Frog Ringer Research Articles

The Effects of Calcitonin Gene-Related Peptide on Heart Function

Calcitonin Gene-Related Peptide (CGRP) is a 37-amino acid peptide produced by peripheral and central neurons. It is found in a variety of organs and systems, regulating important functions in the target tissues. The αCGRP isoform is present in sensory neurons and it has been suggested to play a role in preventing hypertension, pulmonary hypertension, and ultimately acting as a potent vasodilator, improving blood flow distribution and wound healing. Studies have shown that exercise increases the levels of CGRP in skeletal and cardiac muscle cells. Studies in our laboratory suggest that CGRP may increase neurotrophic factor production by cultured cardiac cells (HL-1) in a dose depend manner, especially Glial Cell Line-Derived Neurotrophic Factor (GDNF). Finally, additional studies in our laboratory suggest that CGRP may reduce contraction rate of cultured Hl-1 cells. PURPOSE: To determine the effect of CGRP on heart contractility in frogs. METHODS: 12 frogs were divided into 4 groups. Three treatment groups received different concentrations of CGRP (40nMol, 100nMol, and 400nMol) in frog ringer’s solution. A control group was treated with only frog ringer’s solution. All frogs underwent autopsies to expose their hearts and each heart was connected to a PowerLab data acquisition system, which for collection of electrocardiogram, force of contraction, and heart rate using LabChart software. One-way ANOVA with Tukey post hoc were performed to determine statistical significance. RESULTS: Our results show that CGRP decreases force of contraction in all treatment groups when compared to the control group (Control: +9.20%; 40nM of GGRP: -38.35%; 100nM of CGRP: -34.29%; 400nM of CGRP: -33.10%; P<0.05). In addition, only 400nM of CGRP could, significantly, reduce the heart rate (Control: +3.32%; 400nM of CGRP: -24.01%; P<0.05). Finally, treatment with CGRP did not alter ECG (PQRST) curves in any of the treatment groups. CONCLUSIONS: Treatment with CGRP reduces force of contraction (in all treated groups) and reduces heart rate (400nM) without altering the ECG in frogs.

Direct effects of glucose administration on heart rate, myocardial contraction, and duration of cardiac cycle in frog's heart

Background: Recent and emerging evidences show that glucose ingestion causes prolonged Q-T interval and can trigger arrhythmia. Objective: The objective of this study is to examine the effects of administration of glucose solutions on the heart rate, strength of myocardial contraction, and duration of the cardiac cycle in the frog's heart. Methods: Five pithed frogs with a mean weight of 119 g obtained from the research laboratory, Department of Human Physiology, Bayero University Kano, Nigeria, were dissected and their hearts were exposed; 2–3 drops of frog Ringer's solution were added regularly to keep the heart moist. A kymograph was used to record the frog's myocardial activity. This was also recorded after subsequent addition of 2 ml of 5%, 10%, and 50% dextrose solutions to the heart. Heart rate (b/min), strength of myocardial contraction (mm), and the duration of the cardiac cycle (seconds) were calculated. Data were analyzed using SPSS version 20.0 and the calculated parameters for each glucose solution were compared with that of frog's Ringer's solution using paired t-test. P < 0.05 is significant. Results: The result showed a significant decrease in the heart rate (b/min) from that obtained with Ringer's solution (54.40) after addition of 5%, 10%, and 50% dextrose solutions with mean heart rates of 50.40, 47.60, and 41.00, respectively. The height of myocardial contraction (mm) was found to be significantly decreased after addition of 50% dextrose solution only, with the mean heights for frog's Ringer's, 5%, 10%, and 50% dextrose solutions been 8.65, 8.90, 8.40, and 5.35, respectively. The duration of cardiac cycle in seconds was significantly increased after addition of 10% and 50% dextrose solutions, with the mean duration for the Ringer's, 5%, 10%, and 50% dextrose solutions been 1.11, 1.18, 1.26, and 1.47, respectively. Conclusion: Both 5%, 10%, and 50% dextrose solutions caused a significant decrease in frog's heart rate. Fifty percent dextrose solution caused a significant decrease in the strength of frog's myocardial contraction, and addition of both 10% and 50% dextrose solutions significantly increased the duration of cardiac cycle in frogs.

