Hyperosmotic Urea Research Articles

Osmotic regulation of betaine homocysteine-S-methyltransferase expression in H4IIE rat hepatoma cells

Cell hydration changes critically affect liver metabolism and gene expression. In the course of gene expression studies using nylon cDNA-arrays we found that hyperosmolarity (405 mosmol/l) suppressed the betaine-homocysteine methyltransferase (Bhmt) mRNA expression in H4IIE rat hepatoma cells. This was confirmed by Northern blot and real-time quantitative RT-PCR analysis, which in addition unraveled a pronounced induction of Bhmt mRNA expression by hypoosmotic (205 mosmol/l) swelling. Osmotic regulation of Bhmt mRNA expression was largely paralleled at the levels of Bhmt protein and enzymatic activity. Like hyperosmotic NaCl, hyperosmotic raffinose but not hyperosmotic urea suppressed Bhmt mRNA expression, suggesting that cell shrinkage rather than increased ionic strength or hyperosmolarity per se is the trigger. Hypoosmolarity increased the expression of a reporter gene driven by the entire human BHMT promoter, whereas destabilization of BHMT mRNA was observed under hyperosmotic conditions. Osmosensitivity of Bhmt mRNA expression was impaired by inhibitors of tyrosine kinases and cyclic nucleotide-dependent kinases. The osmotic regulation of BHMT may be part of a cell volume-regulatory response and additionally lead to metabolic alterations that depend on the availability of betaine-derived methyl groups.

Teleost Fh14-3-3a protein protects Xenopus oocytes from hyperosmolality.

We have previously cloned and characterized a novel 14-3-3 gene from the euryhaline telost Fundulus heteroclitus, Fh14-3-3a (Kültz et al., 2001). The corresponding gene product is osmoregulated and most highly expressed in gill epithelium of this fish. In the present study we have expressed Fh14-3-3a cRNA in Xenopus laevis oocytes and investigated the survival and electrophysiological parameters of Xenopus oocytes in isosmotic and various hyperosmotic media. Xenopus oocytes expressing Fh14-3-3a show no mortality after a 16 hour exposure to hyperosmolality in the form of elevating medium K(+), Na(+), polyethylene glycol, or sorbitol concentrations up to 444 mosmol/kg. In contrast, 16 hours of the same hyperosmolality caused 100% mortality in control Xenopus oocytes injected with water. As a result of hyperosmolality the Xenopus oocyte membrane potential decreased between 10 and 70% in oocytes expressing Fh14-3-3a whereas it was completely abolished in control oocytes. We report that one potential cause for the osmoprotective effect of Fh14-3-3a on Xenopus oocytes could be its inhibition of an endogenous chloride current. Hyperosmotic urea was not as harmful to Xenopus oocytes as hypertonicity and maybe acting through a different mechanism. Coexpression of Fh14-3-3a with a human calcium channel in Xenopus oocytes did not affect the electrophysiological properties of this exogenous channel. Thus, the osmoprotective effect of Fh14-3-3a may prove a valuable tool for the characterization of exogenous ion channels in Xenopus oocytes exposed to hyperosmotic conditions.

Regulatory volume increase is associated with p38 kinase-dependent actin cytoskeleton remodeling in rat kidney MTAL.

The kidney medulla is physiologically exposed to variations in extracellular osmolality. In response to hypertonic cell shrinkage, cells of the rat kidney medullary thick ascending limb of Henle's loop undergo p38 kinase-dependent regulatory volume increase (RVI). In the present study, we investigated the role of actin cytoskeleton reorganization in this process. Addition of hyperosmotic NaCl or sucrose, which activates MAP kinases and reduces cellular volume, induced a sustained actin polymerization occurring after 10 min and concurrently with RVI. In contrast, hyperosmotic urea, which does not modify MAP kinase activity and cellular volume, did not induce sustained actin polymerization. Fluorescence microscopy revealed that hyperosmotic NaCl and sucrose, but not urea, induced the redistribution of F-actin from a dense cortical ring to a diffuse network of actin bundles. Stabilization of actin filaments by jasplakinolide and inhibition of the generation of new actin filaments by swinholide A prevented RVI, whereas depolymerization of actin filaments by latrunculin B attenuated cell shrinkage and enhanced RVI. These actin-interfering drugs did not alter extracellular regulated kinase and p38 kinase activation under hypertonic conditions. Similar to swinholide A, inhibiting p38 kinase with SB-203580 abolished sustained actin polymerization, actin redistribution, and decreased RVI efficacy. We therefore propose that in rat kidney the medullary thick ascending limb of Henle's loop exposed to extracellular hypertonicity, p38 kinase activation induces depolymerization of the F-actin cortical ring and polymerization of a dense diffuse F-actin network that both contribute to increase RVI efficacy.

