Evidence for permanent water channels in the basolateral membrane of an ADH-sensitive epithelium

Summary The transepithelial water permeability in frog urinary bladder is believed to be essentially dependent on the ADH-regulated apical water permeability. To get a better understanding of the transmural water movement, the diffusional water permeability (P _d) of the basolateral membrane of urinary bladder was studied. Access to this post-luminal barrier was made possible by perforating the apical membrane with amphotericin B. The addition of this antibiotic increasedP _d from 1.12±0.10×10^–4 cm/sec (n=7) to 4.08±0.33×10^–4 cm/sec (n=7). The effect of mercuric sulfhydryl reagents, which are commonly used to characterize water channels, was tested on amphotericin B-treated bladders. HgCl₂ (10^–3 m) decreasedP _d by 52% andpara-chloromercuribenzoic acid (pCMB) (1.4×10^–4 m) by 34%. The activation energy for the diffusional water transport was found to increase from 4.52±0.23 kcal/mol (n=3), in the control situation, to 9.99±0.91 kcal/mol (n=4) in the presence of 1.4×10^–4 m pCMB. Our second approach was to measure the kinetics of water efflux, by stop-flow light scattering, on isolated epithelial cells from urinary bladders.pCMB (0.5 or 1.4×10^–4 m) was found to inhibit water exit by 91±2%. These data strongly support the existence of proteins responsible for water transport across the basolateral membrane, which are permanently present.     

Effects of angiotensin-II receptor blocker candesartan cilexetil in rats with dilated cardiomyopathy

We examined effects of an angiotensin-II receptor blockers, candesartan cilexetil, in rats with dilated cardiomyopathy after autoimmune myocarditis. Candesartan cilexetil showed angiotensin-II blocking action in a dose-dependent manner in rats with dilated cardiomyopathy. Twenty-eight days after immunization, surviving Lewis rats were divided into four groups and given candesartan cilexetil at 0.05 mg/kg, 0.5 mg/kg or 5 mg/kg per day (Group-C0.05, n = 15, Group-C0.5, n = 15 and Group-C5, n = 15, respectively) or vehicle alone (Group-V, n = 15). After oral administration for 1 month, the left ventricular end-diastolic pressure and heart weight/body weight ratio were lower in Group-C0.05 (13.3± 1.1 mmHg and 3.7± 0.2 g/kg, respectively), in Group-C0.5 (8.0± 0.9 mmHg and 3.3± 0.1 g/kg, respectively) and in Group-C5 (5.5± 1 mmHg and 3.1± 0.1 g/kg, respectively) than in Group-V (13.5± 1.0 mmHg and 3.8± 0.2 g/kg, respectively). The area of myocardial fibrosis was also lower in Group-C0.05 (25± 3%), in Group-C0.5 (20± 3%), and in Group-C5 (12± 1%) than in Group-V (32± 4%). Furthermore, expressions of transforming growth factor-1 and collagen-III mRNA were suppressed in Group-C0.05 (349± 23% and 395± 22%, respectively), Group-C0.5 (292± 81% and 364± 42%, respectively) and in Group-C5 (204± 63% and 259± 33%, respectively) compared with those in Group-V (367± 26% and 437± 18%, respectively). These results suggest that candesartan cilexetil can improve the function of inefficient heart. (Mol Cell Biochem 269: 137–142, 2005)     

Pre-steady-state analysis of the turn-on and turn-off of water permeability in the kidney collecting tubule

