Effect of ethanol on the response of the rat urinary bladder to in vitro ischemia: Protective effect of α-lipoic acid

Purpose: Ethanol exposure has been used to demonstrate the increase of oxidative stress to a variety of tissues. We studied the effect of ethanol on the response of isolated strips of rat bladder to In vitro hypoxia in the absence of glucose (In vitro ischemia). Secondly, we determined if -lipoic acid (LA) could alter the response to ethanol + In vitro ischemia.Methods: Sixty-four rats were used for the these experiments. Each rat was anesthetized and its urinary bladder excised. The bladder body was cut into two longitudinal strips and each strip mounted in individual baths filled with oxygenated Tyrodes solution containing glucose at 37 C. Ethanol (0.3%, 1%, or 3%) was placed in the first six baths (two strips at each concentration). The last two baths did not receive ethanol. Each strip was incubated for 1 h and then stimulated with field stimulation at 2, 8, and 32 Hz. Each strip was stimulated with 10 M carbachol, washed three times with fresh oxygenated buffer and ethanol re-added to their respective baths. Each strip was then stimulated with 120 mM KCl and washed three times as before. Strips were then subjected to 1 h In vitro ischemia (incubation in the absence of glucose with Tyrodes equilibrated with nitrogen instead of oxygen). During the ischemic period, each strip was stimulated for 5 s every 10 min by 32 Hz FS to simulate hyperreflexia. At the end of the hour, the tissues were incubated for an additional hour in the presence of oxygen + glucose and subjected to a second series of stimulations as before. At all times, ethanol was maintained in baths 1–6. In set 2, 1% ethanol was added to the first six baths. LA was added to every other bath, and the experiments performed as mentioned earlier.Results: (a) Ethanol at 0.3% or 1% had no effect on the contractile responses prior to exposure to In vitro ischemia; 3% was inhibitory. (b) In vitro ischemia mediated a significant decrease in the contractile responses to all forms of stimulation except for carbachol. (c) Ethanol mediated a dose-response enhancement of the contractile dysfunctions caused by In vitro ischemia. (d) LA completely reversed the effects of ethanol on contractile responses following In vitro ischemia except for carbachol.Conclusions: The results demonstrate that direct exposure to ethanol significantly enhanced contractile dysfunctions mediated by In vitro ischemia followed by re-oxygenation and that the presence of LA significantly inhibits this effect of ethanol. (Mol Cell Biochem 271: 133–138, 2005)This material is based upon work supported in part by the Office of Research and Development, Department of Veterans Affairs, and NIH RO-1-DK067114     

Interpretation of current-voltage relationships for “active” ion transport systems: I. Steady-state reaction-kinetic analysis of class-I mechanisms

Summary This paper develops a simple reaction-kinetic model to describe electrogenic pumping and co- (or counter-) transport of ions. It uses the standard steady-state approach for cyclic enzyme- or carrier-mediated transport, but does not assume rate-limitation by any particular reaction step. Voltage-dependence is introduced, after the suggestion of Läuger and Stark (Biochim. Biophys. Acta 211:458–466, 1970), via a symmetric Eyring barrier, in which the charge-transit reaction constants are written ask ₁₂=k ₁₂ ⁰ exp(zF/2RT) andk ₂₁=k ₂₁ ⁰ exp(–zF/2RT). For interpretation of current-voltage relationships, all voltage-independent reaction steps are lumped together, so the model in its simplest form can be described as a pseudo-2-state model. It is characterized by the two voltage-dependent reaction constants, two lumped voltage-independent reaction constants (K ₁₂,K ₂₁), and two reserve factors (r _i,r ₀) which formally take account of carrier states that are indistinguishable in the current-voltage (I–V) analysis. The model generates a wide range ofI–V relationships, depending on the relative magnitudes of the four reaction constants, sufficient to describe essentially allI–V data now available on active ion-transport systems. Algebraic and numerical analysis of the reserve factors, by means of expanded pseudo-3-, 4-, and 5-state models, shows them to be bounded and not large for most combinations of reaction constants in the lumped pathway. The most important exception to this rule occurs when carrier decharging immediately follows charge transit of the membrane and is very fast relative to other constituent voltage-independent reactions. Such a circumstance generates kinetic equivalence of chemical and electrical gradients, thus providing a consistent definition of ion-motive forces (e.g., proton-motive force, PMF). With appropriate restrictions, it also yields both linear and log-linear relationships between net transport velocity and either membrane potential or PMF. The model thus accommodates many known properties of proton-transport systems, particularly as observed in chemiosmotic or energy-coupling membranes.     

