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PubMed1992

Aminergic and peptidergic elements and actions in a cardiac parasympathetic ganglion.

Correlated histochemical, immunocytochemical, and electrophysiological experiments have been undertaken to identify putative neurotransmitter-neuromodulator substances in cells and fibers in the parasympathetic cardiac ganglion of the mudpuppy, Necturus maculosus, and to determine the action of these agents on the properties of the parasympathetic postganglionic neurons. The mudpuppy cardiac ganglion contains two neuron types: large parasympathetic postganglionic neurons and smaller intrinsic neurons initially identified as small intensely fluorescent cells. We have shown that the postganglionic neurons contain both acetylcholine and a galanin-like neuropeptide. Also, we have demonstrated that the intrinsic neurons contain a number of different biogenic amines such as dopamine and serotonin, as well as neuropeptides including a substance P-like peptide and a galanin-like peptide. The results of these studies indicate that the anatomical and histochemical organization of the mudpuppy cardiac ganglion is more complex than that seen in other amphibians and is very similar to that found in most mammalian species. Previously, we showed that galanin has actions that make it of interest as a potential inhibitory neurotransmitter in the mudpuppy cardiac ganglion. Galanin hyperpolarizes and decreases membrane excitability in most parasympathetic neurons. Here we show that galanin initiates membrane hyperpolarization by activating a voltage- and time-dependent potassium conductance. We also present the initial results of ongoing studies which indicate that calcitonin gene-related peptide can depolarize some of the parasympathetic neurons as well as evidence that serotonin initiates depolarization in many parasympathetic neurons. This serotonin-induced depolarization consists of an initial transient depolarization followed by a longer, more slowly developing depolarization. Action potential activity is stimulated during the initial period of depolarization, but depressed during the later, slow depolarization. The results of these electrophysiological experiments suggest that many of the bioactive substances that have been identified in the different cells and nerve fibers within the cardiac ganglion affect the excitability of the postganglionic neurons. In conclusion, we suggest that the results of the studies summarized in this review demonstrate that the cardiac ganglion in the mudpuppy is not simply a relay station. Rather, the cardiac ganglion has a complex organization and exhibits a diversity of physiological responses, indicating that it very likely is another site of integration for control of cardiac function.

Animals↗

PubMed1993

Membrane currents evoked by excitatory amino acid agonists in ON bipolar cells of the mudpuppy retina.

1. Whole-cell patch-clamp recordings were obtained from ON bipolar cells in a retinal slice preparation of the mudpuppy, Necturus maculosus. The effects of excitatory amino acid (EAA) agonists applied in the presence of cobalt (2-5 mM) were examined. 2. At the holding potential of -50 mV, L-2-amino-4-phosphonobutanoic acid (L-AP4, 5-10 microM) evoked an outward current accompanied by a conductance decrease. The zero current potential of the L-AP4-evoked current was near 0 mV independent of whether the intracellular Ringer solution contained CsCl or CsCH3SO4. The currents evoked by light were also accompanied by a conductance decrease and reversed near 0 mV. Replacing external sodium with choline or N-methyl-D-glucamine generated an outward current and suppressed the response to L-AP4. The response to L-AP4 was enhanced by removing extracellular calcium and suppressed by increasing extracellular calcium. These results indicate that L-AP4 closes nonspecific cation channels that are blocked by extracellular calcium. 3. In 2 mM cobalt, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA, 50-100 microM) evoked membrane currents that were accompanied by a conductance increase. AMPA-evoked currents exhibited a significant chloride dependence and were suppressed by gamma-aminobutyric acid-A (GABAA) antagonists bicuculline and picrotoxin; a GABA uptake blocker, nipecotic acid; and a glycine antagonist, strychnine. AMPA-induced currents were virtually absent in the presence of 5 mM cobalt and nominally 0 mM extracellular calcium. These results indicate that the conductance increase induced by AMPA in the presence of 2 mM cobalt is largely the result of calcium-dependent synaptic inputs onto GABAA and glycine receptors of ON bipolar cells. 4. N-methyl-D-aspartic acid (250 microM) was ineffective when applied in the presence of 100 microM cadmium or 2 mM cobalt. 5. 1S,3R/1R,3S-1-aminocyclopentane-1,3-dicarboxylic acid (100-200 microM) evoked an outward current accompanied by a conductance decrease and appears to be an agonist at the L-AP4 receptor. 6. The findings of this study suggest that the only type of EAA receptor in mudpuppy ON bipolar cells is the L-AP4 receptor and that L-AP4 receptor activation results in the closing of nonspecific cation channels that are blocked by extracellular calcium.