Effects of organic and inorganic compounds of diesel exhaust particles on the mucociliary epithelium: An experimental study on the frog palate preparation

The toxic actions of acute exposition to different diesel exhaust particles (DEPA) fractions on the mucociliary epithelium are not yet fully understood due to different concentrations of organic and inorganic elements. These chemicals elements produce damage to the respiratory epithelium and exacerbate pre-existent diseases. In our study we showed these differences in two experimental studies. Study I (dose-response curve - DRCS): Forty frog-palates were exposed to the following dilutions: frog ringer, intact DEPA diluted in frog-ringer at 3mg/L, 6mg/L and 12mg/L. Study II (DEPF) (DEPA fractions diluted at 12mg/L): Fifty palates - Frog ringer, intact DEPA, DEPA treated with hexane, nitric acid and methanol. Variables analyzed: relative time of mucociliary transport (MCT), ciliary beating frequency (CBF) and morphometric analysis for mucin profile (neutral/acid) and vacuolization. The Results of DRCS: Group DEPA-12mg/L presented a significant increase in the MCT (p<0.05), proportional volume of acid mucus (p<0.05) and decreased proportional volume of neutral mucus and vacuoles (p<0.05). In relation of DEPF: A significant increase in the MCT associated to a decrease in the proportional volume of neutral mucus was founded in nitric acid group. In addition, a significant increase in the proportional volume of acid mucus was found in methanol group. We concluded that: 1) Increasing concentrations of intact DEPA can progressively increase MCT and promote an acidification of intra-epithelial mucins associated to a depletion of neutral mucus. 2) Intact DEPA seem to act as secretagogue substance, promoting mucus extrusion and consequently reducing epithelial thickness. 3) Organic fraction of low polarity seems to play a pivotal role on the acute toxicity to the mucociliary epithelium, by promoting a significant increase in the MCT associated to changes in the chemical profile of the intracellular mucins.

Capillary tone: cyclooxygenase, shear stress, luminal glycocalyx, and hydraulic conductivity (Lp).

Control of capillary hydraulic conductivity (Lp) is the physiological mechanism that underpins systemic hydration. Capillaries form the largest surface of endothelial cells in any species with a cardiovascular system and all capillaries are exposed to the flow-induced force, shear stress (τ). Vasoactive molecules such as prostacyclin (cyclooxygenase product, COX) are released from endothelial cells in response to τ. Little is known about how COX activity impacts capillary Lp. The purpose here was to assess Lp in situ following an acute Δτ stimulus and during COX1/COX2 inhibition. Mesenteric true capillaries (TC) of Rana pipiens (pithed) were cannulated for Lp assessment using the modified Landis technique. Rana were randomized into Control and Test groups. Two capillaries per animal were used (perfusate, 10 mg·mL−1 BSA/frog Ringer's; superfusate, frog Ringer's or indomethacin (10−5 mol·L−1) mixed in frog Ringer's solution). Three distinct responses of Lp to indomethacin (TC2) were demonstrated (TC1 and TC2 medians: Test Subgroup 1, 3.0 vs. 1.8; Test Subgroup 2, 18.2 vs. 2.2; Test Subgroup 3, 4.2 vs. 10.2 × 10−7 cm·sec−1·cm H2O−1). Multiple regression analysis revealed a relationship between capillary Lp and systemic red blood cell concentration or hematocrit, plasma protein concentration, and Δτ (Test Subgroup 1, R2 = 0.59, P < 0.0001; Test Subgroup 2, R2 = 0.96, P = 0.002), but only during COX inhibition. Maintaining red blood cell and plasma protein levels within a normal range may control barrier function in a healthy state. Recovering barrier function may be an unrecognized benefit of transfusions during blood loss or edema formation.

