amplifying steps of phototransduction. For subsaturating flashes of fixed intensity, the fractional responses obtained on backgrounds of different intensity were found to "peel off" from their common initial trajectory in a background-dependent manner: the more intense the background, the earlier the time of peeling off. This behavior is consistent with a background-induced reduction in the effective lifetime of at least one of the three major integrating steps in phototransduction; i.e., an acceleration of one or more of the following: (1) the inactivation of activated rhodopsin (R*); (2) the inactivation of activated phosphodiesterase (E*, representing the complex G(alpha)-PDE of phosphodiesterase with the transducin alpha-subunit); or (3) the hydrolysis of cGMP, with rate constant beta. Our measurements show that, over the range of background intensities we used, beta increased on average to approximately 20 times its dark-adapted value; and our theoretical analysis indicates that this increase in beta is the primary mechanism underlying the measured shortening of time-to-peak of the dim-flash response and the decrease in sensitivity of the fractional response.

Adaptation, Ocular↗
PubMed1979

Synaptic inputs to the ganglion cells in the tiger salamander retina.

The postsynaptic potentials (PSPs) that form the ganglion cell light response were isolated by polarizing the cell membrane with extrinsic currents while stimulating at either the center or surround of the cell's receptive field. The time-course and receptive field properties of the PSPs were correlated with those of the bipolar and amacrine cells. The tiger salamander retina contains four main types of ganglion cell: "on" center, "off" center, "on-off", and a "hybrid" cell that responds transiently to center, but sustainedly, to surround illumination. The results lead to these inferences. The on-ganglion cell receives excitatory synpatic input from the on bipolars and that synapse is "silent" in the dark. The off-ganglion cell receives excitatory synaptic input from the off bipolars with this synapse tonically active in the dark. The on-off and hybrid ganglion cells receive a transient excitatory input with narrow receptive field, not simply correlated with the activity of any presynaptic cell. All cell types receive a broad field transient inhibitory input, which apparently originates in the transient amacrine cells. Thus, most, but not all, ganglion cell responses can be explained in terms of synaptic inputs from bipolar and amacrine cells, integrated at the ganglion cell membrane.

Animals↗
PubMed1979

Temporal patterns and selectivity in the unitary responses of olfactory receptors in the tiger salamander to odor stimulation.

Temporal patterns and selectivity in unitary responses of 100 single olfactory receptors in the tiger salamander to odor stimulation were investigated. An olfactometer which permitted control of stimulus concentration, duration, and flow rate was calibrated with a gas chromatograph. Stimulus pulses were monitored by recording the electroolfactogram from the surface of the olfactory epithelium. Both diphasic and triphasic spikes were recorded extracellularly. No discernible differences in types of responses, reproducibility of responses, and cross-unit distribution of spontaneous rates distinguished diphasic from triphasic units. The cross-unit selectivity in responses to the seven olfactory stimulants used and the range of odorant concentrations which effectively evoked these responses suggest variations in types and number of types of receptive sites on each cell. Temporal patterns in the unitary responses were generally less complex than those observed in the olfactory bulb. Phasic stimulations evoked phasic patterns. Tonic stimulations evoked phasic/tonic patterns. Occasionally poststimulus depressions or elevations in firing rates were observed. The nature of these patterns varied somewhat with odorant concentration for a particular unit.

Action Potentials↗
PubMed1979

Adaptation and cross-adaptation to odor stimulation of olfactory receptors in the tiger salamander.

We have used the effects of self- and cross-adaptation on the unitary responses of olfactory receptors of the tiger salamander to odor stimulation to investigate the stimulus-specific components of these responses and to provide information about the cross-cell variations in the numbers and numbers of types of constitutent receptive sites. An olfactometer delivered sequential odorous pulses, either juxtaposed or separated by a variable time delay. We used four pairs of odorants judged to be similar within a given pair. The unitary response to the test stimulation relative to that of the conditioning stimulation varied from being unchanged to being completely eliminated. We sometimes observed substantial poststimulus increases in the firing rate following stimulation with juxtaposed odorous pulse. Except in the case of one odorant pair, cross-adaptation occurred both with juxtaposed pulses and with pulses separated in time. With the methyl butyrate/ethyl butyrate odorant pair, however, statistically significant cross-adaptation appeared only with juxtaposed pulses. We propose a simple model to aid in explaining these phenomena. The experimental observations in conjunction with this model are used to obtain estimates of the maximal and minimal number of receptive site types available for interaction with the chosen odorants.

