Frog Ringer Research Articles

Glycocalyx and ciliary interconnections in bullfrog vestibular end organs: normal structure and changes after incubation in frog Ringer's solution.

The ultrastructure of ciliary interconnecting systems as well as changes after incubation in frog Ringer's solution of the bullfrog vestibular end organs were investigated by using a scanning electron microscopy. The plasma membrane of the sensory hairs showed rather rough appearance. Side links emerged from the plasma membrane and tightly connected neighboring cilia. The tip links stretched from the tips of the stereocilia to their taller neighbors. These tip links were arranged in a same direction towards the kinocilium. These findings indicate that the side links may keep the cilia arranged in a bundle and the tip links may be involved in sensory cell transduction system. Changes in the surface texture and ciliary interconnecting systems were detected 2 hrs after incubation in frog Ringer's solution. Such changes included an increase in granularity of the surface membrane, fracturing and disappearance of all types of ciliary interconnections, especially side links. The tip links were more durable than side links. This indicates that the physiological studies of bullfrog hair bundle are justified across a 1-hr interval from the time the temporal bones are removed from the animal.

Relationships between ototoxicities and chemical structures of ototoxic drugs

The relationships between degrees of ototoxicity and the chemical structures of ototoxic drugs were investigated using isolated bullfrog semicircular canals. Thirteen derivatives of tuberactinomycin (Tum), a peptide antibiotic with pharmacological characteristics similar to those of kanamycin, were prepared by replacing the R1 branch with various amino acids. The degrees of ototoxicity were measured in these derivatives as well as in different kinds of aminoglycoside (AGs) antibiotics (TOB, GM, NTL, ISP, AMK). In order to measure the degree of ototoxicity, the ampullary nerve action potential of isolated bullfrog posterior semicircular canal, in response to mechanical endolymphatic flow, was recorded in Frog Ringer's solution and in the presence of different amounts of the various drugs. The degree of ototoxicity was determined by the amount of decrease in the maximal spike account. The degrees of ototoxicity of AGs were calculated to be in the order TOB > GM > ISP > AMK > NTL. This result was in agreement with those of previous morphological investigations. The derivatives of Tum with an R1 branch containing an acidic or a basic amino acid showed greater ototoxicities than those containing aliphatic side chains. The degree of ototoxicity of each drug tended to differ with each concentration, in both AG and Tum derivatives. Thus, the result indicate that chemical structure may be closely related to the degree of ototoxicity.

Topical Tolerability of Calcitonin Nasal Preparation, its Excipients and Sodium Taurocholate Studied by SEM and Mucociliary Transport Velocity

In addition to the active drugs, intranasal preparations contain various excipients and preservatives to stabilise the formulation and prevent the growth of contaminating microorganisms during repeated use. This study assessed the topical tolerability of 1–7 Asu eel and salmon calcitonin, the excipients ammonium glycyrrhizinate, methyl and propyl p-hydroxybenzoate and benzalkonium chloride, and sodium taurocholate, which is used in other preparations to promote absorption. The ex vivo frog palate preparation was used to measure mucociliary transport velocity (MTV) as an index of their effects. In addition, scanning electron microscopy (SEM) was used to study the morphology of the mucosal surface of the palate before and after contact with the test substances. Frog Ringer’s solution, 1–7 Asu eel and salmon calcitonin did not significantly change MTV, confirming good tolerability by ciliated epithelium. The same was true for 0.5 and 2% ammonium glycyrrhizinate and 0.01% benzalkonium chloride, while p-hydroxybenzoates produced a small though significant reduction in MTV. Sodium taurocholate, however, led to a significant reduction in MTV for the palate with and without mucus. SEM studies confirmed the results, clearly identifying the damage to epithelial ciliated cells produced by sodium taurocholate.

Volume status influences atrial peptide-induced water conductivity changes in leopard frog mesenteric capillaries.