Transport physiology of the urinary bladder in teleosts: a suitable model for renal urea handling?

The transport physiology of the urinary bladder of both the freshwater rainbow trout (Oncorhychus mykiss) and the marine gulf toadfish (Opsanus beta) was characterized with respect to urea, and the suitability of the urinary bladder as a model for renal urea handling was investigated. Through the use of the in vitro urinary bladder sac preparation urea handling was characterized under control conditions and in the presence of pharmacological agents traditionally used to characterize urea transport such as urea analogues (thiourea, acetamide), urea transport blockers (phloretin, amiloride), and hormonal stimulation (arginine vasotocin; AVT). Na(+)-dependence and temperature sensitivity were also investigated. Under control conditions, the in vitro trout bladder behaved as in vivo, demonstrating significant net reabsorption of Na(+), Cl(-), water, glucose, and urea. Bladder urea reabsorption was not affected by pharmacological agents and, in contrast to renal urea reabsorption, was not correlated to Na(+). However, the trout bladder showed a threefold greater urea permeability compared to artificial lipid bilayers, a prolonged phase transition with a lowered E(a) between 5 degrees C and 14 degrees C, and differential handling of urea and analogues, all suggesting the presence of a urea transport mechanism. The in vitro toadfish bladder did not behave as in vivo, showing significant net reabsorption of Na(+) but not of Cl(-), urea, or water. As in the trout bladder, pharmacological agents were ineffective. The toadfish bladder showed no differential transport of urea and analogues, consistent with a low permeability storage organ and intermittent urination. Our results, therefore, suggest the possibility of a urea transport mechanism in the urinary bladder of the rainbow trout but not the gulf toadfish. While the bladders may not be suitable models for renal urea handling, the habit of intermittent urination by ureotelic tetrapods and toadfish seems to have selected for a low permeability storage function in the urinary bladder.     

Alterations in the expression of the β-cytoplasmic and the γ-smooth muscle actins in hypertrophied urinary bladder smooth muscle

The obstruction of the bladder outlet induces a marked increase in bladder mass, and this is accompanied by reduced contractility of bladder smooth muscle and alteration in the cellular architecture. In this study, we show that the composition of various isoforms of actin, a major component of the contractile apparatus and the cytoskeletal structure of smooth muscle, is altered in response to the obstruction-induced bladder hypertrophy. Northern blot analysis of the total RNA isolated from hypertrophied urinary bladder muscle, using a cDNA probe specific for smooth muscle -actin, shows over 200% increase in the -actin mRNA. However, the estimate of the amount of actin from the 2D gel reveals only a 16% increase in -actin, since the 2D gel electrophoresis does not distinguish -smooth muscle actin from -cytoplasmic actin. The bladder smooth muscle -actin and the smooth muscle -actin mRNA are not altered in response to the hypertrophy. The obstructed bladder also reveals a decrease in the -cytoplasmic actin (37%) and a concomitant diminution in the -cytoplasmic actin mRNA (29%). Hence, the composition of the actin isoforms in bladder smooth muscle is altered in response to the obstruction-induced hypertrophy. This alteration of the actin isoforms is observed at both the protein and mRNA levels.     

Restriction in the conformational flexibility of apoproteins in the presence of organic cosolvents: A consequence of the formation of “native-like conformation”