Action Potentials↗
PubMed1983

Intracellular pH regulation in the renal proximal tubule of the salamander. Na-H exchange.

Using pH-sensitive microelectrodes to measure intracellular pH (pHi) in isolated, perfused proximal tubules of the tiger salamander Ambystoma tigrinum, we have found that when cells are acid-loaded by pretreatment with NH+4 in a nominally HCO3--free Ringer, pHi spontaneously recovers with an exponential time course. This pHi recovery, which is indicative of active (i.e., uphill) transport, is blocked by removal of Na+ from both the luminal and basolateral (i.e., bath) solutions. Re-addition of Na+ to either the lumen or the bath results in a full pHi recovery, but at a lower-than-normal rate; the maximal rate is achieved only with Na+ in both solutions. The diuretic amiloride reversibly inhibits the pHi recovery when present on either the luminal or basolateral sides, and has its maximal effect when present in both solutions. The pHi recovery is insensitive to stilbene derivatives and to Cl- removal. A transient rise of intracellular Na+ activity accompanies the pHi recovery; there is no change of intracellular Cl- activity. These data suggest that these proximal tubule cells have Na-H exchangers in both the luminal and basolateral membranes.

Amiloride↗
PubMed1983

Intracellular pH regulation in the renal proximal tubule of the salamander. Basolateral HCO3- transport.

We have used pH-, Na-, and Cl-sensitive microelectrodes to study basolateral HCO3- transport in isolated, perfused proximal tubules of the tiger salamander Ambystoma tigrinum. In one series of experiments, we lowered basolateral pH (pHb) from 7.5 to 6.8 by reducing [HCO3-]b from 10 to 2 mM at a constant pCO2. This reduction of pHb and [HCO3-]b causes a large (approximately 0.35), rapid fall in pHi as well as a transient depolarization of the basolateral membrane. Returning pHb and [HCO3-]b to normal has the opposite effects. Similar reductions of luminal pH (pHl) and [HCO3-]l have only minor effects. The reduction of [HCO3-]b and pHb also produces a reversible fall in aiNa. In a second series of experiments, we reduced [Na+]b at constant [HCO3-]b and pHb, and also observed a rapid fall in pHi and a transient basolateral depolarization. These changes are reversed by returning [Na+]b to normal. The effects of altering [Na+]l in the presence of HCO3-, or of altering [Na+]b in the nominal absence of HCO3-, are substantially less. Although the effects on pHi and basolateral membrane potential of altering either [HCO3-]b or [Na+]b are largely blocked by 4-acetamido-4-isothiocyanostilbene-2,2'-disulfonate (SITS), they are not affected by removal of Cl-, nor are there accompanying changes in aiCl consistent with a tight linkage between Cl- fluxes and those of Na+ and HCO3-. The aforementioned changes are apparently mediated by a single transport system, not involving Cl-. We conclude that HCO3- transport is restricted to the basolateral membrane, and that HCO3- fluxes are linked to those of Na+. The data are compatible with an electrogenic Na/HCO3 transporter that carries Na+, HCO3-, and net negative charge in the same direction.

Animals↗
PubMed1985

Intracellular calibration of a pH-sensitive dye in isolated, perfused salamander proximal tubules.

We evaluated the dye 4',5'-dimethyl-5-(and -6-) carboxyfluorescein (Me2CF) for determining the intracellular pH(pHi) of isolated, perfused proximal tubules of the salamander. The intracellular absorbance spectrum, corrected for the intrinsic absorbance of the tubule, was obtained once per second. The dye was incorporated into tubule cells by exposing them to the membrane-permeable precursor 4',5'-dimethyl-5- (and -6-) carboxyfluorescein diacetate. The introduction of the dye had no significant effect on either pHi or cell voltage transients. Compared with dye contained in a cuvette, intracellular dye had a peak absorbance that was red-shifted by approximately 5 nm, and an apparent pK that was increased by approximately 0.3. These differences precluded an accurate calculation of pHi by the comparison of intracellular spectra with in vitro calibration spectra. However, when Me2CF was calibrated intracellularly, using the K-H exchanger nigericin to equalize external pH and pHi, the dye-derived, steady state pHi was within approximately 0.1 of the value obtained with pH-sensitive microelectrodes. Furthermore, when pHi was simultaneously measured with dye and microelectrodes during rapid pHi transients, the pHi time courses measured by the two methods were very similar. We conclude that the intracellular absorbance spectrum of Me2CF can be used to measure steady state pHi and rapid pHi transients reliably, provided the dye is calibrated intracellularly.