Amino Acids↗

PubMed1989

Characterization of a phosphorylation-activated Cl-selective channel in isolated Necturus enterocytes.

1. The cell-attached and excised inside-out configurations of the patch-clamp technique were employed to probe isolated enterocytes of Necturus maculosus for the presence of Cl(-)-selective channels. 2. Chloride-selective channels were rarely observed unless cells were previously stimulated by agonists that raise cyclic AMP. In cell-attached patches forskolin (20 microM) or dibutyryl cyclic AMP 2 mM) evoked single-channel activity that reversed, depending on the cell, between 9 and 27 mV positive to the spontaneous membrane potential. This is close to the Cl- equilibrium potential in those cells; the single-channel current-voltage relationship was linear with a unitary slope conductance between 17 and 25 pS (pipettes filled with 100 mM-NaCl). 3. Large depolarizing voltage steps also activated Cl- channels in excised inside-out membrane patches that were previously quiescent. This mode of activation produced a distinctive single-channel current-voltage relationship with strong outward rectification at depolarizing membrane potentials. Single-channel cord conductance at negative potentials was 15-18 pS and increased to 45 pS at + 100 mV. 4. Altering the Cl- concentration in the bathing solution of excised inside-out patches displaced the observed reversal potential (Erev) to values predicted for Cl- equilibrium potential. Replacement of K+ for Na+ was without effect. 5. The effect of different anions upon Erev was used to determine the channel anion selectivity in excised inside-out patches. The permeability sequence was SCN- greater than I- greater than Br- greater than Cl- greater than F- greater than HCO3- greater than gluconate which corresponds to Eisenman's sequence 1. Neither ionic size nor diffusion rates determine the permeation of ions through the channel. 6. In channels activated by depolarization the open probability (Po) was insensitive to changes in the Ca2+ concentration (less than 10(-8)-10(-3) M) bathing the cytoplasmic face of excised inside-out patches. Depolarization was also without marked effect on Po. 7. Chloride channels in excised inside-out patches were inhibited by stilbene and diphenylamine-2-carboxylate derivatives. 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS, 5 x 10(-5) M) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB, 1 x 10(-5) M) caused an irreversible 'flickery' blockade without altering single-channel current. 3'5-Dichlorodiphenylamine-2-carboxylic acid (DDPC, 5 x 10(-5) M) reduced the currents at every voltage without apparent effects on gating properties of the channel.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed2002

Renal physiology between two wars: the contributions of Dr. Harvey Lester White.

Harvey Lester White (1896-1977) graduated from Washington University (St. Louis) School of Medicine and subsequently spent his entire professional career in the School's Department of Physiology. White's interest in the function of the kidney was evident early in his academic career when he pioneered research related to renal physiology. His first papers dealt with studies of renal tubular function (1923). He performed what appear to represent the first micropuncture studies in the kidney of Necturus maculosus (1926) and thus confirmed the observations of Wearn and Richards regarding glomerular filtration. Through these studies, he was the first to show that glucose reabsorption occurs in the proximal tubules of Necturus. It also appears that White et al. were the first to demonstrate that volume expansion increases bicarbonate excretion in dogs (1926). He studied the influence of posture on renal "activity" in man (1926). Intracapsular pressure determinations in Necturus kidney (1928) were done using micropuncture techniques. White and co-workers presented (1933) a comparison of clearances of creatinine and various sugars confirming the work of Jolliffe, Shannon and Smith (1932) who had proposed the clearances of non-metabolizable sugars to be a measure of glomerular filtration. Between 1932 and 1936, in an effort to better understand the process of glomerular filtration, he began a series of studies on streaming potentials, surface conductance, electro-endosmosis and other related topics. In 1937, White began work on yet another aspect of renal physiology--endocrine influences on renal function. He would immerse himself in these investigations throughout the remainder of his scientific career. We hope that this account will reveal at least a small dimension of the man and his contributions to renal physiology.