Summary Water transport across the mammalian collecting tubule is regulated by vasopressin-dependent water channel insertion into and retrieval from the cell apical membrane. The time course of osmotic water permeability (P _f ) following addition and removal of vasopressin (VP) and 8-Br-cAMP was measured continuously by quantitative fluorescence microscopy using an impermeant fluorophore perfused in the lumen. Cortical collecting tubules were subjected to a 120 mOsm bath-to-lumen osmotic gradient at 37°C with 10–15 nl/min lumen perfusion and 10–20 ml/min bath exchange rate. With addition of VP (250 U/ml), there was a 23±3 sec (sem,n=16) lag in whichP _f did not change, followed by a rise inP _f (initial rate 1.4±0.2×10^–4 cm/sec²) to a maximum of 265±10×10^–4 cm/sec. With addition of 8-Br-cAMP (0.01–1mm) there was an 11±2 sec lag. For [8-Br-cAMP]=0.01, 0.1 and 1mm, the initial rate ofP _f increase following the lag was (units 10^–4 cm/sec²): 1.1±0.1, 1.2±0.1 and 1.7±0.3. MaximumP _f was (units 10^–4 cm/sec): 64±4, 199±9 and 285±11. With removal of VP,P _f decreased to baseline (12×10^–4 cm/sec) with aT _1/2 of 18 min; removal of 0.1 and 1mm 8-Br-cAMP gaveT _1/2 of 4 and 8.5 min. These results demonstrate (i) a brief lag in theP _f response, longer for stimulation by VP than by 8-Br-cAMP, representing the transient build-up of biochemical intermediates proximal to the water channel insertion step, (ii) similar initialdP _f /dt (water channel insertion) over a wide range of [8-Br-cAMP] and steady-stateP _f values, and (iii) more rapidP _f decrease with removal of 8-Br-cAMP than with VP. These pre-steady-state results define the detailed kinetics of the turn-on and turn-off of tubuleP _f and provide kinetic evidence that the rate-limiting step for turn-on ofP _f is not the step at which VP regulates steady-stateP _f . If water channel insertion is assumed to be the rate-limiting step in the turn-on ofP _f , these results raise the possibility that water channels must be activated following insertion into the apical membrane.     

Postsynaptic Currents in Dorsal Root Ganglion Neurons Co-cultured with Spinal Cord Neurons

In co-cultured dorsal root ganglion (DRG) neurons and spinal cord neurons from newborn rats, using a voltage-clamp technique in the whole-cell configuration enabled us to observe in DRG neurons the effects evoked by extracellular local electrical stimulation of cells corresponding to spinal cord neurons in their morphological characteristics. Such stimulation caused the appearance of postsynaptic currents (PSC) in DRG neurons in 9% of the cases. The mean delay of these currents (measured from the stimulus leading edge) was 4.7 ± 0.29 msec, the mean time to peak was 2.6 ± 0.77 msec, and the decay time constant = 14.5 ± 1.04 msec. The reversal potential of evoked PSC (ePSC) was close to the equilibrium potential for chloride ions estimated by the Nernst equation. Application of 20 M bicuculline induced practically complete and reversible ePSC block. The conclusion was drawn that these currents arise due to activation of the chloride channels operated by GABA receptors and, hence, represent an inhibitory PSC. Thus, one may deem it proved that spinal cord neurons can establish functional inhibitory synapses with DRG neurons.     

Effect of osmotic gradient on ADH-induced intramembranous particle aggregates in toad bladder

Summary Paired toad urinary bladders were prepared without or with an osmotic gradient (175 mosm) across them, stimulated for 2.5 (n=6), 5 (n=6), 30 (n=6) or 60 (n=6) min with ADH (20 mU/ml), and studied by freeze-fracture electron microscopy. Water permeability at these times was assessed in additional bladders (n=6 for each case) after tissue fixation according to the technique of Eggena. After both 60 and 30 min of ADH stimulation, the presence of a gradient compared with the absence of one was associated with fewer aggregates (242±35vs. 382±14 ×235 m^–2 at 60 min,P<0.01; 279±36vs. 470±51 ×235 m^–2 at 30 min,P<0.01) and lower water permeability (8.4±1.1vs. 18.8±1.8g×min^–1×cm^–1 ×mosm ^–1 at60min,P<0.005; 9.2±1.0vs. 22.0±2.1 g ×min^–1×cm^–2×mosm ^–1 at 30 min,P<0.001). In addition, with a gradient both maximum water permeability and maximum aggregate frequency were reached nearly together; a similar correspondence occurred without a gradient. We conclude that in the presence of an osmotic gradient both the ADH-associated aggregates and the water permeability response to ADH are prevented from reaching the higher levels observed in bladders not exposed to a gradient.     

