Chlorinated hydrocarbon concentrations in plasma of the Lake Erie water snake (Nerodia sipedon insularum) and northern water snake (Nerodia sipedon sipedon) from the Great Lakes basin in 1998.

From the Great Lakes basin, concentrations of 59 congener-specific polychlorinated biphenyls (PCBs) and 14 organochlorine pesticides were measured in blood plasma of northern water snake (Nerodia sipedon sipedon) and Lake Erie water snake (Nerodia sipedon insularum), which is endangered in Canada. In 1998, four male adult Lake Erie water snakes were sampled from Pelee Island, western Lake Erie; four male northern water snakes were sampled at Little Lake, about 20 km north of Parry Sound in central Ontario; and four adult gravid female northern water snakes were sampled from Garden Island, eastern Lake Ontario. The blood plasma was pooled by site for a total of three samples analyzed. The Pelee Island sample from male Lake Erie water snakes contained less than half the lipid concentration (0.349%) than samples from the other sites, but it was the most contaminated with PCBs, even on a wet weight basis. Summed concentration of individual PCBs in the Pelee Island sample was 167 ng/g (wet weight), which was 14-fold higher than the next most contaminated sample, which was from Little Lake. The plasma sample from Little Lake contained 12 ng/g (WW) and was four times more contaminated with PCBs than the sample from female snakes from Garden Island, Lake Ontario. Organochlorine pesticide concentrations in plasma were relatively similar among sites. None of the pesticides was found above trace concentrations (0.1-0.9 ng/g) except pp'-DDE, which occurred at 2-5 ng/g among sites. PCB congener patterns in the Lake Erie water snakes were compared to PCB patterns in plasma of common snapping turtle (Chelydra serpentina serpentina) from Lake Ontario, herring gull eggs (Larus argentatus) from western Lake Erie, and mudpuppy eggs (Necturus maculosus) from the Detroit River. The PCB patterns in water snake and herring gull sample were most similar, followed by the pattern in snapping turtle plasma. The presence of more lower-chlorinated chlorobiphenyls in the mudpuppy eggs relative to the other species made this sample distinct from the water snake, gull, and turtle.

Towards molecular understanding of species differences in dioxin sensitivity: initial characterization of Ah receptor cDNAs in birds and an amphibian.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related planar halogenated aromatic hydrocarbons (PHAHs) are highly toxic to most vertebrate animals, but there are dramatic species differences in sensitivity, both within and among vertebrate classes. For example, studies in cultured avian hepatocytes have revealed differential sensitivity of birds to PHAHs [Kennedy et al. (1996). Toxicol. Appl. Pharmacol., 141, 214-230]. Differences in the characteristics or expression of the aryl hydrocarbon receptor (AHR) could contribute to these species differences in PHAH responsiveness. To investigate the molecular mechanism of differential PHAH sensitivity, we have begun to characterize the AHR in white leghorn chicken (Gallus gallus), Pekin duck (Anas platyrhynchos), and common tern (Sterna hirundo), as well as an amphibian, mudpuppy (Necturus maculosus). Partial AHR cDNAs encompassing the helix-loop-helix and PAS domains were cloned and sequenced. Comparison of amino acid sequences in this region indicated a high degree of sequence conservation among the bird species (97% amino acid identity). The percent identity between bird sequences and either mouse or mudpuppy was lower (79%); the mudpuppy AHR was 74% identical to the mouse AHR. Phylogenetic analysis of these and other AHR amino acid sequences showed that the bird and mudpuppy AHRs were more closely related to mammalian and fish AHR1 forms than to fish AHR2. Future studies include the in vitro expression and functional characterization of AHRs from these and other non-mammalian vertebrates.

Colocalization of glycolytic enzyme activity and KATP channels in basolateral membrane of Necturus enterocytes.

