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Basolateral K+ conductances in surface epithelium of Necturus antrum: effects of Ca2+ and divalent cations.

Intracellular microelectrode techniques were used to characterize basolateral membrane K+ conductances in isolated Necturus antral mucosa. Exposure of tissues to progressively higher levels of serosal K+ (4, 20, 40, or 60 mM) resulted in progressively greater depolarizations of basolateral membrane potentials and decreases in membrane resistance, consistent with the presence of a significant K+ conductance. Ba2+ (2 mM) partially blocks these conductances. Exposure of tissues to increased levels of serosal Ca2+ (from 1.8 to 6.8 mM) elicited significant hyperpolarization of basolateral potentials and decreases in basolateral resistance. These effects are also elicited by Sr2+ (5 mM), but not by Mg2+ (5 mM). Ba2+ (5 mM) elicits complex and time-dependent effects, but transiently elicits an effect similar to high Ca2+. Ion substitutions in the serosal perfusate suggest that the Ca(2+)-induced effects are due to enhancement of basolateral K+ conductances. Further work is necessary to identify the processes that mediate this increase in basolateral K+ conductance and to evaluate the physiological significance of this change in membrane permeability to K+.

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Nonelectrolyte permeability of the paracellular pathway in Necturus proximal tubule.

Micropuncture experiments were performed on Necturus proximal tubule using stationary microperfusion and microrecollection techniques. The transepithelial movement of the extracellular marker, sucrose, was used to investigate the passive permeability of the paracellular shunt pathway under steady-state conditions, during spontaneous reabsorption and water flow induced by an external osmotic gradient. Measurements were made of the sucrose permeability (P-s) efflux, net flux, and of net volume flow. True P-s determined in the absence of net volume flow and transepithelial gradient was 0.96 10-6 cm s-1. Both ouabain and isotonic volume expansion decreased shunt P-s. During reabsorption, solute-coupled water flow increased apparent P-s and net sucrose flux equalled efflux. Osmotic water flow from lumen to plasma decreased apparent P-s, with net sucrose flux equal to efflux; whereas osmotic flow from plasma to lumen increased apparent P-s but no net flux was observed. It is concluded that changes in P-s can be interpreted as relative alterations of the tight junction and the lateral spaces and that a portion of the volume flow from lumen to plasma proceeds via the tight junction.

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Estimate of relative thickness of peritubular interstitial space in Necturus kidney.

Electrophysiological techniques were used to evaluate volume changes of compartments interposed between the peritubular membrane of proximal tubue cells and capillary wall or peritoneal surface of Necturus kidney. The time courseof potassium-induced peritubular membrane depolarization was measured during a switch from 3- to 90-meq potassium sulfate solution in superfusion as well as in vascular perfusion experiments. When superfusion fluids contained 6, 2, and 0 g dextran/100 ml, mean half times of depolarization in normal kidneys were 4.1, 1.9, and 5.8 s. Decreasing the colloid osmotic pressure of superfusion fluids resulted in a significant increase in half-time values. Similar results were obtained in saline expansion. In controls the mean half time was 4.5s; in saline expansion it was 7.7 s. In vascular perfusions, mean half-time values were 225, 350, 355, and 450 s for dextran concentrations of 6, 4, 2, and 0 g/100 ml, respectively. These results indicate that the half time of depolarization increases significantly as the colloid osmotic pressure of perfusion fluids decreases. Estimates of effective unstirred-layer thickness on the peritubular side of proximal tubular epithelium indicate a decrease by about 25% when vascular colloid osmotic pressure is increased from 0 to 54 mmHg.

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Effects of peptide hormone structure on H+ secretion by Necturus gastric mucosa.