Membrane bilayer assembly in neural tissue of rat and squid as a critical phenomenon: Influence of temperature and membrane proteins

Summary Cell membrane bilayers have been reconstructed in vitro utilizing total lipid extracts from rat neural tissue (forebrain, cerebellum, brainstem and spinal cord) and from the optic lobe and fin nerve of the squidLoligo pealei. In agreement with the critical state theory of bilayer assembly (Gershfeld, N.L. 1986.Biophys. J. 50:457–461; Gershfeld, N.L. 1989.J. Phys. Chem. 93:5256–5261), these lipid extracts spontaneously formed purely unilamellar structures in aqueous dispersion, but only at a critical temperature,T ^*, which was species dependent. For all the rat tissuesT ^*=37±1°C; for squid neural extractsT ^*=15.5±1.4°C. These values correspond to physiological temperatures for both organisms, implying that their lipid metabolism is geared to permit spontaneous assembly of unilamellar membranes at the ambient temperature in the tissues. Membrane protein composition had little or no effect on critical bilayer formation.     

pH i -Dependent membrane conductance of proximal tubule cells in culture (OK): Differential effects on K+- and Na+-conductive channels

Summary Confluent monolayers of the established opossum kidney cell line were exposed to NH₄Cl pulses (20 mmol/liter) during continuous intracellular measurements of pH, membrane potential (PD _m ) and membrane resistance (R _m) in bicarbonate-free Ringer. The removal of extracellular NH₄Cl leads to an intracellular acidification from a control value of 7.33±0.08 to 6.47±0.03 (n=7). This inhibits the absolute K conductance (g _K+), reflected by a decrease of K⁺ transference number from 71±3% (n=28) to 26±6% (n=5), a 2.6±0.2-fold rise ofR _m, and a depolarization by 24.2±1.5 mV (n=52). In contrast, intracellular acidification during a block ofg _K+ by 3 mmol/liter BaCl₂ enhances the total membrane conductance, being shown byR _m decrease to 68±7% of control and cell membrane depolarization by 9.8±2.8 mV (n=17). Conversely, intracellular alkalinization under barium elevatesR _m and hyperpolarizes PD _m . The replacement of extracellular sodium by choline in the presence of BaCl₂ significantly hyperpolarizes PD _m and increasesR _m, indicating the presence of a sodium conductance. This conductance is not inhibited by 10^–4 mol/liter amiloride (n=7). Patch-clamp studies at the apical membrane (excised inside-out configuration) revealed two Na⁺-conductive channels with 18.8±1.4 pS (n=10) and 146 pS single-channel conductance. Both channels are inwardly rectifying and highly selective towards Cl^–. The low-conductive channel is 4.8 times more permeable for Na⁺ than for K⁺. Its open probability rises at depolarizing potentials and is dependent on the pH of the membrane inside (higher at pH 6.5 than at pH 7.8).     

Light-induced changes in extracellular calcium concentration in the compound eye of Calliphora,Locusta and Apis

Summary Ion-selective microelectrodes inserted into the compound eyes of Calliphora, Locusta and Apis were used to monitor the changes in extracellular concentration of Ca²⁺ (Ca_o) brought about by a 1-min exposure to white light (maximal luminous intensity ca. 10³ cd/m²).In the blowfly retina such stimulation causes a decrease in Ca_o. At high light intensities the Ca_o signal is phasic, falling over about 6 s to a transient light-induced minimum (Ca_o= -6.2% ± 0.4%, n = 20, SE) and then rising to an approximately stable plateau (-3.3% ± 0.6%). In migratory locusts the light-induced minimum corresponds to a Ca_o of -13.8% ± 1.6% (n = 10), and at the plateau the Ca_o decrease is-13.2% ± 1.5%. In honey-bees Ca_o at first decreases only slightly, by -2.6% ± 1.0% (n = 10); by the end of the 1-min stimulus the extracellular concentration averages 33.6% ± 14.6% above the dark level.The results suggest a relationship between the position of the characteristic curve of the photoreceptor in the dark-adapted state, the occurrence of quantum bumps, and light-induced increases or decreases in Ca_o. Therefore the species differences might be interpreted as a consequence of differences in the intracellular dark concentration of Ca²⁺.Abbreviations Ca_i intracellular Ca²⁺ concentration - Ca_o extracellular Ca²⁺ concentration     