86Rb fluxes through ATP-regulated K+ (KATP) channels in membrane vesicles derived from basolateral membranes of Necturus small intestinal epithelial cells as well as the activity of single KATP channels reconstituted into planar phospholipid bilayers are inhibited by the presence of ADP plus phosphoenolpyruvate in the solution bathing the inner surface of these channels. This inhibition can be prevented by pretreatment of the membranes with 2, 3-butanedione, an irreversible inhibitor of pyruvate kinase (PK) and reversed by the addition of 2-deoxyglucose plus hexokinase. The results of additional studies indicate that PK activity appears to be tightly associated with this membrane fraction. These results, together with considerations of the possible ratio of Na+-K+ pumps to KATP channels in the basolateral membrane, raise the possibility that "cross talk" between those channels and pumps (i.e., the "pump-leak parallelism") may be mediated by local, functionally compartmentalized ATP-to-ADP ratios that differ from those in the bulk cytoplasm.

Ion, fluid and charge transport across Necturus intestinal epithelium in response to alanine.

Fluxes of Na, Cl and volume were followed across Necturus small intestine under zero voltage clamp. 20 mM L-alanine doubles the net Na and fluid transfer. Although there is a ouabain-sensitive Na pump present in Necturus a major fraction of the net Na flux can be measured for an hour after application of 10(-3) M ouabain. Collected fluid transferred by the epithelium is quasi-isotonic over a range of luminal osmolarities from 100 to 250 milliosmolar in alanine saline. The net Na fluxes account for the Na found in this transported fluid. Fluid transfer also shows a large ouabain-insensitive fraction after the addition of alanine. Compartmental analysis of 22Na-loaded epithelium was used to separate cellular and paracellular fluxes. The estimated Na concentration in the cell derived from its Na content is 9-10 mM, in agreement with that determined with microelectrodes. The Na efflux from cell to serosa is stimulated by alanine, but this increase accounts for only a quarter of the simultaneous rises in Na, fluid and current flow across the epithelium. The increase of Na efflux from the cell induced by alanine is apparently insensitive to ouabain although the cell Na content rises to circa 20 mM but no higher even after 20 hr. From the initial rate of rise of Na in the cell on treatment with ouabain the activity of the Na pump can be estimated to be approximately 92 pM/cm2 . sec, a value much smaller than the transepithelial net flux. The results are not consistent with the standard model in which Na-alanine influx stimulates the Na pump and enhances fluid transport by osmotic coupling in the lateral interspace system. A scheme is proposed based upon that for absorption in Necturus gallbladder by which alanine stimulates an active paracellular fluid transfer driven by motile elements of the junction.

Fluid recirculation in Necturus intestine and the effect of alanine.

Fluid absorption by Necturus small intestine has been studied using radiolabeled dextrans as molecular probes of the paracellular pathway under voltage-clamped conditions. Fluxes of H3-dextrans of MW up to 20K were followed in both directions between mucosal (M) and serosal (S) baths by fractionating those that passed the epithelium as a function of molecular radius. Consideration of the unstirred layers in the baths and the surface geometry rules out any contribution made by solute polarization. The geometry of the paracellular system was measured by light microscopy, TEM and SEM, and values were used in conjunction with a program that calculates convective-diffusive coupling in the tight junctions, intercellular spaces and subepithelium in series. The results indicate that the net fluxes are due to the convection of fluid through two opposing paracellular fluid circuits with different size selectivity, resulting in net absorption at small radii. Alanine at 20 mM stimulates fluid and salt uptake by a factor of 2. Its effect on the two convective components is to increase the M to S flux and decrease the S to M. The selectivities are not significantly different from those before alanine treatment. The volume absorption predicted from the net probe fluxes is very close to that measured gravimetrically across the epithelium.

Role of basolateral Na(+)-K(+)-Cl- cotransport in HCl secretion by amphibian gastric mucosa.