ウシガエル循環障害モデルにおける末梢前庭器の形態変化

An inner ear disorder is one of the main causes of vertigo. Though it has not been proven, many of the inner ear disorders are probably induced by ischemia or virus infection. It is not easy to evaluate the circulation of the vestibule because of its anatomical location. The objectives of the current study were to establish a vestibular ischemia model using the bullfrog, and to investigate changes in the vestibular apparatus in the ischemic condition. Bullfrogs (Rana catesbeiana) were used. Under anesthesia with ether, the unilateral temporal bone was opened from the oral cavity by drilling to expose the vestibular nerve. The vestibular artery on the nerve was cut (vestibular ischemia model). In some cases, the vestibular artery was not cut (sham surgery). The intact temporal bones served as controls. Two to 7 days later, the semicircular canals were removed. The cupulae were stained with India ink and investigated in frog Ringer solution. The ampullae were fixed, cut into 4μm slices and stained with hematoxylin-eosin. Neuroepithelia of the ampullae were investigated under the light microscope. In the vestibular ischemia model, 30-50% of cupulae in each semicircular canal were damaged to various degrees. In the sham surgery and control groups, no cupula was damaged. The neuroepithelia of the ampulla were damaged in 50-80% of the vestibular ischemia model and 1 sham surgery, and were intact in controls. Cupulae were damaged easily when the damage to the neuroepithelia was extensive. Vestibular ischemia can damage both the neuroepithelia and cupulae.

Physiological Polarity of the Frog Utricular Macula

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.

Evidence for functional interaction of plasma membrane electron transport, voltage-dependent anion channel and volume-regulated anion channel in frog aorta

Frog aortic tissue exhibits plasma membrane electron transport (PMET) owing to its ability to reduce ferricyanide even in the presence of mitochondrial poisons, such as cyanide and azide. Exposure to hypotonic solution (108 mOsmol/kg H2O) enhanced the reduction of ferricyanide in excised aortic tissue of frog. Increment in ferricyanide reductase activity was also brought about by the presence of homocysteine (100 microM dissolved in isotonic frog Ringer solution), a redox active compound and a potent modulator of PMET. Two plasma-membrane-bound channels, the volume-regulated anion channel (VRAC) and the voltage-dependent anion channel (VDAC), are involved in the response to hypotonic stress. The presence of VRAC and VDAC antagonists-tamoxifen, glibenclamide, fluoxetine and verapamil, and 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), respectively-inhibited this enhanced activity brought about by either hypotonic stress or homocysteine. The blockers do not affect the ferricyanide reductase activity under isotonic conditions. Taken together, these findings indicate a functional interaction of the three plasma membrane proteins, namely, ferricyanide reductase (PMET), VDAC and VRAC.

Extracellular Loops 2 and 4 of GLYT2 Are Required for N-Arachidonylglycine Inhibition of Glycine Transport

Concentrations of extracellular glycine in the central nervous system are regulated by Na(+)/Cl(-)-dependent glycine transporters, GLYT1 and GLYT2. N-Arachidonylglycine (NAGly) is an endogenous inhibitor of GLYT2 with little or no effect on GLYT1 and is analgesic in rat models of neuropathic and inflammatory pain. Understanding the molecular basis of NAGly interactions with GLYT2 may allow for the development of novel therapeutics. In this study, chimeric transporters were used to determine the structural basis for differences in NAGly sensitivity between GLYT1 and GLYT2 and also the actions of a series of related N-arachidonyl amino acids. Extracellular loops 2 and 4 of GLYT2 are important in the selective inhibition of GLYT2 by NAGly and by the related compounds N-arachidonyl-gamma-aminobutyric acid and N-arachidonyl-d-alanine, whereas only the extracellular loop 4 of GLYT2 is required for N-arachidonyl-l-alanine inhibition of transport. These observations suggest that the structure of the head group of these compounds is important in determining how they interact with extracellular loops 2 and 4 of GLYT2. Site-directed mutagenesis of GLYT2 EL4 residues was used to identify the key residues Arg(531), Lys(532), and Ile(545) that contribute to the differences in NAGly sensitivity.