Histological,immunohistochemical and morphometric changes in lung tissue in juvenile mice experimentally exposed to Stachybotrys chartarum spores

Stachybotrys chartarum is an important toxigenic fungus often associated with chronically wet cellulose-based building materials. The purpose of this study was to evaluate some histological, immunohistochemical and morphometric changes in mouse lung tissues exposed intratracheally to either 50 l of 1.4 × 10⁶ S. chartarum spores (35 ng toxin/kg BW), isosatratoxin-F (35ng/kg BW),50 l of 1.4 × 10⁶ Cladosporium cladosporioides spores, or 50 l saline. Exposure of lung tissues to S. chartarum or C. cladosporioides spores resulted in granuloma formation at the sites of spore impaction. Some of the lung tissues impacted by S. chartarum spores also showed erythrocyte accumulation in the alveolar air space, dilated capillaries engorged with erythrocytes, and hemosiderin accumulation at spore impaction sites, which were features not noted in the C. cladosporioides-spore treated animals. Immunohistochemistry revealed reduced collagen IV distribution in lung granulomas in S. chartarum-treated animals especially at 48 and 72 hr post-exposure compared to that in lungs of mice with C. cladosporioides-spore induced granulomas. Quantitative analysis of pooled S. chartarum and C. cladosporioides spore impacted lungs revealed significant depression (P < 0.05) of alveolar air space from 71.4 ± 6.1 in untreated animals to 56.04 ± 6.1 in the S. chartarum- and 60.24 ± 5.5% in the C. cladosporioides-spore treated animals. It also revealed that alveolus air space in S. chartarum treated animals declined significantly from 63.74 ± 3.1% at12 hr post-exposure to 42.94 ± 7.9% at 72 hr post-exposure and was increased to 54.84 ± 5.2% at 96 hr post-exposure. Alveolus air space in C. cladosporioidestreated animals also decreased significantly from 64.84 ± 7.1% at 12 hr exposure to 54.94 ± 5.4% at 48 hr post-exposure and was increased to 64.64 ± 10.1% at 96 hr post-exposure. It also revealed significant (P <0.05) alveolar accumulation of erythrocytes from 1.24 ± 1.4% in the untreated animals to 3.44 ± 1.5% in the pooled S. chartarum spore treated animals. Erythrocyte abundance in S. chartarum treated animals increased significantly (P <0.001) from 2.14 ± 1. 7% at 12 hr post-exposure to 5.54 ± 1.5% at 72 hr and 4.94 ± 1.4% at 96 hr post-exposure. These results further reveal that exposure to S. chartarum spores elicit tissue responses in vivo significantly different from those associated with exposure to pure trichothecene toxin and to spores of a non-toxigenic fungus.This revised version was published online in October 2005 with corrections to the Cover Date.     

The effect of carbonic anhydrase inhibitors on secretion by the parotid and mandibular glands of red kangaroos Macropus rufus