In amphibians and mammals, luminal H+ secretion by the stomach requires Cl-. It is widely accepted that a basolateral Cl-/HCO3- exchanger in the acid-secreting oxyntic cell restores the Cl- deficit resulting from apical HCl secretion. In this study, we used reverse transcriptase-polymerase chain reaction (RT-PCR) to generate a 1,200-bp fragment specific to a basolateral isoform of the Na(+)-K(+)-Cl- cotransporter in the gastric fundus of Necturus maculosus. By Northern analysis, we observed that gastric mucosa expresses greater than fivefold higher levels of mRNA encoding this cotransporter than any other tissue in the gastrointestinal tract. Feeding results in > 100% increases in mRNA levels in acid-secreting fundic mucosa but does not alter mRNA levels in the neighboring and non-acid-secreting antral mucosa or duodenum. Flux measurements using in vitro fundic mucosae indicate that acid secretion requires Na+ in the nutrient (i.e., serosal side) perfusate, is modulated by changes in nutrient K+ levels, and is inhibited by nutrient solutions containing 50 microM bumetanide, a recognized blocker of Na(+)-K(+)-Cl- cotransport. These findings suggest that this basolateral transporter plays a dominant and previously unsuspected role in secretion of HCl across the apical membrane.

A mechanism for isotonic fluid flow through the tight junctions of Necturus gallbladder epithelium.

During isotonic fluid flow, Necturus gallbladder epithelium mediates net fluxes of paracellular probes by a convective process. We show here that the paracellular system is modeled by permeation through three populations of channels: (i) convective parallel-sided ones of width 7.7 nm (ii) small diffusive ones of radius approximately 0.6 nm, and (ii) large diffusive ones of radius exceeding 50 nm. The reflexion coefficient of the convective channels is very low and the calculated osmotic flow rate is close to zero when compared with the observed fluid absorptive rate of 2 x 10(-6) cm/sec. Analysis reveals that the convective channels behave as though closed to back-diffusion of probes; if this is due to solvent drag then very high fluid velocities are required, acting through minute areas. There are no transjunctional gradients that could drive the flow, and so the fluid must be propelled through the channel by components of the junction. We propose a mechanism based upon an active junctional peristalsis which allows discrimination on the basis of molecular size, in which the channels are always occluded at some point and so back-diffusion cannot occur. There is no local gradient of salt distal to the junctions and therefore the osmotic permeability of the membranes is irrelevant. High fluid velocities are not required, and the flow can occur over a substantial fraction of the junction. The mechanism must involve motile and contractile elements associated with the junction for which there is already considerable evidence.

Merkel-like basal cells in Necturus taste buds contain serotonin.

Several types of cells have been identified in vertebrate taste buds, including dark cells, light cells, intermediate cells, type III cells, and basal cells. The physiological roles of these cell types are not well understood, especially those of basal cells. In this paper we show that there are two types of basal cells in taste buds from Necturus maculosus. One type of basal cell is an undifferentiated cell, presumably a stem cell. By combining light microscopic immunocytochemistry with electron microscopy, we show that the other type of basal cell is positive for serotonin-like immunoreactivity and that these cells have ultrastructural features similar to those found in cutaneous Merkel cells. Based on these findings, and the fact that the Merkel-like taste cells have been shown to make synaptic contacts with adjacent taste cells and with innervating nerve fibers, we conclude that these Merkel-like basal taste cells are serotonergic interneurons.

Influence of temperature and photoperiod on plasma melatonin in the mudpuppy, Necturus maculosus.

A melatonin (MEL) radioimmunoassay employed previously only in mammals was used to estimate plasma MEL in a salamander, Necturus maculosus. Validation procedures included thin-layer chromatography of plasma extracts, parallel inhibition curves of authentic MEL and serially diluted plasma and plasma extracts and quantitative recovery of authentic MEL added to pooled Necturus plasma. A diel cycle of plasma MEL was demonstrated in mudpuppies acclimatized for a minimum of 3 weeks under a 12L:12D photoperiod and 15 +/- 1 degrees and sampled in late March. The MEL cycle persisted under a reversed photoperiod (lights on, 1800 hr), but the amplitude of the MEL peak was diminished, and the peak was more than 180 degrees out of phase with the corresponding peak under a normal photoperiod. In animals acclimated to 5 degrees in mid-June under a 12L:12D photoperiod, the diel cycle of plasma MEL continued, but both midphotophase and midscotophase concentrations were reduced compared with 15 degrees controls. The diel cycle was also present in animals acclimated to 25 degrees, but the decrease in MEL concentrations was less marked than that in 5 degrees animals. Photoperiod is apparently the primary cue for cycles in plasma MEL in Necturus, but the cycle can be influenced by temperature.