Hyperosmotic urea activates basolateral NHE in proximal tubule from P-gp null and wild-type mice.

Using the pH-sensitive fluorescent dye BCECF, we compared the effects of hyperosmotic urea on basolateral Na(+)/H(+) exchange (NHE) with those of hyperosmotic mannitol in isolated nonperfused proximal tubule S2 segments from mice lacking both the mdr1a and mdr1b genes (KO) and wild-type (WT) mice. All the experiments were performed in CO(2)/HCO-free HEPES solutions. Osmolality of the peritubular solution was raised from 300 to 500 mosmol/kgH(2)O by adding mannitol or urea. NHE activity was assessed by the Na(+)-dependent acid extrusion rate (J(H)) after an acid load with NH(4)Cl prepulse. In WT mice, hyperosmotic mannitol had no effect on J(H) at over the entire range of intracellular pH (pH(i)) studied (6.20-6.90), whereas in KO mice it increased J(H) at a pH(i) range of 6.20-6.45. In contrast, in both WT and KO mice, hyperosmotic urea increased J(H) at a pH(i) range of 6.20-6.90. In KO mice, J(H) in a hyperosmotic urea solution were similar to those in a hyperosmotic mannitol solution at a pH(i) range of 6.20-6.40 but were greater than in a hyperosmotic mannitol solution at a pH(i) range of 6.45-6.90. In WT mice, hyperosmotic urea caused an increase in V(max) without changing K(m) for peritubular Na(+). Staurosporine (the PKC inhibitor) inhibited hyperosmotic mannitol-induced NHE activation in KO mice, whereas it had no effect on hyperosmotic urea-induced NHE activation in WT or KO mice. Genistein (the tyrosine kinase inhibitor) inhibited hyperosmotic urea-induced NHE activation in WT and KO mice, whereas it caused no effect on hyperosmotic mannitol-induced NHE activation in KO mice. We conclude that hyperosmotic urea activates basolateral NHE via tyrosine kinase in tubules from both WT and KO mice, whereas hyperosmotic mannitol activates it via PKC only in tubules from KO mice.

Expression and regulation of the Na +/K +/2Cl − cotransporter NKCC1 in rat liver and human HuH-7 hepatoma cells

The expression of sodium potassium chloride cotransporter 1 (NKCC1) was studied in different liver cell types. NKCC1 was found in rat liver parenchymal and sinusoidal endothelial cells and in human HuH-7 hepatoma cells. NKCC1 expression in rat hepatic stellate cells increased during culture-induced transformation in the myofibroblast-like phenotype. NKCC1 inhibition by bumetanide increased α 1-smooth muscle actin expression in 2-day-cultured hepatic stellate cells but was without effect on basal and platelet-derived-growth-factor-induced proliferation of the 14-day-old cells. In perfused rat liver the NKCC1 made a major contribution to volume-regulatory K + uptake induced by hyperosmolarity. Long-term hyperosmotic treatment of HuH-7 cells by elevation of extracellular NaCl or raffinose concentration but not hyperosmotic urea or mannitol profoundly induced NKCC1 mRNA and protein expression. This was antagonized by the compatible organic osmolytes betaine or taurine. The data suggest a role of NKCC1 in stellate cell transformation, hepatic volume regulation, and long-term adaption to dehydrating conditions.