Animals↗

PubMed2002

Electrophysiology of Necturus taste cells.

Taste buds are sensory end organs that detect chemical substances occurring in foodstuffs and relay the relative information to the brain. The mechanisms by which the chemical stimuli are converted into biological signals represent a central issue in taste research. Our understanding of how taste buds accomplish this operation relies on the detailed knowledge of the biological properties of taste bud cells-the taste cells-and of the functional processes occurring in these cells during chemostimulation. The amphibian Necturus maculosus (mudpuppy) has proven to be a very useful model for studying basic cellular processes of vertebrate taste reception, some of which are still awaiting to be explored in mammals. The main advantages offered by Necturus are the large size of its taste cells and the relative accessibility of its taste buds, which can therefore be handled easily for experimental manipulations. In this review, I summarize the functional properties of Necturus taste cells studied with electrophysiological techniques (intracellular recordings and patch-clamp recordings). My focus is on ion channels in taste cells and on their role in signal transduction, as well as on the functional relationships among the cells inside Necturus taste buds. This information has revealed to be well suited to outline some of the general physiological processes occurring during taste reception in vertebrates, including mammals, and may represent a useful framework for understanding how taste buds work.

Action Potentials↗

PubMed1984

Studies on cytochrome P-450-dependent microsomal enzymes of testicular androgen and estrogen biosynthesis in a urodele amphibian, Necturus.

The microsomal fraction isolated from the testis of the urodele amphibian, Necturus maculosus, is very rich in cytochrome P-450 and three cytochrome P-450-dependent steroidogenic enzyme activities, 17 alpha-hydroxylase, C-17, 20-lyase, and aromatase. In this study, we investigated aspects of these reactions using both spectral and enzyme techniques. In animals obtained at different points in the annual cycle, Necturus testis microsomal P-450 concentrations ranged from 0.6-1.8 nmol/mg protein. Substrates for the three enzymes generated type I difference spectra; progesterone and 17 alpha-hydroxyprogesterone appeared to bind to one P-450 species while the aromatase substrates, androstenedione, 19-hydroxyandrostenedione, and testosterone, all bound to another P-450 species. Spectral binding constants (Ks) for these interactions were determined. Michaelis constants (Km) and maximum velocities were determined for progesterone 17 alpha-hydroxylation, 17 alpha-hydroxyprogesterone side-chain cleavage, and for the aromatization of androstenedione, 19-hydroxyandrostenedione, and testosterone. Measured either by spectral or kinetic methods, progesterone, androstenedione, and 19-hydroxyandrostenedione were high affinity substrates (Ks or Km less than 0.3 microM), while 17 alpha-hydroxyprogesterone and testosterone were low affinity substrates (Ks or Km = 0.6-4.8 microM). As evidence for the participation of cytochrome P-450 in these reactions, carbon monoxide was found to inhibit each of the enzyme activities studied. The activity of NADPH-cytochrome c reductase, a component of cytochrome P-450-dependent reactions, was also high in Necturus testis microsomes.

Androgens↗

PubMed1991

Small angle x-ray scattering of chromatin. Radius and mass per unit length depend on linker length.