Summary The effects of carbonic anhydrase inhibitors on secretion by macropodine parotid and mandibular glands were investigated using anaesthetized red kangaroos. In the parotid gland, acetazolamide (500 mol·l^-1) reduced a stable acetylcholine-evoked, half-maximal flow rate of 2.02±0.034 to 0.27±0.023 ml·min^-1 (87% reduction). Concurrently, salivary bicarbonate concentration and secretion fell (129.4±1.46 to 80.9±1.63 mmol·l^-1 and 264.8±7.96 to 22.3±2.30 mol·min^-1, respectively), phosphate and chloride concentrations rose (14.0±0.79 to 27.6±0.85 mmol·l^-1 and 5.6±0.25 to 27.5±1.32 mmol·l^-1, respectively), sodium concentration and osmolality were unaltered, and potassium concentration fell (8.8±0.33 to 6.4±0.29 mmol·l^-1). High-rate cholinergic stimulation during acetazolamide blockade was unable to increase salivary flow beyond 11±0.9% of that for equivalent unblocked control stimulation. However, superimposition of isoprenaline infusion on the acetylcholine stimulation caused a three-fold increase in the blocked flow rate. These treatments were accompanied by small increases in salivary phosphate and chloride concentrations but not bicarbonate concentration. Methazolamide infusion caused similar changes in parotid secretion. In the mandibular gland, acetazolamide infusion had no effect on salivary flow rate during either low- or high-level acetylcholine stimulation. Acetazolamide caused no alterrations in salivary electrolyte secretion at low flow rates, but curtailed the rise in bicarbonate concentration associated with high-level acetylcholine stimulation. Acetazolamide administration did not affect the increase in salivary flow rate associated with isoprenaline infusion, but did block the concomitant increase in bicarbonate concentration and secretion substantially. It was concluded that neither cholinergic nor adrenergic stimulation of mandibular fluid secretion depends on secretion of bicarbonate derived from catalysed hydration of CO₂, but a substantial proportion of the increase in bicarbonate secretion during isoprenaline administration, which is probably ductal in origin, is so dependent. In contrast to other salivary glands, including the ovine parotid, fluid secretion by the kangaroo parotid gland during cholinergic stimulation is largely dependent (about 90%) on secretion of bicarbonate derived from hydration of CO₂ catalysed by glandular carbonic anhydrase. Fluid secretion during adrenergic stimulation is not bicarbonate dependent.Abbreviations b.w. body weight - PAH p-aminohippurate - PCO₂ partial pressure carbon dioxide - PCO₂ partial pressure of oxygen     

The electrogenic sodium pump of the frog retinal pigment epithelium

Summary It was previously shown that ouabain decreases the potential difference across anin vitro preparation of bullfrog retinal pigment epithelium (RPE) when applied to the apical, but not the basal, membrane and that the net basal-to-apical Na⁺ transport is also inhibited by apical ouabain. This suggested the presence of a Na⁺–K⁺ pump on the apical membrane of the RPE. In the present experiments, intracellular recordings from RPE cells show that this pump is electrogenic and contributes approximately –10 mV to the apical membrane potential (V _AP). Apical ouabain depolarizedV _AP in two phases. The initial, fast phase was due to the removal of the direct, electrogenic component. In the first one minute of the response to ouabain,V _AP depolarized at an average rate of 4.4±0.42 mV/min (n=10, mean ±sem), andV _AP depolarized an average of 9.6±0.5 mV during the entire fast phase. A slow phase of membrane depolarization, due to ionic gradients running down across both membranes, continued for hours at a much slower rate, 0.4 mV/min. Using a simple diffusion model and K⁺-specific microelectrodes, it was possible to infer that the onset of the ouabain-induced depolarization coincided with the arrival of ouabain molecules at the apical membrane. This result must occur if ouabain affects an electrogenic pump. Other metabolic inhibitors, such as DNP and cold, also produced a fast depolarization of the apical membrane. For a decrease in temperature of 10°C, the average depolarization of the apical membrane was 7.1±3.4 mV (n=5) and the average decrease in transepithelial potential was 3.9±0.3 mV (n=10). These changes in potential were much larger than could be explained by the effect of temperature on anRT/F electrodiffusion factor. Cooling the tissue inhibited the same mechanism as ouabain, since prior exposure to ouabain greatly reduced the magnitude of the cold effect. Bathing the tissue in 0mm [K⁺] solution for 2 hr inhibited the electrogenic pump, and subsequent re-introduction of 2mm [K⁺] solution produced a rapid membrane hyperpolarization. We conclude that the electrogenic nature of this pump is important to retinal function, since its contribution to the apical membrane potential is likely to affect the transport of ions, metabolites, and fluid across the RPE.     