Component analysis of change in fluid shear stress (Δτ) stimulus and basal capillary hydraulic conductivity (Lp)

One δτ stimulus consists of two components: flow increase and flow plateau. Previously, we reported lower Lp following acute, ramp versus square wave δτ, consistent with rate sensing capability (Microvasc Res 66:2003). Here, basal capillary Lp following δτ was evaluated in terms of rate and magnitude of change. We hypothesized that rate and magnitude of δτ would correlate with Lp. Frogs (Rana pipiens, N=26) were pithed and the mesentery was exteriorized and superfused with frog Ringer's (14‐16°C). Either one or two true capillaries/frog were cannulated at 10 cm H2O and perfused with 10 mg.ml−1 BSA dissolved in Ringer's. After 2 min of low flow each capillary was stimulated with ramp δτ (steady infusion pump) and a plateau was established (2 min). Lp was assessed at 30 cm H2O using the modified Landis technique. Range for acceleration (a) was 27.1 to 202.6 μm▸s−2 and 9.5 to 82.6 dynes▸cm−2 for δτ. Mean±SE Lp was 3.1±0.2 x 10−7 cm.s−1.cm H2O−1 (n=31). Lp correlated negatively with a (slope=‐0.020; R2=0.45; P=0.0002) and positively with magnitude of δτ (slope=0.048, R2=0.37, P=0.0003). δτ /a strengthened the relationship (slope=2.95, R2=0.63, and P&lt;0.0001). These data are consistent with a transducer/signaling mechanism in the capillary wall that senses both components of δτ, integrates them, and adjusts Lp accordingly. Both rate and magnitude of change in blood flow likely impact tissue hydration. Supported by RO1 HL63125

Evaluating criteria for intact capillaries in Rana pipiens using hydraulic conductivity (Lp), rolling and sticking white blood cells (WBC), and an index of systemic inflammation

WBC rolling/sticking along capillary walls have been used for decades to identify inflammation and exclude microvessels from studies of basal capillary function in vivo. For microcirculatory preparations a broader view of animal health state also is required, but often remains undefined. We measured activation of WBC obtained from a peripheral blood sample as an index of systemic inflammation in Rana pipiens and then measured Lp on individual capillaries always devoid of rolling/sticking WBC. We hypothesized that capillary Lp following our standard protocol (abrupt change in shear stress (Δτ)) would be higher in frogs with activated WBC (oxidative burst) compared to control. Frogs (Rana pipiens, N=38) were pithed and mesentery was exteriorized and superfused with frog Ringer's (14–16°C). True capillaries were cannulated at 10 cm H2O, perfused with 10 mg·ml−1BSA/Ringer's, stimulated with Δτ, and Lp assessed at 30 cm H2O (modified Landis technique). Mean±SE Lp for control capillaries (no activated WBC; within 95% confidence intervals (CI) of Δτ/Lp response curve) was 2.7±0.3 × 10−7 cm·s−1·cm H2O−1 (n=15). In frogs with activated WBC, Lp was higher (11.4±1.6 × 10−7, n=9; P&lt;0.0001) compared to control. A subset of frogs with no activated WBC displayed Lp values outside CI that averaged 8.9±1.1 × 10−7 (n=12), were higher than control, and did not differ from the activated WBC group. These data suggest that capillary rolling/sticking WBC may not provide comprehensive information concerning inflammation of intact capillaries in Rana pipiens. Systemic activated WBC accounted for half of the identified outliers. Supported by RO1 HL63125.