1. Three sets of North American leopard frogs (Rana pipiens) were housed on damp sand (DS, n = 19), in fresh water (FW, n = 19), or with free access to both damp sand and fresh water (FA, n = 23) for a period of 5-6 days. At the end of this period pithed frogs were prepared for measurement of capillary hydraulic conductivity and samples of whole-blood, lymph and body cavity fluid were collected for measurement of haematocrit and protein content. 2. To measure hydraulic conductivity (Lp), individual capillaries of the mesenteries of the three groups were cannulated and perfused with a minimum of two, and a maximum of three, frog Ringer solutions. The first solution, common to all experiments, contained dialysed bovine serum albumin (BSA) at a concentration of 10 mg ml-1. The second and third solutions also contained either 10 nM-atrial peptide (AP) or 1 microM-sodium nitroprusside (SNP). Lp was determined from measurements of fluid filtration rate at a minimum of two different capillary pressures (Michel, Mason, Curry, Tooke & Hunter, 1974). 3. In vessels from damp sand-stored frogs (DS), the paired Lp in the presence of 10 nM-atrial peptide (Lp, AP) was 3.2 +/- 0.4-fold higher than Lp, BSA (n = 25). By contrast, in the FW group, Lp, AP did not differ significantly from Lp, BSA (Lp, AP-FW/Lp, BSA = 1.3 +/- 0.2; n = 23). In frogs given free access to water and dry terrain the response to atrial peptide was similar to that observed in the DS group (Lp, AP-FA/Lp, BSA = 3.6 +/- 07; n = 23). Thus, storage in fresh water appears to have inhibited the acute Lp response to atrial peptide observed in vessels from the FA or DS animals. 4. Atrial peptide and sodium nitroprusside elevate intracellular cyclic GMP levels by membrane receptor-dependent and -independent processes, respectively. No difference was observed in the Lp response to 1 microM-perfusate sodium nitroprusside (Lp, SNP) in DS (n = 7), FW (n = 8) or FA (n = 30) frogs (Lp, SNP/Lp, BSA ratios were 3.5 +/- 1.5-, 2.7 +/- 0.8- and 2.6 +/- 0.5-fold, respectively). Thus, the Lp response to sodium nitroprusside was not affected by storage conditions. 5. The mean haematocrit of skin blood samples from the DS groups was 26.5 +/- 1.0% (+/- S.E.M.) compared to 19.1 +/- 1.2% from the FW groups.(ABSTRACT TRUNCATED AT 400 WORDS)

Enhanced fluid uptake in frog mesenteric capillaries associated with plasmin perfusion.

1. We have measured the permeability of single capillaries of the mesenteries of decerebrated frogs, before and during perfusion with solutions containing the fibrinolytic enzyme, plasmin. 2. The hydraulic permeability (Lp) and the effective oncotic pressure exerted across the vessel walls (sigma delta pi) were measured using the method of Michel (1980). The vessels were sequentially perfused with a control frog Ringer solution containing either Ficoll 70 or bovine serum albumin (BSA) at concentrations of 40 mg ml-1 or a mixture of Ficoll 70 (40 mg ml-1) and BSA (10 mg ml-1), and then with a second Ringer perfusate containing plasmin (1 mg ml-1) but in all other respects identical to the control solution. 3. In sixteen out of seventeen experiments, perfusion with plasmin increased sigma delta pi. In eleven of these experiments the increase was very large such that sigma delta pi exceeded the in vitro value for perfusate oncotic pressure. 4. In the same seventeen vessels plasmin perfusion was associated with a fall in Lp from a mean value of 10.3 x 10(-7) cm s-1 cmH2O-1 to one of 7.7 x 10(-7) cm s-1 cmH2O-1. The fall in Lp was not significant. 5. In five of the seventeen vessels, a second control perfusion was made after exposure to plasmin. There was no evidence that Lp had increased above or sigma delta pi had fallen below the initial control value. 6. In a further six experiments, the effects of plasmin were investigated in the absence of other perfusate macromolecules. No significant changes in Lp or sigma delta pi were observed. 7. In a further eight vessels, the effects of plasmin on fluid filtration were investigated with the tissues cold and then at room temperature. In all eight vessels plasmin reduced filtration or increased fluid reabsorption to a greater extent when the tissue temperature was 17 degrees C than when it was 4 degrees C. 8. The large increases in sigma delta pi which we have observed during perfusion of single vessels with plasmin-containing solutions are consistent with the development of substantial local osmotic gradients at the capillary wall following the enzyme's action upon substrates at the endothelial cell surface, one of which could be fibrin. Alternatively, plasmin might stimulate endothelial cells to liberate molecules which locally amplify the oncotic pressure exerted by the perfusate macromolecules. These effects are more marked at room temperature than at 4 degrees C.