The mechanism of nitrate transport across the tonoplast of barley root cells

Nitrate-selective microelectrodes were used to measure not only nitrate activity in the cytoplasm and vacuole of barley (Hordeum vulgare L.) root cells, but also the tonoplast electrical membrane potential. For epidermal cells, the mean cytoplasmic and vacuolar pNO₃ (-log₁₀ [NO₃]) values were 2.3±0.04 (n=19) and 1.41±0.03 (n=35), respectively, while for cortical cells, the mean cytoplasmic and vacuolar nitrate values were 2.58±0.18 (n=4) and 1.17±0.06 (n=13), respectively. These results indicate that the accumulation of nitrate in the vacuole must be an active process. Proton-selective microelectrodes were used to measure the proton gradient across the tonoplast to assess the possibility that nitrate transport into the vacuole is mediated by an H⁺/NO ₃ ^– antiport mechanism. For epidermal cells, the mean cytoplasmic and vacuolar pH values were 7.12±0.06 (n=10) and 4.93±0.11 (n=22), respectively, while for cortical cells, the mean cytoplasmic and vacuolar pH values were 7.24±0.07 (n=3) and 5.09±0.17 (n=7), respectively. Calculations of the energetics for this mechanism indicate that the observed gradient of nitrate across the tonoplast of both epidermal and cortical cells could be achieved by an H⁺/NO ₃ ^– antiport with a 11 stoichiometry.Abbreviations and Symbols G/F free-energy change for H⁺/NO ₃ ^– antiport - F Faraday constant - pH_c cytoplasmic pH - pH_v vacuolar pH - p[NO₃]_c log₁₀ (cytoplasmic [NO ₃ ^– ]) - P[NO₃]_v -log₁₀ (vacuolar [NO₃]) We wish to thank Dr. K. Moore for assistance with statistical analysis.     

Regulation of basolateral membrane potential after stimulation of Na+ transport in proximal tubules

Summary We have previously shown that stimulation of apical Na-coupled glucose and alanine transport produces a transient depolarization of basolateral membrane potential (V _bl) in rabbit proximal convoluted tubule (PCT. Sl segment). The present study is aimed at understanding the origin of the membrane repolarization following the intial effect of addition of luminal cotransported solutes. Luminal addition of 10–15mMl-alanine produced a rapid and highly significant depolarization ofV _bl (20.3±1.1 mV,n=15) which was transient and associated with an increase in the fractional K⁺ conductance of the basolateral membrane (t _K) from 8 to 29% (P<0.01,n=6). Despite the significant increase int _K, the repolarization was only slightly reduced by the presence of basolateral Ba²⁺ (2mM,n=6) or quinine (0.5 mM,n=5). The repolarization was greatly reduced in the presence of 0.1 mM 4-acetamino-4isothiocyamostilbene-2,2-disulfonic acid (SITS) and blunted by bicarbonate-free solutions. Intracellular pH (pH _i ) determined with the fluorescent dye 2, 7-bis-2-carboxyethyl-5(and-6)-carboxyfluorescein (BCECF), averaged 7.39±0.02 in control solution (n=9) and increased to 7.50±0.03 in the first 15 sec after the luminal application of alanine. This was followed by a significant acidification averaging 0.16±0.01 pH unit in the next 3 min. In conclusion, we believe that, contrary to other leaky epithelia, rabbit PCT can regulate its basolateral membrane potential not only through an increase in K⁺ conductance but also through a cellular acidification reducing the basolateral HCO ₃ ^– exit through the electrogenic Na-3(HCO₃) cotransport mechanism.     

Myocardial content of selected elements in experimental anthracycline-induced cardiomyopathy in rabbits