Determinants of Anion-Proton Coupling in Mammalian Endosomal CLC Proteins

Many proteins of the CLC gene family are Cl(-) channels, whereas others, like the bacterial ecClC-1 or mammalian ClC-4 and -5, mediate Cl(-)/H(+) exchange. Mutating a "gating glutamate" (Glu-224 in ClC-4 and Glu-211 in ClC-5) converted these exchangers into anion conductances, as did the neutralization of another, intracellular "proton glutamate" in ecClC-1. We show here that neutralizing the proton glutamate of ClC-4 (Glu-281) and ClC-5 (Glu-268), but not replacing it with aspartate, histidine, or tyrosine, rather abolished Cl(-) and H(+) transport. Surface expression was unchanged by these mutations. Uncoupled Cl(-) transport could be restored in the ClC-4(E281A) and ClC-5(E268A) proton glutamate mutations by additionally neutralizing the gating glutamates, suggesting that wild type proteins transport anions only when protons are supplied through a cytoplasmic H(+) donor. Each monomeric unit of the dimeric protein was found to be able to carry out Cl(-)/H(+) exchange independently from the transport activity of the neighboring subunit. NO(3)(-) or SCN(-) transport was partially uncoupled from H(+) countertransport but still depended on the proton glutamate. Inserting proton glutamates into CLC channels altered their gating but failed to convert them into Cl(-)/H(+) exchangers. Noise analysis indicated that ClC-5 switches between silent and transporting states with an apparent unitary conductance of 0.5 picosiemens. Our results are consistent with the idea that Cl(-)/H(+) exchange of the endosomal ClC-4 and -5 proteins relies on proton delivery from an intracellular titratable residue at position 268 (numbering of ClC-5) and that the strong rectification of currents arises from the voltage-dependent proton transfer from Glu-268 to Glu-211.

Modulatory effect of auxiliary β1 subunit on Nav1.3 voltage-gated sodium channel expressed in Xenopus oocyte

homologous and form single transmembrane glycoproteins with short intracellular loops and immunoglobulin-like extracellular segments. The association of the various α subtypes with different combinations of auxiliary β subunits creates the possibility of additional molecular and functional complexity for neuronal sodium channels. Nav1.1, Nav1.2, Nav1.3 and Nav1.6 are predominantly expressed in the central nervous system while Nav1.7, Nav1.8 and Nav1.9 are principally found in the peripheral nervous system. 2 The Nav1.3 α subtype has attracted interest recently because its expression is dramatically up-regulated in nociceptive sensory neurons after peripheral nerve damage, perhaps contributing to the pathophysiology of neuropathic pain. 3 Theoretically, no matter how the expression of the Nav1.3 α subunit is changed in neuropathic pain, the function of the Nav1.3 α subtype should be modulated by β subunits. The aim of this study was to examine the effects of cloned rat β1 subunit on Nav1.3 function.

P2X5 Subunit Assembly Requires Scaffolding by the Second Transmembrane Domain and a Conserved Aspartate

Functional homomeric and heteromeric ATP-gated P2X receptor channels have been shown to display a characteristic trimeric architecture. Of the seven different isoforms (designated P2X(1)-P2X(7)), P2X(5) occurs in humans primarily as a non-functional variant lacking the C-terminal end of the ectodomain and the outer half of the second transmembrane domain. We show that this truncated variant, which results from the splice-skipping of exon 10, is prone to subunit aggregation because the residual transmembrane domain 2 is too short to insert into the membrane. Alleviation of the negative hydrophobic mismatch by the addition of a stretch of moderately hydrophobic residues enabled formation of a second membrane-spanning domain and strictly parallel homotrimerization. Systematic mutagenesis identified only one transmembrane domain 2 residue, Asp(355), which supported homotrimerization in a side chain-specific manner. Our results indicate that transmembrane domain 2 formation contributes 2-fold to hP2X(5) homotrimerization by tethering the end of the ectodomain to the membrane, thereby topologically restricting conformational mobility, and by intramembrane positioning of Asp(355). While transmembrane domain 2 appears to favor assembly by enabling productive subunit interactions in the ectodomain, Asp(355) seems to assist by simultaneously driving intramembrane helix interactions. Overall, these results indicate a complex interplay between topology, helix-helix interactions, and oligomerization to achieve a correctly folded structure.