Analyses of low angle x-ray scattering from chromatin, isolated by identical procedures but from different species, indicate that fiber diameter and number of nucleosomes per unit length increase with the amount of nucleosome linker DNA. Experiments were conducted at physiological ionic strength to obtain parameters reflecting the structure most likely present in living cells. Guinier analyses were performed on scattering from solutions of soluble chromatin from Necturus maculosus erythrocytes (linker length 48 bp), chicken erythrocytes (linker length 64 bp), and Thyone briareus sperm (linker length 87 bp). The results were extrapolated to infinite dilution to eliminate interparticle contributions to the scattering. Cross-sectional radii of gyration were found to be 10.9 +/- 0.5, 12.1 +/- 0.4, and 15.9 +/- 0.5 nm for Necturus, chicken, and Thyone chromatin, respectively, which are consistent with fiber diameters of 30.8, 34.2, and 45.0 nm. Mass per unit lengths were found to be 6.9 +/- 0.5, 8.3 +/- 0.6, and 11.8 +/- 1.4 nucleosomes per 10 nm for Necturus, chicken, and Thyone chromatin, respectively. The geometrical consequences of the experimental mass per unit lengths and radii of gyration are consistent with a conserved interaction among nucleosomes. Cross-linking agents were found to have little effect on fiber external geometry, but significant effect on internal structure. The absolute values of fiber diameter and mass per unit length, and their dependencies upon linker length agree with the predictions of the double-helical crossed-linker model. A compilation of all published x-ray scattering data from the last decade indicates that the relationship between chromatin structure and linker length is consistent with data obtained by other investigators.

Animals↗

PubMed1989

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.

Acetylcholinesterase↗

PubMed1997

Identification and functional assay of an extracellular calcium-sensing receptor in Necturus gastric mucosa.

In mammals and amphibians, increases in extracellular Ca2+ can activate bicarbonate secretion and other protective functions of gastric mucosa. We hypothesized that the recently cloned extracellular Ca(2+)-sensing receptor (CaR) is functioning in the gastric mucosa. In Necturus maculosus gastric mucosa, reverse transcription-polymerase chain reaction using primers based on previously cloned CaR sequences amplified a 326-bp DNA fragment that had 84% nucleotide sequence identity with the rat kidney CaR. Immunohistochemical localization of the CaR using specific anti-CaR antiserum revealed its presence on the basal aspect of gastric epithelial cells. In microelectrode studies of Necturus antral mucosa, exposure to elevated Ca2+ (4.8 mM) and the CaR agonists NPS-467 and neomycin sulfate resulted in significant hyperpolarizations of basal membrane electrical potentials and increases in apical-to-basal membrane resistance ratios. Circuit analysis revealed that these changes reflected specific decreases in basolateral membrane resistance. Inhibition of prostaglandin synthesis using indomethacin significantly attenuated these effects. We conclude that the CaR is present and functioning in Necturus gastric antrum.

Amiloride↗

PubMed1985

3,5,3'-Triiodothyronine receptors and thyroxine 5'-monodeiodinating activity in thyroid hormone-insensitive amphibia.

Adult anuran Amphibia and neotenous urodeles, such as Necturus maculosus, rarely respond to thyroid hormone (TH). In this study, the possibility was examined that this lack of response is due either to an inability to convert thyroxine (T4) to 3,5,3'-triiodothyronine (T3) or to the absence of nuclear TH receptors. Following injection of [125I]T4, significant amounts of [125I]T3, analyzed by chromatography, were detected in the serum and liver of Necturus and Rana catesbeiana frog indicating that both possesses a T4 5'-monodeiodinating system. Nuclear binding of T3 was studied in suspensions of purified hepatic nuclei and intact red blood cells (RBC). Analysis of binding data revealed that frog liver nuclei contained two sets of saturable T3 binding sets with affinities comparable to those of the two sets of sites previously demonstrated in tadpole liver nuclei (Galton and Schaafsma, 1983). However, the number of sites per nuclei was small compared to tadpole; expressed as pmol/mg DNA, the maximum binding capacity of the high affinity set of sites (MBC1) was 2.2 +/- 0.31 versus 12.9 +/- 1.80 and MBC2 was 24.9 +/- 4.5 versus 42.2 +/- 54. Receptor number in RBC nuclei was also smaller in frog than tadpole: 90 +/- 26 versus 882 +/- 56 sites/nucleus (Kd less than 10(-11) M in both groups). No comparable high affinity binding sites were detected in Necturus liver, but some sites were found in Necturus RBC. These cells contained 2111 +/- 120 sites/nucleus, more than twice the number found in tadpole.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1977