The influence of n-propanol on the overall α-helical conformation of β-globin, apocytochrome C, and the functional domain of streptococcal M49 protein (pepM49) and its consequence on the proteolysis of the respective proteins has been investigated. A significant amount of α-helical conformation is induced into these proteins atpH 6.0 and 4°C in the presence of relatively low concentrations of n-propanol. The induction of α-helical conformation into the proteins increased as a function of the propanol concentration, the maximum induction occurring around 30% n-propanol. In the case of α-globin, the fluorescence of its tryptophyl residues also increased as a function of n-propanol concentration, the midpoint of this transition being around 20% n-propanol. Furthermore, concomitant with the induction of helical conformation into these proteins, the proteolysis of their polypeptide chain by V8 protease also gets restricted. The α-helical conformation induced into α- and β-globin by n-propanol decreased as the temperature is raised from 4 to 24°C. In contrast, the α-helical conformation of both α- and β-chain (i.e., globin with noncovalently bound heme) did not exhibit such a sensitivity to this change in temperature. However, distinct differences exist between the n-propanol induced “α-helical conformation” of globins and the “α-helical conformation” of α- and β-chains. A cross-correlation of the n-propanol induced increase in the fluorescence of β-globin with the corresponding increase in the α-helical conformation of the polypeptide chain suggested that the fluorescence increase represents a structural change of the protein that is secondary to the induction of the α-helical conformation into the protein (i.e., an integration of the helical conformation induced to the segments of the polypeptide chain to influence the microenvironment of the tryptophyl residues). Presumably, the fluorescence increase is a consequence of the packing of the helical segments of globin to generate a “native-like structure.” The induction of α-helical conformation into these proteins in the presence of n-propanol and the consequent generation of “native-like conformation” is not unique to n-propanol. Trifluoroethanol, another helix-inducing organic solvent, also behaves in the same fashion as n-propanol. However, in contrast to the proteins described above, n-propanol could neither induce an α-helical conformation into performic acid oxidized RNAse-A nor restrict its proteolysis by proteases. Thus, the high sensitivity of apoproteins and the protein domains to assume α-helical conformation in the presence of low concentration of n-propanol with a concomitant restriction of the proteolytic susceptibility of their polypeptide chain appears to be unique to those proteins that exhibit high α-helical propensities. Apparently, this phenomenon of helix induction and the restriction of proteolysis reflects the formation of rudimentary tertiary interaction of the native protein and is unique to apoproteins or structural domains of α-helical proteins. Consistent with this concept, the induction of α-helical conformation into shorter polypeptide fragments of 30 residues, (e.g., α_1-30, which exists in an α-helical conformation in hemoglobin) is very low. Besides, this peptide exhibited neither the high sensitivity to the low concentrations of n-propanol seen with the apoproteins/protein domains nor the resistance toward proteolysis. The results suggest that the organic cosolvent induced decrease in the conformational flexibility of the apoprotein, and the consequent restriction of their proteolytic cleavage provides an opportunity to develop new strategies for protease catalyzed segment condensation reactions.     

Balancing acts: the role of TGF-β in the mucosal immune system

The gastrointestinal mucosal immune system faces unique challenges in dealing not only with fed antigens but also both commensal and pathogenic bacteria. It is tasked with digesting, transporting and using nutritional antigens while protecting the host from pathogenic organisms. As such, mechanisms that mediate effective immunity and immune tolerance are active within the gut environment. To accomplish this, the mucosal immune system has evolved sophisticated mechanisms that safeguard the integrity of the mucosal barrier. Transforming growth factor-β (TGF-β) emerges as a key mediator, balancing the tolerogenic and immunogenic forces at play in the gut. In this review, we discuss the role of TGF-β in the generation and functioning of gut lymphocyte populations. We highlight recent findings, summarize controversies, outline remaining questions and provide our personal perspectives.     

Amplification of optical activity by crystallization in the presence of tailor-made additives. The “inversion rule”

Experiments are described in which enantiomerically pure dimers, trimers and oligomers, generated from non chiral monomers by crystallization and topochemical reaction in suitably designed chiral crystals, induce preferential crystallization of the sarent monomer in the enantiomorphous chiral phase of opposite absolutute configuration. (We coin the term inversion rule for this effect). A general mechanism of amplification of optical activity by crystallization is proposed on the basis of these results, involving selective adsorption of resolved impurities on the surface of chiral crystals of similar stereochemistry, resulting in a decrease of the growth rate of the affected enantiomer and consequent preferential crystallization of the antipode. The implications of this mechanism to the generation and amplification of chiralily in a closed system are discussed.This paper was originally published in the Proceedings of the VIth International Conference on the Origins of Life, Jerusalem, June 1980, Ed. Y. Wolman, Reidel Dordrecht, pp. 355–364.     

Regulation of IGF-1 but not TGF-β1 by NGF in the smooth muscle of the inflamed urinary bladder

Intraperitoneal injection of cyclophosphamide (CYP) causes hemorrhagic cystitis with excess growth of muscular layer leading to bladder hypertrophy; this could be attributable to changes in the expression profiles of growth factors in the inflamed urinary bladder. The growth factors characterized in the current study include nerve growth factor (NGF), insulin-like growth factor (IGF)-1, and transforming growth factor (TGF)-β1. We found that following CYP injection for 8 h and 48 h, the mRNA levels of all three factors were increased in the inflamed bladder when compared to control. The level of NGF mRNA was mainly increased in the urothelium layer while the levels of IGF-1 mRNA and TGF-β1 mRNA were increased in the smooth muscle layer. The level of NGF high affinity receptor TrkA mRNA was also increased in both the urothelium and the smooth muscle layers during bladder inflammation. When we blocked NGF action with NGF neutralizing antibody in vivo, we found that the up-regulation of IGF-1 in the inflamed bladder was reversed while the up-regulation of TGF-β1 was not affected by NGF neutralization. The effect of NGF on regulating IGF-1 expression was further confirmed in bladder smooth muscle culture showing that exogenous NGF increased the mRNA level of IGF-1 after 30 min to 1 h stimulation. These results suggested that bladder inflammation induced region-specific changes in the expression profiles of NGF, IGF-1 and TGF-β1. The up-regulation of NGF in the urothelium may have a role in affecting bladder smooth muscle cell physiology by regulating IGF-1 expression.     