Extra- and intra-cellular ice formation in Stage I and II Xenopus laevis oocytes

We are currently investigating factors that influence intracellular ice formation (IIF) in mouse oocytes and oocytes of the frog Xenopus. A major reason for choosing these two species is that while their eggs normally do not possess aquaporin channels in their plasma membranes, these channels can be made to express. We wish to see whether IIF is affected by the presence of these channels. The present Xenopus study deals with control eggs not expressing aquaporins. The main factor studied has been the effect of a cryoprotective agent [ethylene glycol (EG) or glycerol] and its concentration. The general procedure was to (a) cool the oocytes on a cryostage to slightly below the temperatures at which extracellular ice formation occurs, (b) warm them to just below the melting point, and (c) then re-cool them to −50 °C at 10 °C/min. In the majority of cases, IIF occurs well into step (c), but a sizeable minority undergo IIF in steps (a) or (b). The former group we refer to as low-temperature flashers; the latter as high-temperature flashers. IIF is manifested as abrupt blackening of the egg, which we refer to as “flashing.” Observations on the Linkam cryostage are restricted to Stage I and II oocytes, which have diameters of 200–300 μm. In the absence of a cryoprotective agent, that is in frog Ringers, the mean flash temperature for the low-temperature freezers is −11.4 °C, although a sizeable percentage flash at temperatures much closer to that of the EIF (−3.9 °C). When EG is present, the flash temperature for the low-temperatures freezers drops significantly to ∼−20 °C for EG concentrations ranging from 0.5 to 1.5 M. The presence of 1.5 M glycerol also substantially reduces the IIF temperature of the low-temperature freezers; namely, to −29 °C, but 0.5 and 1 M glycerol exert little or no effect. The IIF temperatures observed using the Linkam cryostage agree well with those estimated by calorimetry [F.W. Kleinhans, J.F. Guenther, D.M. Roberts, P. Mazur, Analysis of intracellular ice nucleation in Xenopus oocytes by differential scanning calorimetry, Cryobiology 52 (2006) 128–138]. The IIF temperatures in Xenopus are substantially higher than those observed in mouse oocytes [P. Mazur, S. Seki, I.L. Pinn, F.W. Kleinhans, K. Edashige, Extra- and intracellular ice formation in mouse oocytes, Cryobiology 51 (2005) 29–53]. Perhaps that is a reflection of their much larger size.

Caspase-3 Cleaves and Inactivates the Glutamate Transporter EAAT2

EAAT2 is a high affinity, Na+-dependent glutamate transporter with predominant astroglial localization. It accounts for the clearance of the bulk of glutamate released at central nervous system synapses and therefore has a crucial role in shaping glutamatergic neurotransmission and limiting excitotoxicity. Caspase-3 activation and impairment in expression and activity of EAAT2 are two distinct molecular mechanisms occurring in human amyotrophic lateral sclerosis (ALS) and in the transgenic rodent model of the disease. Excitotoxicity caused by down-regulation of EAAT2 is thought to be a contributing factor to motor neuron death in ALS. In this study, we report the novel evidence that caspase-3 cleaves EAAT2 at a unique site located in the cytosolic C-terminal domain of the transporter, a finding that links excitotoxicity and activation of caspase-3 as converging mechanisms in the pathogenesis of ALS. Caspase-3 cleavage of EAAT2 leads to a drastic and selective inhibition of this transporter. Heterologous expression of mutant SOD1 proteins linked to the familial form of ALS leads to inhibition of EAAT2 through a mechanism that largely involves activation of caspase-3 and cleavage of the transporter. In addition, we found evidence in spinal cord homogenates of mutant SOD1 ALS mice of a truncated form of EAAT2, likely deriving from caspase-3-mediated proteolytic cleavage, which appeared concurrently to the loss of EAAT2 immunoreactivity and to increased expression of activated caspase-3. Taken together, our findings suggest that caspase-3 cleavage of EAAT2 is one mechanism responsible for the impairment of glutamate uptake in mutant SOD1-linked ALS.