Cardiotoxicity represents the main drawback of clinical usefulness of anthracycline antineoplastic drugs. In this study, a content of selected elements (Ca, Mg, K, Se, Fe) in the post-mortem removed samples of the myocardial tissue was studied in three groups of rabbits: 1) control group (i.v. saline; n = 10); 2) daunorubicin-receiving animals (DAU; 3 mg/kg, i.v; n=11); 3) animals receiving cardioprotective iron-chelating agent dexrazoxane (DEX; 60 mg/kg, i.p.; n=5) prior to DAU. Drugs were administered once weekly for 10 weeks. 5–7 days after the last administration, cardiac left ventricular contractility (dP/dt_max) was significantly decreased in DAU-treated animals (745 ± 69 versus 1245 ± 86 kPa/s in the control group; P < 0.05), while in the DEX+DAU group it was insignificantly increased (1411 ± 77 kPa/s). Of the myocardial elements content studied, a significant increase in total Ca against control (16.2 ± 2.4 versus 10.6 ± 0.9 g/g of dry tissue; P < 0.05) was determined in the DAU-group, which was accompanied with significant decreases in Mg and K. In the heart tissue of DEX-pretreated animals, no significant changes of elements content were found as compared to controls, while the Ca content was in these animals significantly lower than in the DAU group (9.1 ± 0.4 versus 16.2 ± 2.4 g/g; P < 0.05). Hence, in this study we show that systolic heart failure induced by chronic DAU administration is primarily accompanied by persistent calcium overload of cardiac tissue and the protective action of DEX is associated with the restoration of normal myocardial Ca content.Published online: March 2005     

Clasper control in the round stingray,Urolapkus halleri lower sensor motor pathways

Synopsis The innervation of the clasper has been studied in the round stingray,Urolophus halleri. Several large myelinated nerves (diameters approx. 0.7 mm; # 60–64 counting from the vagus) innervate the clasper muscles and skin. Low level electrical stimulation (<100A) of the nerves evokes clasper movements including: elevation, medial and lateral extension, rotation and opening. Stimulation of the spinal cord in the area of the roots of the clasper nerves also evoked the movements (<100A). Retrograde labeling of the clasper nerves using either cobalt-lysine or horseradish peroxidase (HRP) confirmed that motor neurons and sensory components of the nerves are at the levels indicated by stimulation. The motor neurons have large multipolar cell bodies (50–70) and occupy a discrete segment of the spinal cord.     

Visualization of the NMDA recognition site in rat and mouse spinal cord by [3H]CGS 19755in vitro autoradiography

Summary The possibility to visualize the NMDA recognition site with [³H]CGS 19755in vitro autoradiography was evaluated in rat brain and spinal cord sections and thereafter used to study the distribution of the NMDA recognition site in rat and mouse spinal cord. The [³H]CGS 19755 binding was concentrated to the dorsal horn, in particular to the substantia gelatinosa. Notable binding was also seen in the intermediate area and ventral horn, while some binding was observed in the funiculi. No major differences were seen in [³H]CGS 19755 binding at various levels of the rat or mouse spinal cord, although a more dense binding was seen in the mouse. A similar distribution of the [³H]CGS 19755 specific binding and the NMDA receptor associated ion-channel site, labeled with [³H]TCP, was found in the mouse spinal cord. Taken together, our data indicate that the NMDA recognition site can be visualized in rat and mouse spinal cord byin vitro [³H]CGS 19755 autoradiography.Abbreviations NMDA N-methyl-D-aspartate - CGS 19755 Cis-4-phosphonomethyl-2-piperidine carboxylic acid - D-AP5 D(—)-2-Amino-5-phosphonopentanoic acid - TCP N-(1-2-thienylcyclohexyl)-3,4-piperidine - MK-801 (±)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate - AMPA -Amino-3-hydroxy-5-methyl-isoxazolepropionic acid - kainate 2-Carboxy-3-carboxymethyl-4-isopropenyl pyrrolidine - CGP 39653 D,L-(E)-2-amino-4-propyl-5-phosphonopentenoic acid     

Apical membrane endocytosis via coated pits is stimulated by removal of antidiuretic hormone from isolated,perfused rabbit cortical collecting tubule

Summary Antidiuretic hormone increases the water permeability of the cortical collecting tubule and causes the appearance of intramembrane particle aggregates in the apical plasma membrane of principal cells. Particle aggregates are located in apical membrane coated pits during stimulation of collecting ducts with ADHin situ. Removal of ADH causes a rapid decline in water permeability. We evaluated apical membrane retrieval associated with removal of ADH by studying the endocytosis of horseradish peroxidase (HRP) from an isotonic solution in the lumen. HRP uptake was quantified enzymatically and its intracellular distribution examined by electron microscopy. When tubules were perfused with HRP for 20 min in the absence of ADH, HRP uptake was 0.5±0.3 pg/min/m tubule length (n=6). The uptake of HRP in tubules exposed continuously to ADH during the 20-min HRP perfusion period was 1.3±0.8 pg/min/m (n=8). HPR uptake increased markedly to 3.2±1.1 pg/min/m (n=14), when the 20-min period of perfusion with HRP began immediately after removal of ADH from the peritubular bath. Endocytosis of HRP occurred in both principal and intercalated cells via apical membrane coated pits. We suggest that the rapid decline in cortical collecting duct water permeability which occurs following removal of ADH is mediated by retrieval of water permeable membrane via coated pits.     