Different responses to acute or progressive osmolarity increases in the mIMCD3 cell line

Cells from the kidney medulla are able to survive and function when exposed to high concentrations of NaCl and urea. In vitro, cultured epithelial cells from the kidney medulla are able to survive stronger acute hyperosmotic shocks when both solutes are present. However, in vivo, increases in osmolarity are not acute. In this study, we compared the survival of a murine renal epithelial cell line during acute or progressive (two step) adaptation to hypertonic NaCl and/or urea. Increasing osmolarity to 700 mOsm/l with NaCl or urea in a single step led to massive cell death ( 50% in 24 hours). However, genomic DNA of dying cells was not degraded, and electron microscopy revealed weak condensation of chromatin, absence of membrane blebbing, and no nuclear indentation. Pre-adaptation to permissive concentrations of NaCl (200 mOsm/l giving a final osmolarity of 500 mOsm/l) protected cells against subsequent increases in osmolarity, allowing adaptation to final osmolarities as high as 900 mOsm/l. In contrast, pre-adaptation to permissive concentrations of urea (200 mOsm/l) did not lead to enhanced cell survival after a subsequent 200 mOsm/l step. Cell death was as rapid as after an acute shock, but was more typical of apoptosis (genomic DNA laddering, strong chromatin condensation, nuclear indentation, and blebbing of the membrane giving rise to apoptotic bodies). Thus, acute hyperosmolarity induces cell death with essentially similar responses to NaCl and urea. In contrast, progressive adaptation of mIMCD3 cells to NaCl allows cell survival, whereas progressive adaptation to hyperosmotic urea triggers a cell death pathway different from the one triggered by acute hyperosmotic shocks.

ERK activation by urea in the renal inner medullary mIMCD3 cell line.

Urea- and NaCl-inducible extracellular signal-regulated kinase (ERK) phosphorylation exhibited dissimilar kinetics. Among cell lines examined, the effect of urea was unique to mIMCD3 inner medullary collecting duct cells and MDCK cells. Urea-inducible ERK activation was approximately 10-fold less sensitive to the MEK inhibitor, PD-98059, than was that of NaCl. This difference did not appear to be accounted for by differential activation of MEK isoforms. Interestingly, the inhibitor of p38 activation, SB-203580, abrogated the effect of both urea and NaCl upon both ERK and MEK activation; however, the former was much less sensitive to the inhibitor. Consistent with this observation, NaCl was much more effective than urea at inducing p38 phosphorylation. The effect of hypertonic stress (e.g., sorbitol 100 mM) could be blocked by appropriate medium dilution such that isotonicity was maintained. In marked contrast, the effect of hyperosmotic urea could not be blocked in this fashion, implying the absence of dependence upon cell volume. Together, these data suggest that cells of the renal inner medulla are potentially uniquely responsive to urea and that urea and hypertonic stressors induce ERK activation through distinct mechanisms.

Effects of hyperosmolality on Na, K-ATPase gene expression in vascular smooth muscle cells.