Electrophysiological effects of extracellular ATP on Necturus gallbladder epithelium.

The effects of addition of ATP to the mucosal bathing solution on transepithelial, apical, and basolateral membrane voltages and resistances in Necturus gallbladder epithelium were determined. Mucosal ATP (100 microM) caused a rapid hyperpolarization of both apical (Vmc) and basolateral (Vcs) cell membrane voltages (delta Vm = 18 +/- 1 mV), a fall in transepithelial resistance (Rt) from 142 +/- 8 to 122 +/- 7 omega.cm2, and a decrease in fractional apical membrane resistance (fRa) from 0.93 +/- 0.02 to 0.83 +/- 0.03. The rapid initial hyperpolarization of Vmc and Vcs was followed by a slower depolarization of cell membrane voltages and a lumen-negative change in transepithelial voltage (Vms). This phase also included an additional decrease in fRa. Removal of the ATP caused a further depolarization of membrane voltages followed by a hyperpolarization and then a return to control values. fRa fell to a minimum after removal of ATP and then returned to control values as the cell membrane voltages repolarized. Similar responses could be elicited by ADP but not by adenosine. The results of two-point cable experiments revealed that ATP induced an initial increase in cell membrane conductance followed by a decrease. Transient elevations of mucosal solution [K+] induced a larger depolarization of Vmc and Vcs during exposure to ATP than under control conditions. Reduction of mucosal solution [Cl-] induced a slow hyperpolarization of Vmc and Vcs before exposure to ATP and a rapid depolarization during exposure to ATP. We conclude that ATP4- is the active agent and that it causes a concentration-dependent increase in apical and basolateral membrane K+ permeability. In addition, an apical membrane electrodiffusive Cl- permeability is activated by ATP4-.

Galanin immunoreactivity in the mudpuppy cardiac ganglion.

The source of galanin-immunoreactive fibers in the cardiac ganglion and on cardiac muscle in mudpuppy (Necturus maculosus) has been determined utilizing immunohistochemical techniques. The galanin-immunoreactive fibers are not processes of afferent fibers originating in either the rostral four dorsal root ganglia or vagal sensory ganglia. Following colchicine treatment, all of the postganglionic parasympathetic neurons and a subpopulation of the small intrinsic neurons in the cardiac ganglion exhibit galanin immunoreactivity. The majority of the galanin-immunoreactive fibers that form complexes on the parasympathetic postganglionic neurons are derived from galanin-immunoreactive small intrinsic neurons, although some of these connections may represent collateral processes from other parasympathetic postganglionic neurons. All of the galanin-immunoreactive processes that innervate cardiac muscle are derived from postganglionic parasympathetic neurons in the cardiac ganglion.

Na+/H+ exchange in glial cells of Necturus optic nerve.

Single and double-barreled pH-sensitive electrodes were used to study intracellular pH (pHi) regulation in glial cells of Necturus optic nerve in the nominal absence of HCO3-/CO2. After the cells were acidified by the addition and withdrawal of NH4+, the pHi recovered toward the original steady-state pHi. The recovery from acidification was Na+-dependent and inhibited by 1 mM amiloride. These results suggest the existence in intact vertebrate glial cells of a Na+/H+ exchanger which functions in acid extrusion.

Electron-cytochemical localization of alkaline phosphatase to G cells of Necturus maculosus antrum.