Subtle influence of solution breakdown products on capillary barrier function revealed by hydraulic conductivity (L p )/change in shear stress (ΔSS) relationship

The relationship between ΔSS and Lp can be used to assess the impact of experimental protocols on capillary barrier function. The purpose of the present study was to determine how much of the Lp response to shear stress following pronase digestion could be accounted for by the vehicle solution. We hypothesized that vasoactive breakdown products in the pipette solution would account only partially for the effect on barrier function demonstrated following pronase digestion. Frog mesenteric true capillaries were cannulated and exposed to either control (n=18, 10 mg.ml−1 BSA in frog Ringer’s), filtered pronase (n=7, 30 min incubation, 10,000 MWCO filter), or pronase (n=12, 0.1 mg.ml−1, 1 min). Lp was assessed on the first occlusion using the modified Landis technique and strict inclusion and exclusion criteria were applied. Mean Lp was 2.6 ± 0.2 x 10−7 for control, 5.3 ± 1.5 x 10−7 for filtered pronase, and 14.3 ± 2.9 x 10−7 cm.s−1.cm H2O−1 after pronase. The slope of the ΔSS/Lp relationship was 0.05(±0.01) (R2=0.60, P=0.001) for control, 0.20(±0.02) (R2=0.96, P=0.003) for filtered pronase, and 1.13(±0.09) (R2=0.97, P<0.0001) for pronase. The 4.0-fold response to filtered pronase accounted partially for the 22.6-fold response of individual capillaries to pronase. These effects were demonstrated most clearly by the ΔSS/Lp curves while means underestimated both responses. The ΔSS/Lp relationship has proven to be more sensitive than mathematical averages for deciphering the mechanisms of how mechanical flow stimuli change capillary barrier function. Supported by NIH RO1 HL63125.

Analysis of intracellular ice nucleation in Xenopus oocytes by differential scanning calorimetry

Intracellular ice formation (IIF) plays a central role in cell damage during cryopreservation. We are investigating the factors which trigger IIF in Xenopus oocytes, with and without aquaporin water channels. Here, we report differential scanning calorimeter studies of Xenopus control oocytes which do not express aquaporins. Stage I to VI oocytes (which increase progressively in size) were investigated with emphasis on stage I and II because they are translucent and can also be studied under the cryomicroscope. Measurements were made in 1, 1.5, and 2 M ethylene glycol (EG) in frog Ringers plus SnoMax. A multistep freezing protocol was used in which the samples were cooled until extracellular ice formation (EIF) occurred, partially remelted, slowly recooled through the EIF temperature, and then rapidly (10 °C/min) cooled. EIF in the 1, 1.5, and 2 M EG occurred at −6.4, −7.8, and −8.9 °C, respectively. Freezing exotherms of individual stage I–VI oocytes were readily visible. A general trend was observed in which the IIF temperature of the early stage oocytes (I–III) was well below T EIF while the later stages (IV–VI) froze at temperatures much closer to T EIF. Thus, in 1.5 M EG, T IIF was −21.1, −25, and −26.6 °C in stages I–III, but was −17 and −8.5 °C for stage IV and V–VI. Concurrently, the percentage of oocytes in which IIF was observed fell dramatically from a high of 40 to 72% in early stages (I–III) to a low of only 7% in stage V–VI because, particularly in the later stages, IIF was hidden in the EIF exotherm. We conclude that early stage oocytes are a good model system in which to investigate modulators of IIF, but that late stage oocytes are damaged during EIF and infrequently supercool.