Electrophysiological characterization of a new member of the RCK family of rat brain K + channels

A novel member of the RCK family of rat brain K + channels, called RCK2, has been sequenced and expressed in Xenopus oocytes. The K + currents were voltage-dependent, activated within 20 ms (at 0 mV), did not inactivate in 5 s, and had a single channel conductance in frog Ringers of 8.2 pS. Compared to other members of the RCK family the pharmacological profile of RCK2 was unique in that the channel was resistant to block (IC 50 = 3.3 μM) by charybdotoxin [(1988) Proc. Natl. Acad. Sci. USA 85, 3329–3333] but relatively sensitive to 4-aminopyridine (0.3 mM), tetraethylammonium (1.7 mM), α-dendrotoxin (25 nM), noxiustoxin (200 nM), and mast cell degranulating peptide (200 nM). Thus, RCK2 is a non-inactivating delayed rectifier K + channel with interesting pharmacological properties.

Permeability of frog mesenteric capillaries after partial pronase digestion of the endothelial glycocalyx.

1. The proteolytic enzyme pronase, which degrades the endothelial cell glycocalyx, was perfused through single capillaries of frog mesentery. Hydraulic conductivity (Lp) of each vessel was determined before and after pronase perfusion. In three vessels in which Lp increased, the ultrastructure of interendothelial clefts was examined. In a separate group of frogs the effect of pronase on the endothelial glycocalyx was assessed by using cationized ferritin to label the capillary luminal surface. 2. Control Lp was 2.0 x 10(-7) cm s-1 cmH2O-1 (10 mg ml-1 bovine serum albumin, BSA, in frog Ringer solution). Vessels were then perfused with a solution containing 0.1 mg ml-1 pronase and 10 mg ml-1 BSA for 1 min. Lp measured in the same eleven vessels increased to 4.9 x 10(-7) cm s-1 cmH2O-1 (P less than 0.005). 3. Transverse sections of three of these vessels were examined by transmission electron microscopy at eight sites along each vessel. In these sections a total of 156 interendothelial cell clefts were found and photographed. No morphological features, such as fenestrations, transendothelial channels, or intercellular gaps associated with inflammation, were found which might account for the increases in Lp. 4. Measurement of cleft dimensions yielded a harmonic mean cleft depth (delta x) of 0.32 microns and an arithmetic mean cleft depth of 0.64 microns. Mean width (w) of the clefts outside the tight regions was 0.012 microns and the cleft length per unit area (L) was 1330 cm-1. The mean fractional pore area of vessel wall per unit cleft depth, Ap/delta x, calculated as Lw/delta x, was 48.7 cm-1. 5. There was less cationic ferritin (CF) labelling of the luminal glycocalyx in pronase-perfused than in control capillaries. On average, the proportion of the luminal surface covered by CF was 85% in controls and 42% in pronase-treated capillaries (P less than 0.01). In some vessels the CF pattern was greatly disrupted, indicating large changes in the glycocalyx structure. 6. It is concluded that the moderate increases in Lp induced by pronase perfusion are associated with partial digestion of the endothelial glycocalyx but are not accompanied by changes in the dimensions of the intercellular cleft. These observations support the fibre matrix hypothesis of capillary permeability and suggest that the endothelial glycocalyx contributes as much as 60% of the hydraulic resistance of the capillary wall.