Length and interspersion of repetitive and non repetitive DNA sequences in four amphibian species with different genome sizes.

The interspersion period of repetitive and unique sequences was analyzed by two different methods, electron microscopy and agarose gel electrophoresis, for four Amphibian species with different nuclear DNA content, namely the Anura Xenopus laevis (3 pg DNA per haploid genome) and Bufo bufo (7 pg) and the Urodela Triturus cristatus (23 pg) and Necturus maculosus (52 pg). Within each of the two subclasses it has been found that interspecific differences, in DNA content, due to variations in the amount of repetitive sequences, do not involve variations in length of the interspersed repetitive sequences. They remain about 380 base pairs. Furthermore, the unique sequences length has been found to be shorter in Bufo (760 base pairs) than in Xenopus (1600) and in Necturus (880) than in Triturus (1340). A study of the interspersion period has shown that the great difference in DNA content between Anura and Urodela, which had been previously shown not to have involved changes in the relative amounts of the various sequence classes, does not involve changes in the interspersion period.

Animals↗

PubMed1981

2-amino-4-phosphonobutyric acid: a new pharmacological tool for retina research.

Information processing in the vertebrate retina occurs in two separate channels known as ON and OFF channels. When intracellular electrophysiological recordings were obtained from the perfused retina-eyecup preparation of the mud-puppy (Necturus maculosus), the addition of 2-amino-4-phosphonobutyric acid to the bathing medium blocked all responses in the ON channel but left intact the OFF responses including OFF ganglion cell discharge. 2-Amino-4-phosphonobutyric acid blocks the light response of the ON bipolar cell by mimicking the endogenous photoreceptor transmitter.

Action Potentials↗

PubMed1997

A cyclic nucleotide-dependent chloride conductance in olfactory receptor neurons.

Whole-cell membrane currents were recorded from olfactory receptor neurons from the neotenic salamander Necturus maculosus. Cyclic nucleotides, released intracellularly by flash photolysis of NPE-caged cAMP or NPE-caged cGMP, activated a transient chloride current. The chloride current could be elicited at constant voltage in the absence of extracellular Ca2+ as well as in the presence of 3 mM intracellular Ca2+, suggesting that the current did not require either voltage or Ca2+ transients for activation. The current could be elicited in the presence of the protein kinase inhibitors H-7 and H-89, and in the absence of intracellular ATP, indicating that activation was independent of protein kinase A activity. These results suggest that Necturus olfactory receptor neurons contain a novel chloride ion channel that may be directly gated by cyclic nucleotides.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed2000

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.

Animals↗

PubMed1991

Mediation of responses to calcium in taste cells by modulation of a potassium conductance.

Calcium salts are strong taste stimuli in vertebrate animals. However, the chemosensory transduction mechanisms for calcium are not known. In taste buds of Necturus maculosus (mud puppy), calcium evokes depolarizing receptor potentials by acting extracellularly on the apical ends of taste cells to block a resting potassium conductance. Therefore, divalent cations elicit receptor potentials in taste cells by modulating a potassium conductance rather than by permeating the cell membrane, the mechanism utilized by monovalent cations such as sodium and potassium ions.

Animals↗

PubMed1988

Apical localization of K+ channels in taste cells provides the basis for sour taste transduction.