Spontaneous Thermal Motion of the GABAA Receptor M2 Channel-lining Segments

The gamma-aminobutyric acid type A (GABA(A)) receptor channel opening involves translational and rotational motions of the five channel-lining, M2 transmembrane segments. The M2 segment's extracellular half is loosely packed and undergoes significant thermal motion. To characterize the extent of the M2 segment's motion, we used disulfide trapping experiments between pairs of engineered cysteines. In alpha1beta1 gamma2S receptors the single gamma subunit is flanked by an alpha and beta subunit. The gamma2 M2-14' position is located in the alpha-gamma subunit interface. Gamma2 13' faces the channel lumen. We expressed either the gamma2 14' or the gamma2 13' cysteine substitution mutants with alpha1 cysteine substitution mutants between 12' and 16' and wild-type beta1. Disulfide bonds formed spontaneously between gamma2 14'C and both alpha1 15'C and alpha1 16'C and also between gamma2 13'C and alpha1 13'C. Oxidation by copper phenanthroline induced disulfide bond formation between gamma2 14'C and alpha1 13'C. Disulfide bond formation rates with gamma2 14'C were similar in the presence and absence of GABA, although the rate with alpha1 13'C was slower than with the other two positions. In a homology model based on the acetylcholine receptor structure, alphaM2 would need to rotate in opposite directions by approximately 80 degrees to bring alpha1 13' and alpha1 15' into close proximity with gamma2 14'. Alternatively, translational motion of alphaM2 would reduce the extent of rotational motion necessary to bring these two alpha subunit residues into close proximity with the gamma2 14' position. These experiments demonstrate that in the closed state the M2 segments undergo continuous spontaneous motion in the region near the extracellular end of the channel gate. Opening the gate may involve similar but concerted motions of the M2 segments.

Luffa Operculata Affects Mucociliary Function of the Isolated Frog Palate

Luffa operculata is a medicinal plant used in homeopathic and alternative medicine. In the United States, it is sold in a purified spray form, whereas a homemade L. operculata dry fruit infusion (DFI) is commonly used in Latin America. The L. operculata DFI is applied intranasally, inducing profuse mucous secretion and relieving nasal symptoms. Nevertheless, this medication may cause irritation of the nasal mucosa, as well as epistaxis or anosmia. Given the growing popularity of alternative medicine, a decision was made to evaluate the effects of this substance on mucous membranes. The effects of L. operculata DFI on mucociliary transport velocity, ciliary beat frequency, and transepithelial potential difference (PD) were evaluated in an isolated frog palate preparation. We tested 46 palates immediately before immersion and again at 5 and 20 minutes after immersion. Four groups (n = 10) were tested in frog Ringer: control; L. operculata DFI, 60 mg/L; 600 mg/L; and 1200 mg/L. An additional group was tested using L. operculata DFI prepared with water (600 mg/L of H2O, n = 6). Epithelial samples were harvested for ultrastructural study. In treated palates, mucociliary transport velocity and ciliary beat frequency decreased significantly (p < 0.001 and p < 0.008, respectively). There was a dose-dependent decrease in PD modulus (p < .007). Our PD findings indicated ion-fluid transport abnormalities, which were confirmed by transmission electron microscopy that showed enlargement of interepithelial spaces. In this ex vivo model, the L. operculata DFI infusion promoted significant changes in the mucociliary function of the epithelium, suggesting that it is potentially noxious to human nasal mucosa.

Luffa operculata effects on the epithelium of frog palate: histological features

SummaryLuffa operculata is the botanical name of buchinha-do-norte or cabacinha, which is a medicinal plant widely used for the treatment of rhinitis and rhinosinusitis. In Europe and USA, it is available in homeopathic medicines. In Brazil, Luffa operculata dry fruit infusion is inhaled or instilled into the nose releasing profuse mucous secretion, thus relieving nasal symptoms. Nevertheless, this often may cause irritation, epistaxis or anosmia.Study designExperimental.Material and MethodThe effects of Luffa operculata were evaluated in different concentration infusions, in isolated frog palate preparation, testing 46 palates after immersion. Four groups (n = 10) were tested with the infusion prepared with frog Ringer (isotonic): control; 60 mg/L; 600 mg/L; and 1200 mg/L. An additional group was tested using the infusion with water (600 mg/L H2O, n = 6). Epithelial samples were harvested to be studied under light microscopy and electron transmission microscopy.ResultsIn treated palates, light microscopy findings were dose-dependent standard toxic changes. Electron transmission microscopy showed enlargement of intercellular spaces and tight junctions disruption, pointing to ion-fluid transport abnormalities. Conclusions: Luffa operculata infusion in currently used doses can promote significant structural and ultrastructural changes in the epithelium of this ex vivo model of respiratory mucosa.