Evidence for cholinergic regulation of microvessel hydraulic conductance during tissue hypoxia.

Cholinergic regulation of single-vessel hydraulic conductivity (Lp) during normoxia and hypoxia was tested in single mesenteric vessels of pithed frogs (Rana pipiens). Capillaries were cannulated in situ and perfused with frog Ringer's solution containing 10 mg/ml albumin and human erythrocytes while the mesentery was continuously superfused with frog Ringer's solution (15 degrees C). Lp was first measured under normoxic (room air equilibrated) conditions by the modified Landis microocclusion method. Repeated measurements of filtration coefficient under control conditions, for periods up to 80 minutes, demonstrated that Lp did not change with time in normoxic vessels (n = 18). After initial control measurement (Lpo), perfusion with 1 microM acetylcholine increased Lp by 4.6 +/- 1.0-fold (mean +/- SEM, n = 6). The response to acetylcholine was antagonized by the addition of 10 microns atropine to the perfusate (Lp/Lpo = 1.8 +/- 0.4). Perfusion with atropine alone reduced Lp in three of six capillaries Lp/Lpo = 0.56 +/- 0.04); Lp in the remaining three vessels was unaffected. Tissue hypoxia was simulated by exposing the mesentery to deoxygenated superfusate Po2 less than or equal to 10 mm Hg) for 10-15 minutes. Tissue hypoxia had no effect on Lp in atropine-treated vessels (n = 8). Without atropine, tissue hypoxia increased Lp by 2.3 +/- 0.7-fold, whereas the addition of atropine completely antagonized this response (n = 5). In contrast to the inhibitory action of atropine during tissue hypoxia, Lp rose 5.2 +/- 1.6-fold (n = 4) in vessels simultaneously exposed to deoxygenated perfusate.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential sensitivity of exchange vessel hydraulic conductivity to atrial natriuretic peptide

Acute plasma volume reduction by atrial natriuretic peptide (ANP) may be mediated, at least in part, by increased exchange vessel water conductivity (Lp). The present study tests the hypothesis that physiological levels of ANP acutely and reversibly elevate single capillary Lp. Paired, in situ measurements of Lp were obtained using the modified Landis technique in individually perfused mesenteric capillaries of the frog, Rana pipiens. Control Lp measurements ranged from 0.1 to 40 x 10(-7) cm.s-1.cmH2O-1 (with a median value of 2.5 x 10(-7] in 81 microvessels perfused with frog Ringer solution containing dialyzed bovine serum albumin. Vessels were recannulated and perfused with one or more concentrations of human ANP (hANP) spanning the physiopathological range: 0.01, 0.1, 1, 10, and 100 nM. When possible, a final recannulation and Lp determination was performed in the absence of hANP. A median 2.2-fold rapid Lp increase was observed compared with control at each peptide concentration. Lp changes persisted for the duration of exposure to hANP, returning to control levels on withdrawal of the peptide. True and venular capillaries exhibited similar Lp responses: median 2.2- and 2.4-fold elevations, respectively. Sixty percent of true and venular capillaries exhibited twofold or greater hANP-induced increases in Lp, whereas only 25% of arteriolar capillaries exhibited such a response. Thus a differential sensitivity to the peptide exists across the exchange vessel network. The observations of this study demonstrate that physiological levels of ANP are capable of modulating exchange vessel Lp, one means by which the peptide may acutely alter plasma volume.(ABSTRACT TRUNCATED AT 250 WORDS)

Membrane currents elicited by prostaglandins, atrial natriuretic factor and oxytocin in follicle‐enclosed Xenopus oocytes.