Cultured vascular smooth muscle cells (VSMC) from rat thoracic aortas were exposed to hyperosmotic media to determine the effects on Na, K-ATPase alpha1- and beta1-mRNA expression. Hyperosmotic media (500 mOsm/kgH2O) supplemented with glucose or mannitol increased alpha1-mRNA levels threefold at 24 hr and beta1-mRNA levels sevenfold at 12 hr. In sharp contrast, hyperosmotic urea medium had no effect at any time. Both the protein synthesis inhibitor cycloheximide and the RNA transcription inhibitor actinomycin D reduced alpha1- and beta1-mRNA upregulation induced by hyperosmotic glucose or mannitol media. Protein kinase C (PKC) inhibitors (staurosporine A or calphostin C) or tyrosine kinase (TK) inhibitors (genistein or herbimycin A) had no effect on the alpha1-mRNA upregulation induced by hyperosmotic glucose or mannitol media. Hyperosmotic glucose or mannitol media (500 mOsm/kgH2O) significantly increased alpha1- and beta1-subunit protein levels and Na, K-ATPase activity, whereas hyperosmotic urea medium had no effect. Transfection experiments with the 5'-flanking sequences of the alpha1- or beta1-subunit genes linked to the luciferase reporter gene revealed that hyperosmolar glucose medium increased luciferase activity 2.9- and 3.7-fold, respectively. Similarly, hyperosmotic mannitol medium increased such activity 2.7- and 3.4-fold, respectively. These results demonstrate that: (i) hyperosmolality induced by the poorly permeating solutes (glucose and mannitol) stimulates alpha1- and beta1-mRNA accumulation, alpha1- and beta1-subunit protein accumulation, and Na, K-ATPase activity, whereas the rapidly permeating solute (urea) has no effect; (ii) the upregulation of alpha1- and beta1-mRNA in response to hyperosmotic glucose or mannitol media requires, at least in part, de novo synthesis of intermediate regulatory proteins; (iii) the hyperosmolality-induced alpha1-mRNA upregulation occurs through PKC- and TK-independent mechanisms, whereas the hyperosmolality-induced beta1-mRNA upregulation occurs through activation of PKC and TK; and (iv) hyperosmolality induced by glucose or mannitol increases promoter activities of the alpha1- and beta1-subunit genes.

Induction of tyrosine phosphorylation and Na+/H+ exchanger activation during shrinkage of human neutrophils.

The ubiquitous isoform of the Na+/H+ exchanger (NHE1) is essential for the regulation of cellular volume. The underlying molecular mechanism, which is poorly understood, was studied in human polymorphonuclear leukocytes (PMN). Suspension of PMN in hypertonic media induced rapid cellular shrinkage and activation of NHE1, which is measurable as a cytosolic alkalinization. Concomitantly, hypertonic stress also induced extensive tyrosine phosphorylation of several proteins. Pretreatment of PMN with genistein, a tyrosine kinase inhibitor, prevented not only the tyrosine phosphorylation in response to a hypertonic shock but also the activation of NHE1. The signal elicited by hyperosmolarity that induces activation of tyrosine kinases and NHE1 was investigated. Methods were devised to change medium osmolarity without altering cell volume and vice versa. Increasing medium and intracellular osmolarity in normovolemic cells failed to activate tyrosine kinases or NHE1. However, shrinkage of cells under iso-osmotic conditions stimulated both tyrosine phosphorylation and NHE1 activity. These findings imply that cells detect alterations in cell size but not changes in osmolarity or ionic strength. The identity of the proteins that were tyrosine-phosphorylated in response to cell shrinkage was also investigated. Unexpectedly, the mitogen-activated protein kinases SAPK, p38, erk1, and erk2 were not detectably phosphorylated or activated. In contrast, the tyrosine kinases p59(fgr) and p56/59(hck) were phosphorylated and activated upon hypertonic challenge. We propose that cells respond to alterations in cell size, but not to changes in osmolarity, with increased tyrosine phosphorylation, which in turn leads to the activation of NHE1. The resulting changes in ion content and cytosolic pH contribute to the restoration of cell volume in shrunken cells.

NaCl but not urea activates p38 and jun kinase in mIMCD3 murine inner medullary cells.

The mitogen-activated protein kinases (MAPKs), p38 and jun kinase (JNK), are activated by diverse stressors in cells of nonrenal medullary origin. Epithelial cells of the renal medulla are among the very few cells of higher eukaryotes routinely subjected to hyperosmotic stress, composed of principally NaCl and urea. Hyperosmotic NaCl activated p38 and JNK in a time- and dose-dependent fashion in cells of the murine terminal inner medullary collecting duct cell line (mIMCD3) as determined by immune complex kinase assay. Hyperosmotic urea exerted a minimal effect upon only p38 activation, which was evident only at 5 min. The NaCl effect was dose dependent to 800 mosmol/kgH2O; 800 mosmol/kgH2O urea, in contrast, exerted no effect. Consistent with these observations, NaCl (800 mosmol/kgH2O) but not urea (800 mosmol/kgH2O) increased tyrosine phosphorylation of p38 and JNK at 10 min. Therefore, even in the extremely osmotolerant renal medullary mIMCD3 cell line, derived from a tissue adapted for routine exposure to elevated osmolality, hypertonic NaCl activated two stress-responsive MAPKs. Urea, in contrast, exerted virtually no effect; therefore, cellular protection from urea stress operates through a mechanism distinct from the stress-responsive MAPKs.