The actions of human synthetic gastrin I(G), the C-terminal tetrapeptide of gastrin (T), and the C-terminal octapeptide of cholecystokinin (OP) on acid secretion and transepithelial potential difference (PD) of the isolated Necturus gastric mucosa were determined. All three peptides induced H+ secretion, but the maximum H+ output was less with OP than with G or T. G and OP produced their maximum H+ output at lower molar concentrations than T. G- and OP-stimulated secretion was long sustained, but T-stimulated secretion rapidly returned to basal levels. T- and G-stimulated secretion was partially inhibited by the addition of OP. Evidence is presented that T rapidly disappears from solutions exposed to gastric mucosa, whereas G does not. Washing sensitized the mucosa to subsequent addition of T. The results suggest that the action of the common C-terminal tetrapeptide of G, T, and OP is modified by the preceding amino acid sequences, and that T, the smallest of the three peptides, is rapidly degraded by gastric tissue in vitro. The implications of the work for the study of gastrointestinal hormone structure-function relationships in isolated tissue preparations are discussed.

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Tracer Na fluxes in Necturus proximal tubule.

Steady-state bidirectional sodium fluxes were measured across Necturus proximal tubules. New methods for capillary perfusion and collection of venous effluent enabled flux determination to be made from the appearance of luminal tracer in the capillaries. Fluxes and permeability were measured in the absence of net fluid reabsorption. The sodium permeability measured in the plasma-to-lumen direction was 3 X 10(-6) cm/s. The flux ratio (lumen-to-plasma/plasma-to-lumen) was about twice the passive value calculated from the measured concentrations and potentials. Estimates for the permeability and flux across the shunt pathway were obtained from nonsteady-state flux determinations. The shunt pathway appeared to be the most significant route for passive sodium movement from plasma-to-lumen. Nonsteady-state tracer measurements also enabled an estimate to be made of the lumenal cell membrane permeability and unidirectional sodium flux. Two-thirds of the lumen-to-plasma flux was calculated to traverse the cellular path and the remainder through the shunt. Approximately one-third of the intracellular sodium was found to exchange rapidly with tracer.

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Permeability changes in Necturus proximal tubule during volume expansion.

The permeability of Necturus proximal tubule to hydrophilic nonelectrolytes of varying molecular size was studied under control conditions and during isotonic expansion of the animal's extracellular volume. Transepithelial permeability was measured in perfused tubular segments under conditions of zero net water flux. During volume expansion, tubular permeability to urea increased slightly, whereas mannitol decreased slightly and permeability to sucrose was significantly decreased. Volume expansion had a greater effect on osmotic flow parameters; the NaCl reflection coefficient decreased from 0.64 to 0.47 (summer animals) and from 0.41 to 0.27 (winter animals). Osmotic water flux and hydraulic conductivity increased but only in the lumen-to-capillary direction. Reflection coefficients of nonelectrolytes measured at the apical surface were reduced during volume expansion for probing molecules greater than 3 A in radius and were unchanged for smaller molecules, less than 3 A, suggesting two pore populations. We propose that an increase in tight-junction permeability can account for modification of osmotic flow parameters, whereas the whole thickness of the epithelium, particularly the intercellular space, plays the dominant role in regulation of diffusional permeability.

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Luminal Na+ entry into Necturus proximal tubule cells.

The dependence of intracellular Na+ activity on the electrical driving force across the luminal membrane and the presence of Cl- in the luminal perfusate was studied in Necturus proximal tubule. Intracellular Na+ and K+ activities were measured with microelectrodes filled with liquid ion exchanger. Perfusion of the tubule lumen with a NaCl-free solution caused cell Na+ activity to fall from the control value of 29.7 to 6.6 mM. In the absence of luminal driving force across the luminal membrane in accordance with simple diffusion of Na+ across this membrane. When the tubule lumen contained Na+ and Cl-, an electrically neutral component of Na+ entry into cells from the lumen appeared in addition to the diffusional component of Na+ entry.

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Studies on the bile acids of the gall bladder bile of a mud-puppy, Salamander necturus.