1. Membrane currents were recorded from voltage clamped Xenopus laevis oocytes, still surrounded by follicular cells, theca and enveloping inner ovarian epithelia (ovarian follicles). 2. Superfusing follicles with frog Ringer solution containing E-series prostaglandins (PGE1 or PGE2) or oxytocin (0.5-2 microM) generated slow membrane currents arising from an increase in membrane conductance to K+. 3. Follicles taken from different frogs varied greatly in responsiveness to PGE and oxytocin. For example, enclosed oocytes with good sensitivity to prostaglandins responded to 1 nM-PGE, whereas follicles from some frogs failed to respond at 5 microM. 4. Oocytes with good responsiveness to PGE also produced K+ currents to PGA1, PGA2, PGB1, 11-deoxy-PGE1 and 11-beta-PGE2, whereas PGF2 alpha, PGI2, PGD2 and 8-iso-PGE1 generally failed to elicit membrane currents. 5. Responses to PGE and oxytocin were mimicked by the adenylate cyclase activator forskolin or by intraoocyte pressure injection of cyclic nucleotides. Responses were potentiated by the phosphodiesterase inhibitors theophylline and 3-isobutyl-1-methylxanthine (IBMX). In IBMX (0.5 mM), human atrial natriuretic factor (ANF) (10-60 nM) elicited a similar K+ conductance. This all implied that cyclic nucleotides played a role in the receptor-channel coupling mechanism of these responses. 6. Defolliculating oocytes effectively abolished responses to prostaglandins, oxytocin and ANF, suggesting that the currents arise in follicular cells. 7. The responses of PGE, oxytocin and ANF thus resembled currents elicited by catecholamines, adenosine, gonadotrophins and vasoactive intestinal peptide (VIP). However, PGE, oxytocin and ANF responses were not blocked by catecholaminergic or purinergic antagonists. Moreover, when comparing follicles isolated from different frogs, the sensitivity to PGE and oxytocin varied independently of that to gonadotrophin or VIP. These experiments suggest that Xenopus ovarian follicles contain specific and distinct receptors for PGE, oxytocin and ANF. 8. Acetylcholine attenuated the cyclic nucleotide-mediated K+ responses, including currents elicited by PGE, oxytocin and ANF. Attenuation was not dependent on, or mimicked by, activation of the inositol phosphate-diacylglycerol messenger pathways located in the oocyte itself, nor was it appreciably blocked by loading follicle-enclosed oocytes with 0.1-1.5 mM-EGTA.

Effects of hydroxyethyl rutosides upon the permeability of single capillaries in the frog mesentery.

1. We have investigated the effects of a standardised mixture of hydroxyethyl rutosides (HR) upon the permeability of the walls of single capillaries and venules of the frog mesentery. 2. In each experiment a single vessel was perfused via a micropipette with frog Ringer solutions containing bovine serum albumin (10 mg ml-1) and Ficoll 70 (40 mg ml-1) first in the absence of HR and then with HR added to the perfusate. The permeability of the vessel walls was assessed during each perfusion by using a development of the Landis micro-occlusion technique to estimate their hydraulic permeability (Lp) and the effective osmotic pressure (sigma delta pi) exerted across them by the perfusate macromolecules. 3. Measurements were made both in vessels which appeared to be healthy and in vessels showing signs of stasis or inflammation before perfusion. 4. HR at concentrations of 1.0, 0.1 and 0.01 mg ml-1 reduced hydraulic permeability to approximately half of its value in the absence of HR. It increased sigma delta pi to macromolecules at concentrations of 10, 1.0, 0.1, 0.01 and 0.001 mg ml-1. The effects of HR upon permeability were not reversed within 10 min of perfusion with an HR-free solution. 5. Ultrastructural examination of a number of vessels in which initial high values of permeability were reduced to values within the normal range of permeabilities by HR, showed clear signs of damage to the endothelium, with large gaps between adjacent endothelial cells. 6. These observations suggest that HR does reduce microvascular permeability both in healthy vessels and vessels showing of inflammation. The reduction in permeability of inflamed vessels does not appear to be the result of closure of the gaps between adjacent endothelial cells.