The effect of hyperosmotic treatment on the functioning of ampullary electroreceptor organs.

The condition of the microvilli of the apical membrane in ampullary electroreceptor organs of the freshwater catfish, Ictalurus nebulosus, was manipulated by administration of hyperosmotic solutions (500 mM) in order to investigate to what extent the apical surface area determines the sensitivity and the shape of the frequency curves. Electrophysiological recordings demonstrated that hyperosmotic sucrose and glucose solutions reduce the sensitivity by 80%, and alter the shape of the frequency curves. The synaptic delay and the average spontaneous activity remained almost unchanged. Calculations with an equivalent electric circuit of an electroreceptor cell indicate that a reduction in apical surface area can account for these effects. Apparently, the sensitivity and the frequency curves in ampullary organs in freshwater catfish depend on the apical surface area. A hyperosmotic urea solution proved to be less suitable to investigate the effect of manipulations of the apical surface area. The almost total loss in sensitivity caused by administration of urea is caused by both shunting of the stimulus by leaky tight junctions and a reduced synaptic efficacy. The repetitive activity observed after exposure to glucose or sucrose must be attributed to malfunctioning of postsynaptic parts of the receptor organ.

Urea signaling in cultured murine inner medullary collecting duct (mIMCD3) cells involves protein kinase C, inositol 1,4,5-trisphosphate (IP3), and a putative receptor tyrosine kinase.

Urea, in concentrations unique to the renal medulla, increases transcription and protein expression of several immediate-early genes (IEGs) including the zinc finger-containing transcription factor, Egr-1. In the present study, the proximal 1.2 kb of the murine Egr-1 5' -flanking sequence conferred urea-responsiveness to a heterologous luciferase reporter gene when transiently transfected into renal medullary mIMCD3 cells,and this effect was comparable with that of the extremely potent immediate-early gene inducer, O-tetradecanoylphorbol 13-acetate (TPA). Urea inducibility of Egr-1 expression was protein kinase C (PKC)-dependent because staurosporine and calphostin C abrogated the urea effect, and down-regulation of PHC through chronic TPa treatment inhibited both urea-inducible Egr-1 protein expression and gene transcription. In addition, hyperosmotic urea increased inositol 1,4,5-trisphosphate (IP3) release from mIMCD3 cells and induced tyrosine phosphorylation of the receptor tyrosine kinase-specific phospholipase C (PLC) isoform, PLC-gamma. Importantly, urea-inducible Egr-1 expression was strongly genistein-sensitive, to a much greater extent than the comparable TPA-inducible Egr-1 expression. These data suggest that urea-inducible Egr-1 expression is a consequence of sequential PLC-gamma activation, IP3 release, and PKC activation. Urea-inducible PLC-gamma activation, in conjunction with the genistein-sensitivity of urea-inducible Egr-1 expression suggest the possibility of a cell surface or cytoplasmic urea-sensing receptor tyrosine kinase.

Hyperosmotic urea increases transcription and synthesis of Egr-1 in murine inner medullary collecting duct (mIMCD3) cells.