Effect of guanosine 3′:5′-monophosphate on the hydroosmotic activity of adenosine 3′:5′-monophosphate in toad urinary bladder

Guanosine 3′:5′-monophosphate has a slight hydroosmotic effect on toad urinary bladder. Furthermore, this nucleotide strongly inhibits the responses to 3′:5′-adenosine monophosphate and oxytocin. The response to an increase in medium tonicity is not modified by the guanosine nucleotide. A role for guanosine 3′:5′-monophosphate in the regulation of water permeability in toad urinary bladder is proposed.     

Formation of the functional organization of the cerebral cortex at rest in young schoolchildren varying in the maturity of cerebral regulatory systems: II. Analysis of EEG α-rhythm coherence

Coherence (COH) of rhythmic components of the EEG α rhythm at rest was analyzed to demonstrate that the maturation of deep regulatory systems (RSs) of the brain at different levels substantially affected the functional organization of the cerebral cortex and the time course of the formation of intercentral connections in young schoolchildren. The specific effect of fronto-thalamic regulatory system immaturity (FRSI) was a decrease in the α-rhythm COH predominantly in neighboring left-hemispheric derivations of foci in the anterior temporal area. A deficit of nonspecific activation had the strongest effect on the α-rhythm integration of right-hemispheric areas, although this effect was less distinct than that of FRSI and remained only as a tendency by the age of nine to ten years. Children with normal and functionally immature cerebral RSs differed from each other with respect to age-related changes in the corticocortical connections, especially in the left hemisphere. In the norm, intense growth of functional connections in the left hemisphere ceased in the period between seven to eight and nine to ten years of age; in contrast, children with RS immaturity exhibited a trend towards an increasingly greater amount of these connections in both hemispheres, which, apparently, corresponded to an earlier stage of ontogeny.     

Immunocytochemical evidence for the presence of “mammalian” neurohormonal peptides in neurones of the tapeworm Diphyllobothrium dendriticum

Summary In the nervous system of the obligatory endoparasite Diphyllobothrium dendriticum immunoreactivity (IR) to growth hormone-releasing factor (GRF), peptide histidine isoleucine (PHI), bovine pancreatic polypeptide (BPP), gastrin, gastrin-releasing peptide (GRP), oxytocin, FMRF-amide (FMRF) and serotonin (5HT) was demonstrated by immunocytochemical methods. A very strong GRF-IR was observed in the CNS and PNS of larvae and of the constantly growing adult worms. GRF-IR axon terminals occur beneath the basal lamina of the tegument along the inside of the bothridia, the holdfast organ of the worm. GRF-IR fibres surround the yolk producing vitelline glands and occur in the wall of the vagina. PHI-IR was observed in the CNS and PNS of larvae and adult worms. PHI-IR terminals occur beneath the basal lamina of the tegument along the strobila, the nutrient absorbing surface of the worm. PHI-IR fibres seem to innervate the testicular follicles. FMRF-IR fibres and perikarya occur close to the vitelline glands and the uterine pore and in the male copulatory organ. Numerous large 5HT-IR perikarya with long varicose fibres were observed in the nervous system of the worm. 5HT-IR perikarya occur close to the genital atrium. D. dendriticum is the phylogenetically lowest organism in which IR to PHI has been demonstrated.     