Expression of amino acid transport systems in Xenopus oocytes injected with mRNA of rat small intestine and kidney

Xenopus and Cynops oocytes were injected with exogenous mRNA prepared from rat small intestine and kidney and their electrical responses to amino acids were measured by both the current clamped and the voltage clamped methods. Oocytes injected with mRNA of rat small intestine showed a depolarization response to several neutral and basic amino acids, and almost no response to acidic amino acids. The responses to amino acids increased with incubation time after injection of mRNA, and followed Michaelis-Menten type kinetics. The responses were dependent on both Na + concentration and membrane potential, and were inactivated by a sulfhydryl reagent, 5,5-dithiobis(2-nitrobenzoate). These results are interpreted as due to the expression of Na +/amino acid cotransporter(s) in oocytes injected with rat small intestine mRNA. On the other hand, the oocyte injected with rat kidney mRNA showed a hyperpolarization response to neutral amino acids, a depolarization response to basic ones, and almost no response to acidic ones in frog Ringer solution. These responses were independent of Na + concentration and followed Michaelis-Menten type kinetics. These amino acid response characteristics in oocytes injected with rat kidney mRNA are interpreted as due to the expression of facilitated diffusion carrier protein(s) (uniporter) of amino acids in the oocyte.

O2 modulation of single-vessel hydraulic conductance.

To investigate the response of the microvascular endothelial barrier to O2 lack, hydraulic conductivities (Lp) were compared under control and hypoxic conditions. In 29 decerebrate frogs (Rana pipiens) the mesentery was exposed and continuously superfused with room air-equilibrated frog Ringer (14 degrees C, pH 7.4). Single mesenteric microvessels (15- to 50-microm diam) were cannulated and perfused with frog Ringer containing 5-40 mg/ml bovine serum albumin and 5-10% (vol/vol) human erythrocytes. The modified Landis technique was used to measure transmural water flux per unit area (Jv/S) from marker red cell movement at pressures ranging from 15 to 45 cmH2O. In 21 vessels, a 10-min superfusion with 100% N2-equilibrated Ringer increased Lp 2.4-fold (+/- 0.72 SD) over control (P less than or equal to 0.05). In an additional six vessels, simultaneous exposure to 100% N2-equilibrated perfusate and superfusate resulted in a 10-fold (+/- 3.0) increase in Lp (P less than or equal to 0.05). In 15 of the vessels, a 10- to 75-min reexposure to control conditions decreased Lp, averaging a 77% recovery toward base line. These data support an active role for O2 in the regulation of microvascular membrane permeability, which appears to be sensitive to the severity of O2 lack. The rapid increase in capillary water conductivity following exposure to hypoxic conditions may be the initial process in the events leading to edema formation.

Effect of lipid hydroperoxide on Xenopus oocytes and on neurotransmitter receptors synthesized in Xenopus oocytes injected with exogenous mRNA

The effect of 13- l-hydroperoxylinoleic acid (LOOH) on both Xenopus oocytes and neurotransmitter receptors synthesized in the oocytes was studied by electrophysiological and ion flux measurement. Addition of LOOH to the incubation mixture of the oocytes raised the membrane potential and decreased the membrane resistance of the oocytes. These effects of LOOH on the oocytes were reversed within a few hours by incubation with frog Ringer solution. Addition of LOOH also caused an increase of Li + and 45Ca 2+ uptake into the oocytes. However, production of alkoxy radicals by the addition of FeCl 2 to the incubation mixture containing LOOH did not accelerate the damage to the oocytes by LOOH. So essential toxicity is caused possibly by an increase in the membrane permeability resulting from disturbance of the lipid bilayer arrangement, not from production of active alkoxy radicals during decomposition of LOOH. Nicotinic acetylcholine and γ-aminobutyric acid receptors were synthesized in Xenopus oocytes by injecting mRNA prepared from Electrophorus electricus electroplax and rat brain. LOOH noncompetitively inhibited the function of these receptors and also increased the rate of desensitization of the receptors.

An experimental study demonstrating the physiological polarity of the frog's utricle.