Electron-cytochemical localization of alkaline phosphatase activity was performed on G cells of Necturus maculosus antral mucosa. Alkaline phosphatase activity was localized to the nuclear membrane, the Golgi/endoplasmic reticulum, and the limiting membranes of G cell peptide-secretion vesicles. There was no specific localization of alkaline phosphatase activity to the plasma membrane. Treatment of the tissues with levamisole (an alkaline phosphatase inhibitor) did not markedly reduce the specific alkaline phosphatase activity. Specific lead deposition was reduced by removal of the substrate from the reaction mixture. The results from this study on N. maculosus G cells demonstrate that alkaline phosphatase activity can be found in a non-mammalian gastric endocrine cell and that specific activity was localized primarily to those intracellular structures involved with protein biosynthesis.

The presence and possible role of a galanin-like peptide in the mudpuppy heart.

A correlated histochemical and pharmacological study was undertaken to establish the presence, origin, and possible function of nerve fibers containing a galanin-like peptide in the mudpuppy (Necturus maculosus) heart. Whole mount preparations of septum-sinus venosus or atria and sections of ventricular muscle were prepared for immunocytochemistry. Galanin-immunoreactive fibers were found coursing diffusely across the septum-sinus venosus to form complex networks over cardiac muscle strands. Individual atrial muscle strands were densely innervated by galanin-immunoreactive fibers and galanin-immunoreactive fibers were also observed in the epicardial and myocardial layers of the ventricle. Most of the parasympathetic postganglionic neurons in the cardiac ganglion and many of the small intensely fluorescent-like cells exhibited galanin immunoreactivity. Galanin-immunoreactive fibers were present in the nerve trunks connecting clusters of parasympathetic postganglionic neurons. Close associations between galanin-positive fibers and individual parasympathetic postganglionic neurons were also observed. The presence of the galanin-immunoreactive fibers was similar in preparations taken from animals pretreated with 6-hydroxydopamine to that seen in preparations taken from control animals, indicating that the galanin-positive fibers were not sympathetic postganglionic axons. Moreover, the galanin-immunoreactive nerve fibers were separate from fibers containing substance P and/or calcitonin gene-related peptide that have previously been shown to be processes of afferent fibers. In twitch-tension experiments, galanin in the range 1 x 10(-7) to 1 x 10(-6) M caused cardioinhibition of spontaneously beating isolated septal-sinus venosus preparations. Galanin also produced a concentration-dependent (1 x 10(-7) to 1 x 10(-6) M) decrease in the twitch-tension development of electrically stimulated atrial or ventricular preparations. Local application of galanin produced hyperpolarization of cardiac muscle fibers in both isolated septal-sinus venosus preparations and atrial preparations. The response of individual parasympathetic ganglion cells to local application of galanin varied between neurons; some neurons were depolarized whereas others were hyperpolarized. We conclude that a galanin-like peptide is contained in both the parasympathetic postganglionic neurons and small intensely fluorescent-like cells and their processes. Further, we hypothesize that in the case of the parasympathetic postganglionic neurons, the galanin-like peptide may work in conjunction with acetylcholine to regulate cardiac activity.

Acetylcholinesterase activity in the adrenal chromaffin vesicles of Urodela.

The presence of acetylcholinesterase (AChE) activity in the adrenal chromaffin cells of Necturus maculosus and Ambystoma maculatum (Amphibia, Urodela) has been demonstrated by cytochemical method at the electron microscope level. The enzymatic activity is localized in RER and perinuclear cisternae, on the plasma membrane and within the chromaffin vesicles, both in adrenaline (A) and noradrenaline (N) cells. Moreover N cells appear to be more reactive than A cells and Necturus more reactive than Ambystoma. The possible function of the AChE activity inside the vesicles is discussed as a mechanism of protons donor or as peptidasic activity acting on various peptides present in the vesicle.

Na/H exchange in antral cells isolated from Necturus, rabbit, and guinea pig.