Permeability parameters of the toad isolatedstratum corneum

Summary A technique for isolating thestratum corneum from the subjacent layers of the epithelium was developed which permits studying thestratum corneum as an isolated membrane mounted between half-chambers. The method basically consists of an osmotic shock induced by immersing a piece of skin in distilled water at 50°C for 2 min. When the membrane is bathed on each surface by NaCl-Ringer's solution, its electrical resistance is 14.1±1.3 cm² (n=10). This value is about 1/100 of the whole skin resistance in the presence of the same solution. The hydraulic filtration coefficient (L _p ) measured by a hydrostatic pressure method, with identical solutions on each side of the membrane, is 8.8×10^–5±1.5×10^–5 cm sec^–1 atm^–1 (n=10) in distilled water and 9.2×10^–5±1.4×10^–5 cm sec^–1 atm^–1 (n=10) in NaCl-Ringer's solution. These values are not statistically different and are within the range of 1/80 to 1/120 of the whole skinL _p . Thestratum corneum shows an amphoteric character when studied by KCl diffusion potentials at different pH's. The membrane presents an isoelectric pH of 4.6±0.3 (n=10). Above the isoelectric pH the potassium transport number is higher than the chloride transport number; below it, the reverse situation is valid. Divalent cations (Ca⁺⁺ or Cu⁺⁺) reduce membrane ionic discrimination when the membrane is negatively charged and are ineffective when the membrane fixed charges are protonated at low pH.     

Water and salt permeability of gastric vesicles

Summary Volume-dependent changes in light scatter have been shown to be a linear function of the osmotic gradient imposed upon gastric vesicles purified from hog gastric mucosa. Observation of the light scattered 90° to incident, using the Durrum stop flow system D-110, indicates that the vesicles exposed to hypertonic medium undergo rapid shrinkage due to water loss from the vesicle interior. The rate constant for this water movement is 1.1±0.09 sec^–1 (n=10) and is linearly dependent on temperature between 16 and 36°C. The activation energy of 13.93±0.60 kcal mole^–1 (n=3), calculated from an Arrhenius plot, is inconsistent with water movement facilitated by a large-pore aqueous channel. A slower reswell phase, dependent on solute entry into the intravesicular space, follows the water-dependent shrink phase. KCl entry, studied because of the intravesicular requirement for active K⁺/H⁺ transport, exhibits two entry stages. The faster, described by a single exponential imposed upon a constantly sloping background, has a rate constant of 7.75±0.48×10^–3 sec^–1 (n=15). The slower phase, which typically accounts for 90% of the reswell process, demonstrates a rate constant of 1.94±0.23×10^–4 sec^–1 (n=15). In the presence of valinomycin or nigericin, two fast rate constants and one slow rate constant of swelling are observed. The rate constant of the faster reswell phase is increased from 7.75±0.48×10^–3 sec^–1 (n=15) to 15.74±3.7×10^–3 sec^–1 (n=5) and 17.23±3.4×10^–3 (n=3) by the addition of nigericin (1 g ml^–1) and valinomycin (4.5 m), respectively. The second part of the faster reswell phase is approximately that seen in the control population. Transport-dependent volume changes of significant magnitude can be demonstrated following the addition of ATP to vesicles equilibrated with 150mm KCl. The volume change is a function of HCl leak rate and is abolished by ionophores which eliminate the transport-dependent pH gradient. So ₄ ^–- substitution, which eliminates the overshoot phenomena observed in KCl medium, also eliminates the shrinkage resulting from ATP addition.