The frog Rana nigromaculata was used as an experimental model. The utricle and its nerve were isolated with the anterior and the lateral semicircular canal ampullae in frog Ringer's solution. The utricular otoconia and the otoconial membrane were then carefully removed. The halved anterior canal cupula was next placed in the tip of a glass microelectrode, which was mounted on a micromanipulator. The cupula was placed in the center of either the medial or the lateral part of the macula and was moved toward or away from the striola along the axis vertical to the striola. When the medial part of the macula was stimulated, 5 micron of striolapetal cupular movement elicited an excitatory nerve discharge. When the lateral part of the macula was stimulated, the striolapetal stimulus likewise induced an excitatory response. These results indicate the existence of physiological polarity on the utricular macula.

Effect of albumin on the osmotic pressure exerted by myoglobin across capillary walls in frog mesentery.

1. Individual capillaries in mesenteries of pithed frogs were perfused sequentially with two frog Ringer solutions containing myoglobin at a concentration of 70 mg/ml. The first perfusate solution contained bovine serum albumin at a concentration of 10 mg/ml. The second perfusate contained no albumin. The modified Landis micro-occlusion technique (Michel, Mason, Curry & Tooke, 1974) was used to measure the capillary hydraulic conductivity and the effective osmotic pressure of the myoglobin solution across the capillary wall under conditions where the transcapillary concentration difference was established by steady-state ultrafiltration. 2. In six capillaries the effective osmotic pressure of the myoglobin solution at 20-22 degrees C was 15.9 +/- 2.2 (S.E. of mean) cmH2O when albumin was in the perfusate and 10.1 +/- 1.1 cmH2O after albumin was removed from the perfusate. 3. The hydraulic conductivity of the capillary wall increased from a mean value of 6.5 +/- 1.4 X 10(-7) cm/(s cmH2O) when albumin was in the perfusate to 15.6 +/- 3.8 X 10(-7) cm/(s cmH2O) during perfusion with Ringer solution containing myoglobin alone. 4. Control experiments to measure steady-state filtration rates at pressures 10-30 cmH2O were carried out to check the assumption in the method that the reduction in the measured osmotic pressure of myoglobin was the result of changes in the properties of the molecular sieve within the capillary wall and was not simply the result of solute accumulation on the tissue side of the capillary wall. 5. The measured reduction in the effective osmotic pressure of myoglobin when albumin was removed from the perfusate does not conform to the hypothesis (Crone, 1984) that the tight segment of the intercellular junction is the principal molecular filter for myoglobin in the wall of frog mesenteric capillaries. 6. Our results do conform to the hypothesis that a network of fibrous molecules, reinforced by adsorbed albumin, forms the principal molecular filter at the capillary wall in frog mesenteric capillaries.

Modulation of ryanodine-induced Ca 2+ release in amphibian skeletal muscle

We examined effects of ryanodine on tension in intact and skinned amphibian skeletal muscle. 100 μM ryanodine (RY) alone in the frog Ringer's solution (FR) produced tension in the intact muscle reaching its peak by 1 h: 10 min treatment with RY augmented depolarization-induced tension and prevented a subsequent caffeine-induced contraction. In contrast, RY in Ca 2+-free FR was unable to produce tension, after which caffeine produced irreversible tension. In skinned fibers. RY at pCa 6.5 produced tension and abolished a subsequent caffeine-induced contraction: while RY in 2 mM EGTA did not produce tension. These data indicate that RY, in the presence of Ca 2+, releases Ca 2+ from the SR resulting in subsequent depletion of Ca in the SR.

カエル卵形嚢斑の生理学的極性 クプラによる刺激効果について

The frog utricle and its nerve were isolated with the anterior and the lateral semicircular canal ampullae in frog Ringer's solution. The utricular otoconia and the otoconial membrane were carefully removed. The cupula of the anterior semicircular canal was removed and was sectioned in half. The halved cupula was stuck into the tip of a glass microelectrode which was mounted on a micromanipulator. The base of the cupula was gently placed on the macula. The micromanipulator allowed sliding motion of the cupula on the macula. The cupula was placed in the center of the medial or lateral part of the macula and was moved toward (striolapetal) or away from (striolafugal) the striola along the axis vertical to the striola. When the medial part of the macula was stimulated, 5 μm of striolapetal cupula movement elicited excitatory nerve discharge. When the lateral part of the macula was stimulated, the striolapetal stimulus likewise induced an excitatory response. These results indicate the presence of physiological polarity on the utricular macula.