The major bile acids of gall bladder bile of salamander necturus have been reported and shown to be taurine conjugates. The bile acid conjugates were hydrolysed and the free bile acids were separated by thin layer chromatography and identified through gas liquid chromatography and mass spectrometry (GC-MS) as methyl esters and methyl-ester trimethyl silyl ethers. About 56.3% of the total bus acid was present as allocholic acid whereas 3alpha, 7alpha, l2alpha-dihydroxy-5alpha-cholestanoic acid and cholic acid represented 30.4 and 12.3% respectively. Dihydroxy bile acids such as deoxycholic, allodeoxycholic and allochenodeoxycholic were present in less than 0.5% of the total bile acids.

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Occurrence of polyhydroxy alcohols in the gall bladder bile of a mud-puppy, Salamander necturus.

Bile obtained from the gall bladder of Salamander necturus was investigated for the presence of bile alcohols by a combined technique of Thin Layer Chromatography (TLC), Gas Liquid Chromatography (GLC) and Gas Chromatography-Mass Spectrometry (GC-MS). Dried bile salts were hydrolysed with 2.5N KOH in an autoclave at 121 degrees C. Unsaponifiable matter was extracted with ethyl acetate, three times, washed with distilled water, dried with anhydrous sodium sulphate,evaporated to dryness and analysed for its bile alcohols. Analytical GLC revealed the presence of at least thirteen bile alcohols in the mixture. Two major bile alcohols, i.e. 5alpha-anhydrocyprinol and 5alpha-cyprinol constituted about 73% of the total unsaponifiable matter. Others were identified as 3alpha, 7alpha, 12alpha,trihdroxy-5beta-delta23-homocholene,3alpha,7alpha,12alpha-trihydroxy-5alpha-delta24-nor-27-cholestene,3alpha,7alpha,12alpha-rihydroxy-5alpha-delta24-cholestene,5alpha-bufol and 5alpha-dermophol.

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[Effects of salicylate on the electrical properties of the proximal convoluted tubule of Necturus maculosus].

The effects of peritubular salicylate for chloride substitution were studied in the isolated perfused Necturus kidney. This substitution resulted in changes of cell membrane p.d., varying from tubule to tubule; the withdrawal of the test-anion invariably produced a steep and prolonged depolarization. Exposure of the tissue to salicylate brought about, in addition, electrical uncoupling of junctional membranes, which was not related to concomitant changes of membrane p.d.

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X-like and Y-like ganglion cells in the Necturus retina.

The linearity of spatial summation within the receptive fields of Necturus retinal ganglion cells was examined with a counterphase-modulated split-field test stimulus. Modulation-locked histograms were constructed with the border at a variety of positions relative to the receptive field center. Most cells generated distinct, modulation-locked discharges when the border was 0.10 to 0.25 mm from the receptive field center. The discharges for more central border positions depended upon response type. Sustained-responding cells showed a sharp decrease in total response per cycle at the center position and increasing response for increasingly eccentric positions, i.e., nulling or X-like behavior. In contrast, the total response of transiently responding cells was largely independent of border position. For central positions of the border, these cells generated two bursts of impulses per cycle, i.e., frequency doubling or Y-like behavior.

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Intracellular ion activities in Necturus proximal tubule.

Ion-sensitive microelectrodes were used to measure the intracellular activities of Na, K, and Cl in proximal tubules of the perfused Necturus kidney. Cell Cl was 2-3 times higher than the value predicted for passive distribution during perfusion with normal Ringer; intracellular Na was far below the level for passive distribution. Cell Na and Cl fell to very low values when the lumen was NaCl-free. Cl entry into the tubule cell from the lumen required luminal Na. Na entered the cell across the luminal membrane both by diffusion and by coupled movement with Cl.

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Morphological studies on the mouth cavity of urodeles. IV. The teeth of the upper jaw and the palate in Necturus maculosus (Rafinesque) (Proteidae: Amphibia).

Adults of the neotenic (paedomorph) Necturus maculosus possess in the upper jaw and the palate rather uniform, conical, monocuspid teeth arranged in a single line ("Zahnzeile"; monostichous pattern) and showing a broad dividing zone, which separates the pedicel and the distal crown. This zone consists of globular and fibrous material obviously mineralized. Both premaxillaries are provided with teeth by one continuous dental lamina, whereas the vomer and palatopterygoid are accompanied by two dental laminae on each side of the palate. The results are compared to the conditions observed in other larval, neotenic, and metamorphosed Urodela.