It was previously shown that when cells of renal epithelial origin are exposed to hyperosmotic urea in concentrations unique to the renal medulla, abundance of mRNA encoding the immediate-early gene product Egr-1 is up-regulated. This phenomenon appears restricted to cells of renal epithelial origin. In the present study, the newly isolated murine renal inner medullary cell line mIMCD3 was used to determine whether the urea-induced increase in Egr-1 mRNA abundance is associated with increased expression of functional protein product, and whether this increase is transcriptionally mediated. In Western analysis, urea (200 mM) increased Egr-1 immunoreactivity 3-fold relative to sham-treated cells. [35S]Methionine pulse-labeling followed by immunoprecipitation confirmed that this increased immunoreactivity was associated with increased de novo Egr-1 protein synthesis. Electrophoretic mobility shift assay demonstrated that urea treatment induced a commensurate increase in specific DNA binding activity for the Egr-1 consensus sequence. In addition, the increase in Egr-1 mRNA expression accompanying urea treatment was a consequence of enhanced transcription, as determined by nuclear run-off assay. Taken together, these data indicate that hyperosmotic urea increases both Egr-1 transcription and new protein synthesis in renal epithelial cells in culture, and that this newly synthesized Egr-1 is a functional DNA-binding protein. To our knowledge, this is the first example of urea-inducible gene transcription. In addition, Egr-1 represents the first eukaryotic transcription factor transcriptionally activated by a hyperosmotic stressor.

Toads Taste Sodium With Their Skin: Sensory Function in a Transporting Epithelium

ABSTRACT Amphibians do not normally ingest water orally. Rather, they obtain water by osmotic flow across their skin (Bentley and Yorio, 1979). Previous studies of the desert toad, Bufo punctatus, showed that dehydrated animals placed on a surface moistened with pure water adopted a particular type of behavior, termed the water absorption response (WR), in which toads splayed their hindlimbs and oscillated their body to maximize the area of skin in contact with the surface (Brekke et al. 1991). If the surface was moistened with a hyperosmotic urea solution, the animals moved over the surface as if to search for osmotically available water and soon jumped off the surface without initiating WR behavior. Since the skin was the only part of the animal in contact with the surface, it was concluded that receptors in the skin enabled the animals to avoid WR behavior on surfaces where water was not osmotically available. Similarly, toads did not initiate WR behavior when NaCl was used as the solute. These observations suggested that toads have either cutaneous receptors for detecting osmolality, per se, or cutaneous receptors for specific solutes.

Urea induces Egr-1 and c-fos expression in renal epithelial cells.

The membrane-permeant solute urea, in concentrations present in the mammalian renal medulla, increased expression at the mRNA level of two immediate-early gene (IEG) transcription factors, Egr-1 and c-fos, in a time- and dose-dependent fashion in confluent growth-suppressed Madin-Darby canine kidney (MDCK) cells. This upregulation occurred in the absence of both cytotoxicity and an inhibition of protein synthesis, two potential nonspecific inducers of IEG expression. These findings were of interest because we have previously shown that hyperosmotic stress induced by the functionally membrane-impermeant solute NaCl increased expression of these IEG, whereas hyperosmotic stress induced by the membrane-permeant solute glycerol failed to do so. The urea-induced increase in Egr-1 mRNA expression was not secondary to enhanced message stability as determined by actinomycin D experiments and is therefore likely a consequence of urea-induced transcriptional activation. Augmented Egr-1 expression in response to urea treatment was also observed in another renal epithelial cell line, LLC-PK1, but not in other cell types examined. Therefore cells of renal epithelial origin may be uniquely capable of responding to hyperosmotic urea with increased expression of IEG transcription factors, and this increase is likely transcriptionally mediated.

Urea selectively induces DNA synthesis in renal epithelial cells.

Hyperosmotic stress with the functionally impermeant solute NaCl has been shown by us and others to inhibit cell growth and DNA synthesis. Several lines of evidence suggest that urea, the other principal renal medullary solute, may exert a growth-promoting effect on renal epithelial cells. Among these is the finding that urea upregulates expression at the mRNA level of two growth-associated immediate-early genes, Egr-1 and c-fos. In the present study, urea, in concentrations characteristic of the renal medulla, increased [3H]thymidine incorporation approximately threefold in confluent, growth-suppressed Madin-Darby canine kidney (MDCK) cells, whereas another readily membrane-permeant solute, glycerol, did not. Urea also overcame the inhibitory effect of hyperosmotic NaCl on DNA synthesis. The urea-induced increase in [3H]thymidine incorporation was also evident in the renal epithelial LLC-PK1 cell line, but not in renal nonepithelial and epithelial nonrenal cell types examined. In addition, it was associated with a 15% increase in total DNA content measured fluorometrically at 24 h of treatment. There was, however, no associated increase in cell proliferation as measured by cell number, total protein content, or cell cycle distribution. Urea also failed to induce polyploidy or aneuploidy. Therefore cells of renal epithelial origin may be uniquely capable of responding to hyperosmotic urea with increased DNA synthesis through an undefined and potentially novel mechanism.