Platelet-derived growth factor receptor-α cells in mouse urinary bladder: a new class of interstitial cells

Specific classes of interstitial cells exist in visceral organs and have been implicated in several physiological functions including pacemaking and mediators in neurotransmission. In the bladder, Kit(+) interstitial cells have been reported to exist and have been suggested to be neuromodulators. More recently a second interstitial cell, which is identified using antibodies against platelet-derived growth factor receptor-α (PDGFR-α) has been described in the gastrointestinal (GI) tract and has been implicated in enteric motor neurotransmission. In this study, we examined the distribution of PDGFR-α(+) cells in the murine urinary bladder and the relation that these cells may have with nerve fibres and smooth muscle cells. Platelet-derived growth factor receptor-α(+) cells had a spindle shape or stellate morphology and often possessed multiple processes that contacted one another forming a loose network. These cells were distributed throughout the bladder wall, being present in the lamina propria as well as throughout the muscularis of the detrusor. These cells surrounded and were located between smooth muscle bundles and often came into close morphological association with intramural nerve fibres. These data describe a new class of interstitial cells that express a specific receptor within the bladder wall and provide morphological evidence for a possible neuromodulatory role in bladder function.     

A cytochemical and ultrastructural study of the “S.I.F.” cells in cat sympathetic ganglia

Summary According to the hypothesis of Eccles and Libet, the small intensely fluorescent cells (S.I.F. cells) in the sympathetic ganglion would represent an essential element in the inhibition of the principal neuron. As a contribution to the study of this important problem, we have investigated serial sections in superior cervical (S.C.G.) and celiac (C.G.) ganglia of the cat, a species that has not been extensively studied up to now, both by fluorescence and electron microscopy. We have shown that the S.I.F. cells are three times fewer in the cat S.C.G. than in the rat S.C.G. There are five times more S.I.F. cells in the C.G. of the cat than in the S.C.G. of the same species. Moreover we have described two types of S.I.F. cells.Type I is composed of cells characterized by highly polymorphous large dense-cored vesicles. These cells lack processes and are grouped in clusters centered on fenestrated capillaries. They could be endocrine function cells. Type II is formed of isolated cells which exibit long processes and establish synaptic junctions with the dendrites of the principal neurons. In this case, the dense-cored vesicles are very regular and much smaller. These cells could be equivalent to interneurons. Type I very strongly predominates in the S.C.G. and C.G. of the cat where it represents more than 90% of the S.I.F. cell total observed by fluorescence microscopy. A priori such a quantitative and qualitative heterogeneity hardly consistent with Eccles and Libet's hypothesis based on the existence of dopaminergic interneurons only, allows the question to be raised as to the functional significance of the S.I.F. cells in ganglion physiology. The notion of modulation of ganglionic transmission does not seem to be quiered by these new data but could be founded on different forms of action embodied in the broader conception of the neuromodulation phenomenon.     

Megaspore development in Selaginella. I. “Wicks”, their presence,ultrastructure, and presumed function

Summary Structures have been found in the locular space between the tapetal cells and megaspores in Selaginella argentea and S. kraussiana that enter the megaspore wall and extend to the plasma membrane of the megaspore cytoplasm. We have called these structures wicks. Unless special fixation procedures are used wicks are either very poorly preserved or not apparent. Wicks appear to be routes for the transport of materials from the tapetum to developing megaspores. The entry of the wicks into the megaspore wall and their passage throughout the wall implies that the megaspore wall of Selaginella is a three-dimensional mesh-work of inter-connecting spaces. Wicks have several macromolecular-sized subunits, and the results of our histochemical reactions indicated the presence of glycoprotein and/or mucopolysaccharide. X-ray microanalysis of the S. convoluta exospore showed that silicon is present in rod-shaped structures between units of the exospore in mature megaspores. Because of the size and form of the structures between the exospore units we consider that they are remnants of wicks stabilized by silicon.Present address:Cátedra de Palinologia, Museo de La Plata, Paseo del Bosque s/nro., 1900 La Plata, Argentina.     

Time-resolved fluorescence of 2-aminopurine in DNA duplexes in the presence of the EcoP15I Type III restriction–modification enzyme

EcoP15I is a Type III DNA restriction and modification enzyme of Escherichia coli. We show that it contains two modification (Mod) subunits for sequence-specific methylation of DNA and one copy of a restriction endonuclease (Res) subunit for cleavage of DNA containing unmethylated target sequences. Previously the Mod₂ dimer in the presence of cofactors was shown to use nucleotide flipping to gain access to the adenine base targeted for methylation (Reddy and Rao, J. Mol. Biol. 298 (2000) 597–610.). Surprisingly the Mod₂ enzyme also appeared to flip a second adenine in the target sequence, one which was not subject to methylation. We show using fluorescence lifetime measurements of the adenine analogue, 2-aminopurine, that only the methylatable adenine undergoes flipping by the complete Res₁Mod₂ enzyme and that this occurs even in the absence of cofactors. We suggest that this is due to activation of the Mod₂ core by the Res subunit.