Patch clamp measurements onXenopus laevis oocytes: currents through endogenous channels and implanted acetylcholine receptor and sodium channels

Functional acetylcholine receptor (AChR) and sodium channels were expressed in the membrane of Xenopus laevis oocytes following injection with poly(A)+-mRNA extracted from denervated rat leg muscle. Whole-cell currents, activated by acetylcholine or by depolarizing voltage steps had properties comparable to those observed in rat muscle. Oocytes injected with specific mRNA, transcribed from cDNA templates and coding for the AChR of Torpedo electric organ, expressed functional AChR channels at a much higher density. Single-channel currents were recorded from the oocyte plasma membrane following removal of the follicle cell layer and the vitelline membrane from the oocyte. The follicle cell layer was removed enzymatically with collagenase. The vitelline membrane was removed either mechanically after briefly exposing the oocyte to a hypertonic solution, or by enzyme treatment with pronase. Stretch activated (s.a.) currents were observed in most recordings from cell-attached patches obtained with standard patch pipettes. S.a.-currents were evoked by negative or positive pressure (greater than or equal to 5 mbar) applied to the inside of the pipette, and were observed in both normal and mRNA injected oocytes indicating that they are endogenous to the oocyte membrane. The s.a.-channels are cation selective and their conductance is 28 pS in normal frog Ringer's solution (20 +/- 1 degree C). Their gating is voltage dependent, and their open probability increases toward more positive membrane potentials. The density of s.a.-channels is estimated to be 0.5-2 channels per micron 2 of oocyte plasma membrane. In cell-attached patches s.a.-currents are observed much less frequently when current measurement is restricted to smaller patches of 3-5 micron 2 area using thick-walled pipettes with narrow tips. In outside-out patches s.a.-currents occur much less frequently than in cell-attached or inside-out patches. AChR-channel and sodium channel currents were observed only in a minority of patches from oocytes injected with poly(A)+-mRNA from rat muscle. AChR-channel currents were seen in all patches of oocytes injected with specific mRNA coding for Torpedo AChR. In normal frog Ringer's solution (20 +/- 2 degrees C) the conductance of implanted rat muscle AChR-channels was 38 pS and that of sodium channels 20 pS. The conductance of implanted Torpedo AChR channels was 40 pS. The conductance of implanted channels was similar in cell-attached and in cell-free patches.(ABSTRACT TRUNCATED AT 400 WORDS)

Potential dependence of unidirectional chloride fluxes across isolated frog skin

Isolated frog skins were voltage clamped at transepithelial potentials ( V t) ranging from −60 mV to 60 mV to measure transepithelial 36Cl − fluxes from the apical to the basolateral bathing solution ( J 13) and in the opposite direction ( J 31). The potential dependence of fluxes obtained in Na +-free choline Ringer's indicates the presence of conductive components that probably correspond to fluxes through paracellular and cellular pathways, respectively. Rectification of fluxes with reversal of the potential reflects a structural asymmetry, presumably in surface charge density. The data are consistent with a charge density of one negative charge per 280 Å 2 on the apical side. A new model for passive Cl − transport was developed that includes surface charge asymmetry and specifically accounts for the observed variation of conductance with potential. In normal frog Ringer's, J 13 was larger than J 31 at zero potential (active Cl − transport), J 13 rose exponentially with increasing positive potential to reach a maximum at 40 mV (approximately open-circuit), and the predicted partial Cl − conductance exceeded the measured conductance leading to the conclusion that when J 13 is largely driven by Na + transport, much of the coupling occurs via nonconductive pathways. Theophylline stimulates Cl − transport that also occurs via nonconductive pathways as V t becomes more positive.