Mechanisms of intracellular pH (pHi) regulation were investigated using the pH-sensitive dye BCECF (2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein) to measure pHi in isolated antral cells from Necturus, rabbit, and guinea pig. The BCECF fluorescence was calibrated in terms of pHi by using a nigericin-high potassium concentration method to equilibrate pHi and pHo. Average pHi for the cells of Necturus and rabbit was approximately 7.1 in a HEPES-buffered, HCO3-free Ringer's solution. Average pHi in Necturus cells in CO2/HCO3-buffered Ringer's solution was approximately 7.0. An ammonium-loading technique was used to acidify the cell to measure intrinsic buffering capacity (beta i) and to monitor pHi recovery. Average beta i = 28 mM per pH unit in Necturus and 42 mM per pH unit in rabbit cells in HEPES-buffered Ringer's solution. Recovery from an acid load was sodium-dependent, completely inhibited by 100 microM amiloride, and unaffected by changes in potassium concentration between 6 and 25 mM. The results provide evidence in support of the presence of an amiloride-sensitive Na/H exchanger in antral cells from rabbit, guinea pig, and Necturus.

Membrane properties of isolated mudpuppy taste cells.

The voltage-dependent currents of isolated Necturus lingual cells were studied using the whole-cell configuration of the patch-clamp technique. Nongustatory surface epithelial cells had only passive membrane properties. Small, spherical cells resembling basal cells responded to depolarizing voltage steps with predominantly outward K+ currents. Taste receptor cells generated both outward and inward currents in response to depolarizing voltage steps. Outward K+ currents activated at approximately 0 mV and increased almost linearly with increasing depolarization. The K+ current did not inactivate and was partially Ca++ dependent. One inward current activated at -40 mV, reached a peak at -20 mV, and rapidly inactivated. This transient inward current was blocked by tetrodotoxin (TTX), which indicates that it is an Na+ current. The other inward current activated at 0 mV, peaked at 30 mV, and slowly inactivated. This more sustained inward current had the kinetic and pharmacological properties of a slow Ca++ current. In addition, most taste cells had inwardly rectifying K+ currents. Sour taste stimuli (weak acids) decreased outward K+ currents and slightly reduced inward currents; bitter taste stimuli (quinine) reduced inward currents to a greater extent than outward currents. It is concluded that sour and bitter taste stimuli produce depolarizing receptor potentials, at least in part, by reducing the voltage-dependent K+ conductance.

Rheogenic transport of basic and acidic amino acids across the brush border of Necturus small intestine.

In studies with isolated Necturus intestine, glutamate (Glu-) and Na+ each enhanced the mucosal influx of the other. Measurement of apical membrane potential, Va, with microelectrodes revealed a rapid depolarization with addition of 10 mM mucosal Glu-. This depolarization was Na+ dependent. Upon complete removal of Cl- from the bathing medium Va hyperpolarized and the Glu- -induced depolarization increased significantly. However, removal of Cl- did not alter the total Glu- influx. These data suggest that external Cl- attenuates the rheogenicity of Na+/Glu- cotransport in the apical membrane of the absorptive cells. We have presented a model consistent with these observations in which Cl- competes with one -COO- group of Glu- for its binding site on the carrier. The two complexes which may form, carrier/Glu-/2Na+ or carrier/Glu-/2Na+/Cl-, allow for either electrogenic or electroneutral transport of Glu-, depending on the ratio [Glu-]/[Cl-] in the extracellular fluid. In other experiments, addition of mucosal L-lysine (Lys+) induced a rapid depolarization of Va. In the presence of Na+, the depolarization appeared to be saturable with respect to Lys+ concentration. In Na+-free media, however, the depolarization increased with Lys+ concentration up to a maximum at 10 mM and then decreased to near zero at 30 mM. These data are consistent with a model for Lys+ entry in which an anionic site of the carrier can bind either Na+ or the epsilon-NH3+ group of Lys+. In this model transport of either complex, carrier-/Lys+ or carrier-/Lys+/Na+ (and return of the carrier to the extracellular surface) is rheogenic. However, at higher Lys+ concentrations, the epsilon-NH3+ group of a second Lys+ molecule may bind to the carrier forming a complex, carrier-/2Lys+, which is not transported.