Ambystoma↗
PubMed1987

Effect of electroneutral luminal and basolateral lactate transport on intracellular pH in salamander proximal tubules.

We used microelectrodes to examine the effects of organic substrates, particularly lactate (Lac-), on the intracellular pH (pHi) and basolateral membrane potential (Vbl) in isolated, perfused proximal tubules of the tiger salamander. Exposure of the luminal and basolateral membranes to 3.6 mM Lac- caused pHi to increase by approximately 0.2, opposite to the decrease expected from nonionic diffusion of lactic acid (HLac) into the cell. Addition of Lac- to only the lumen also caused alkalinization, but only if Na+ was present. This alkalinization was not accompanied by immediate Vbl changes, which suggests that it involves luminal, electroneutral Na/Lac cotransport. Addition of Lac- to only the basolateral solution caused pHi to decrease by approximately 0.08. The initial rate of this acidification was a saturable function of [Lac-], was not affected by removal of Na+, and was reversibly reduced by alpha-cyano-4-hydroxycinnamate (CHC). Thus, the pHi decrease induced by basolateral Lac- appears to be due to the basolateral entry of H+ and Lac-, mediated by an H/Lac cotransporter (or a Lac-base exchanger). Our data suggest that this transporter is electroneutral and is not present at the luminal membrane. A key question is how the addition of Lac- to the lumen increases pHi. We found that inhibition of basolateral H/Lac cotransport by basolateral CHC reduced the initial rate of pHi increase caused by luminal Lac-. On the other hand, luminal CHC had no effect on the luminal Lac(-)-induced alkalinization. These data suggest that when Lac- is present in the lumen, it enters the cell from the lumen via electroneutral Na/Lac cotransport and then exists with H+ across the basolateral membrane via electroneutral H/Lac cotransport. The net effect is transepithelial Lac- reabsorption, basolateral acid extrusion, and intracellular alkalinization.

Ambystoma↗
PubMed1991

Net charge transport during sodium-dependent calcium extrusion in isolated salamander rod outer segments.

The light-sensitive current and the current associated with the extrusion of internal Ca2+ in exchange for external Na+ have been recorded from detached rod outer segments from the salamander retina by the use of the whole-cell voltage clamp technique. No significant current-carrying mechanisms are present in the outer segment membrane apart from the light-sensitive conductance and the Na:Ca,K exchange, and exchange currents can therefore be recorded directly without the use of subtraction procedures or pharmacological blockers. The charge moved by the exchange was studied by loading outer segments with a known amount of calcium and then recording the exchange current on return to a Na(+)-containing solution. Calcium is not sequestered to any significant extent in a slowly exchanging internal store, as the charge recovered is unaffected if admission of the Na(+)-containing solution is delayed for 40 s. The number of charges flowing into the cell in exchange for each Ca2+ ion extruded was found not to deviate significantly from one over a wide range of ionic conditions and membrane potentials. These results show that the stoichiometry of the exchange is fixed over a wide range of conditions, and that the size of the inward exchange current is therefore directly proportional to the rate of Ca2+ efflux through the carrier.

1-Methyl-3-isobutylxanthine↗
PubMedDate not supplied

Carbonic anhydrase and red blood cell anion exchange in the neotenic aquatic salamander, Necturus maculosus.

Carbonic anhydrase (CA) activity in blood and other tissues and red blood cell (rbc) anion exchange were measured in the mud puppy, Necturus maculosus, in order to gain insight into the strategy for CO2 transport used by these neotenic salamanders and to further explore evolutionary relationships between rbc CA activity and anion exchange in nonmammalian vertebrates. CA activity was detectable in all of the tissues examined, but CA activity in blood was much lower than that in most vertebrates. There was no indication, however, that additional CA had been incorporated into the membrane fraction of other tissues to compensate for this low blood CA activity. In further contrast to most other animals, low levels of CA activity were also detectable in mud puppy plasma. Preliminary characterization of the rbc CA indicated that the Type II, fast-turnover enzyme was indeed present, but that there are a very low number of active sites in mud puppy rbc's. Further experiments showed that the rbc's were highly permeable to anions and that the relative rate of anion flux could be inhibited by 4, 4-diisothiocyanostilbene-2,2-disulphonic acid. Thus, the process of CO2 transport in the blood of mud puppies probably involves components of the Jacobs-Stewart cycle, as in most other vertebrates.

Adaptation, Physiological↗