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Dimensions of cells and lateral intercellular spaces in living Necturus gallbladder.

The size and shape of the cells and lateral intercellular spaces were measured in living Necturus gallbladder epithelium. Interspace volume was determined as a function of the transepithelial hydrostatic pressure difference. The compliance of the lateral membranes of the gallbladder cells was calculated from the interspace pressure-volume curves in both the presence and absence of fluid transport. Cell and interspace volume were studied when the NaCl in the mucosal bath was substituted by equiosmolar quantities of sucrose. The cells decreased in volume after the removal of mucosal NaCl and increased to control volume when the mucosal perfusate was 100 NaCl Ringer. The interspaces collapsed when fluid transport was inhibited by the removal of mucosal NaCl and reopened when NaCl was reintroduced to the mucosal bath. The rate of change of cell volume was used to calculate the active transport rate and the flux of NaCl across the apical membrane. The magnitude of the cell volume change during NaCl replacement indicated that all intracellular NaCl was readily accessible to be transported out of the cell.

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Retina viability in the Necturus.

Electroretinograms (ERG's) were recorded from the eyes of paralyzed normal and decapitated mudpuppies (Necturus maculosus). Fully dark-adapted responses to a range of stimulus intensities were compiled hourly under both conditions. Statistical analyses indicate no significant change in responsiveness in paralyzed normal animals during periods up to 6 hours. After decapitation, there was a significant decline of sensitivity to light during the first 3 hours. An oxygen-sensitive component similar to the c-wave was observed with DC recording but only in normal eyes. Anoxic mudpuppy eyes show an ERG decline with an average time constant of 1.5 hours.

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The organization of the motoneurons innervating the axial musculature of vertebrates. I. Goldfish (Carassius auratus) and mudpuppies (Necturus maculosus).

The motoneurons innervating different regions of the myomeres in goldfish and mudpuppies were examined by applying HRP to the musculature or to branches of spinal nerves. In goldfish, the populations of motoneurons innervating epaxial or hypaxial muscle occupied similar positions in the motor column and had similar size distributions. There was no relationship between the size or location of a motoneuron in the motor column and the dorsoventral location of the muscle it innervated in the myomeres. Instead, different populations of motoneurons innervated the functionally different red and white musculature. The red muscle was innervated only by small motoneurons that occupied the ventral portion of the motor column. Their small axons passed lateral to the Mauthner axon in the cord, and most of them traveled in a separate branch of each spinal nerve that ran in the horizontal septum to the red muscle. The white muscle was innervated by a population of motoneurons that did not innervate red. They were large and they occupied a characteristic position in the extreme dorsal part of the motor column. Their large axons traveled medial to the Mauthner axon in the cord and entered branches of spinal nerves running deep in the epaxial or hypaxial muscle. The white muscle was probably also innervated by some smaller motoneurons similar to those innervating red; however, these may have been motoneurons whose axons ran through white muscle to reach other muscle. The large motoneurons innervating only white muscle are similar to the primary motoneurons identified in developmental studies in teleosts (Myers: Soc. Neurosci. Abstr. 9:848, '83); the smaller ones, innervating both red and white, are like secondary motoneurons. Therefore, in goldfish, motoneurons having different morphology and developmental history also innervate different regions in the myomeres. The motor column in mudpuppies was, in general respects, similar to the column in goldfish. There were large primary motoneurons and small secondary ones. Though there were slight differences in the locations of motoneurons filled from nerves entering epaxial and hypaxial muscle, their distributions in the cord overlapped substantially. The motor columns in these two anamniotes differ substantially from the motor columns in those amniotes that have been studied. In amniotes, the motoneurons innervating epaxial and hypaxial muscles are spatially segregated in the cord (Smith and Hollyday: J. Comp. Neurol. 220:16-28, '83; Fetcho: J. Comp. Neurol. 249:551-563, '86).(ABSTRACT TRUNCATED AT 400 WORDS)

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