Previous studies have shown that mudpuppy taste receptor cells respond to sour taste stimuli (weak acids) with depolarizing receptor potentials or action potentials that are blocked by the K+ channel blocker tetraethylammonium. Voltage-clamp recordings from isolated taste cells indicated that taste receptor cells exhibit a variety of voltage-dependent conductances and that acids reduce a voltage-dependent K+ current. Since taste stimuli are restricted to the apical surface of the intact tongue, only 1-2% of the taste receptor cell surface is exposed to chemical stimuli. Thus, modification of a K+ conductance would be an effective transduction mechanism in receptor cells only if the majority of K+ channels were located on the apical membrane. We have used a combination of "loose-patch" and whole-cell recording methods to map the distribution of voltage-sensitive K+ and Na+ channels on dissociated Necturus maculosus taste cells. We report here that the K+ conductance is approximately equal to 50-fold greater on apical membrane than on basolateral membrane, whereas the Na+ conductance is distributed evenly. The marked nonuniformity of the voltage-sensitive K+ conductance, together with the block of this conductance by sour stimuli, indicates that K+ current modulation is the mechanism of sour taste transduction.

Animals↗

PubMed1997

Thyroid hormone receptor genes of neotenic amphibians.

Since thyroid hormones play a pivotal role in amphibian metamorphosis we used PCR to amplify DNA fragments corresponding to a portion of the ligand-binding domain of the thyroid hormone receptor (TR) genes in several neotenic amphibians: the obligatory neotenic members of the family Proteidea the mudpuppy Necturus maculosus and Proteus anguinus as well as two members of the facultative neotenic Ambystoma genus: the axolotl Ambystoma mexicanum and the tiger salamander Ambystoma tigrinum. In addition, we looked for TR genes in the genome of an apode Typhlonectes compressicaudus. TR genes were found in all these species including the obligatory neotenic ones. The PCR fragments obtained encompass both the C and E domains and correspond to alpha and beta genes. Their sequences appear to be normal, suggesting that there is no acceleration of evolutionary rates in the TR genes of neotenic amphibians. This result is not surprising for Ambystomatidae, which are known to respond to T3 (3,3',5-triiodothyronine) but is not in agreement with biochemical and biological data showing that Proteidea cannot respond to thyroid hormones. Interestingly, by RT-PCR analysis we observed a high expression levels of TRalpha in gills, intestine, and muscles of Necturus as well as in the liver of Ambystoma mexicanum, whereas TRbeta expression was only detected in Ambystoma mexicanum but not in Necturus. Such a differential expression pattern of TRalpha and TRbeta may explain the neoteny in Proteidea. The cloning of thyroid-hormone-receptor gene fragments from these species will allow the molecular study of their failure to undergo metamorphosis.

Amino Acid Sequence↗

PubMed1999

Volume regulatory responses of basolateral membrane vesicles from Necturus enterocytes: role of the cytoskeleton.

Previous studies from this laboratory have demonstrated that basolateral membrane vesicles isolated from Necturus maculosus small intestinal epithelial cells possess a K(+) channel that is inhibited by ATP. In the present studies, we demonstrate that these vesicles, which are essentially devoid of soluble cytoplasmic contaminants, exhibit volume regulatory responses that parallel those of intact epithelial cells. Thus, suspension of these vesicles in a solution that is hypotonic to the intravesicular solution increases channel activity whereas suspension in a solution that is hypertonic to the intravesicular solution decreases, and may abolish, channel activity. These volume regulatory responses appear to be mediated by the same K(ATP) channel and depend on an intact actin cytoskeletal network. The responses to both hypotonic and hypertonic challenge are abolished by cytochalasin D or by incubating the vesicles under conditions that are known to depolymerize actin. Phalloidin, which is known to stabilize actin filaments, partially prevents the action of cytochalasin D. Thus, the present results indicate that the K(ATP) channel activity of basolateral membrane vesicles from Necturus basolateral membranes respond to hypo- and hypertonic challenge monotonically around an isotonic "set point" and that these responses depend on an intact actin cytoskeleton.

Actins↗