Electrogenic Na/HCO3 cotransport across basolateral membrane of isolated perfused Necturus proximal tubule.

This study was undertaken to determine whether the proximal tubule of the mud puppy Necturus maculosus possesses a basolateral Na/HCO3 cotransporter. We examined the effects on basolateral membrane potential (Vbl) and intracellular pH (pHi) of 1) lowering basolateral [HCO3-] at constant PCO2, and 2) replacing Na+ with N-methyl-D-glucamine. Vbl and pHi were measured with Ling-Gerard and liquid-membrane pH microelectrodes, respectively, in isolated tubules perfused in vitro. We found that decreasing basolateral [HCO3-] from 10 mM (pH 7.5) to 2 mM (pH 6.8) resulted in an immediate depolarization of 14.9 mV, and a pHi decrease of 0.35. SITS (4-acetamido-4'-isothiocyanostibene-2,2'-disulfonic acid, 0.5 mM) inhibited the HCO3-induced depolarization by 87% and inhibited the initial rate of the pHi decrease by 79%. Replacement of basolateral Na+ with N-methyl-D-glucamine resulted in an immediate depolarization of 11.3 mV, and a pHi decrease of 0.36. SITS inhibited the zero Na-induced depolarization by 86% and the initial rate of the pHi decrease by 81%. Nominal removal of basolateral HCO3- (replaced with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) inhibited the zero Na-induced depolarization by 64%, whereas nominal removal of Na+ inhibited the 2 mM HCO3-induced depolarization by 67%. Replacement of all basolateral Cl- with glucuronate did not inhibit the changes in Vbl induced by changing [HCO3-] or [Na+]. Observations similar to those described above have been made previously on Ambystoma proximal tubules, and attributed to an electrogenic Na/HCO3 cotransport mechanism that carries HCO3-, Na+, and net negative charge in the same direction. We conclude that Necturus proximal tubules possess a similar, if not identical, electrogenic Na/HCO3 cotransport mechanism.

Electrophysiology of L-lysine entry across the brush-border membrane of Necturus intestine.

Microelectrode measurements of apical membrane potentials (Va) in absorptive cells of isolated Necturus intestine showed that, in the presence or absence of external Na+, 10 mM lysine added to the mucosal medium caused rapid depolarization followed by slower repolarization of Va. In Na+-free media the effects of 10 mM lysine on Va were abolished by 10 mM leucine which alone had no effect on Va under these conditions. This indicates that uncoupled electrodiffusion of lysine plays little or no role in lysine entry across the brush-border membrane. When external Na+ was greater than 10 mM the maximum depolarization of Va (delta Va') induced by [Lys] ranging from 5 to 30 mM was a simple saturable function of [Lys]. In Na+-free media, the relationship between delta Va' and [Lys] was biphasic. At first, delta Va' increased with increasing [Lys] reaching a maximum at 10 mM lysine. When [Lys] was further increased, delta Va' declined progressively to reach zero or near zero values. A single transport pathway model is proposed to account for rheogenic lysine entry across the brush-border membrane in the presence and absence of Na+. This postulates an amino acid transporter in the membrane with two binding sites. One is an amino acid site specific for the alpha-amino-alpha-carboxyl group. The other is a Na+ site. Neutral amino acids (e.g. leucine) compete with lysine for the amino acid site. The Na+ site has some affinity for the epsilon-amino group of lysine. When external Na+ is high the Na+ site is essentially 'saturated' with Na+ and formation of a mobile complex between an amino acid and the transporter depends in a saturable fashion on amino acid concentration. In Na+-free media or in media containing low [Na+]; at low external [Lys] the epsilon-amino group of a lysine molecule (simultaneously attached to the amino acid site) interacts with the Na+ site to form a mobile complex, as external [Lys] is increased, attachment of different lysine molecules to each site of an increasing number of transporters to form nontransported or poorly transported complexes results in substrate inhibition of the rheogenic lysine transport process.