Direct Inactivation of Human Polymorphonuclear Leukocyte by Hyperosmotic Urea Comparable to the Renal Medulla

Hyperosmolarity in the renal medulla inhibits host defenses against bacterial pyelonephritis. Urea and NaCl contribute most to high osmolarity in the renal medulla. We therefore examined the inhibitory mechanism of urea on superoxide generation by human polymorphonuclear leukocytes. Superoxide production was inhibited by high concentration of urea. This inhibition was found to be direct and immediate. In addition, direct inactivation of NADPH oxidase, the key enzyme complex of superoxide generation, was shown by an NADPH oxidase activity assay using cell lysates of polymorphonuclear leukocytes stimulated by phorbol myristate acetate. The inhibitory effect of urea on NADPH oxidase was reversed by washing urea out of the assay system of cell lysates. Kinetic analysis of the inhibition of NADPH oxidase activity by urea showed decreased Vmax and Km, suggesting uncompetitive inhibition. These findings suggested that urea inactivated polymorphonuclear leukocyte superoxide production through a direct and uncompetitive inhibition of NADPH oxidase.

Inhibition of Chemiluminescent Response of Human Polymorphonuclear Leukocytes by Hyperosmolality Comparable to Renal Medulla and Preservation by Ofloxacin

Hyperosmolality of the urine and renal medulla inhibits polymorphonuclear leukocyte (PMN) functions such as phagocytosis, intracellular killing and superoxide generation. The main factors of hyperosmolality in the urinary tract are urea and NaCl. We studied the chemiluminescent (CL) functions of PMNs in hypersomotic urea and NaCl solutions and the effects of ofloxacin, a new quinolone antimicrobial, for inhibiting the functions of PMNs subjected to hyperosmolality. When PMNs were incubated with hypersomotic urea and NaCl solutions, the CL response was significantly reduced. The reduced CL response was significantly preserved by supplementation with ofloxacin (OFLX). The CL response of PMNs may thus also be reduced in the urinary tract and OFLX may exert strong antimicrobial and preservative effects on PMN.

The influence of central hyperosmotic solutions on drinking and vasopressin release following peripheral hyperosmotic NaCl in the minipig

The drinking and lysine vasopressin (LVP, porcine vasopressin) responses were measured in minipigs given simultaneous intracarotid (i.c.) (1.28 ml/min) and intracerebroventricular (i.c.v.) (50 μl/min) infusions of solutions of differing osmolality and sodium content. Observations were made during, and for a further 15 min after, the combined infusions which lasted 15 min. Drinking in response to i.c. infusion of 2.0 Osm NaCl started with a latency of 7 min and was unaffected by simultaneous i.c.v. infusion of 0.15 M NaCl, was additive with the drinking produced by i.c.v. 1.4 Osm NaCl, sucrose or mannitol, and inhibited by i.c.v. infusion of 1.4 Osm urea. LVP release following i.c. hyperosmotic NaCl was observed as early as the first blood sample, at 2 min, and was attenuated by i.c.v. hyperosmotic urea and attenuated, then slightly augmented, by i.c.v. infusions of the hyperosmotic non-electrolyte solutions. These results show that drinking following peripheral administration of an osmotic stimulus in the minipig can add to that induced by central infusions of solutions that of the cerebrospinal fluid (CSF) but may have increased or decreased CSF sodium concentration. By contrast, the stimulated LVP release was attenuated by solutions that decreased CSF sodium concentration. Thus, in minipigs, separate osmoreceptors appear to mediate osmotically induced drinking and LVP release.

Cerebral vessels cryofixed after hyperosmosis or cold injury in normothermic and hypothermic frogs

Three purported means by which large solutes may penetrate the blood-brain barrier are: permeabilized tight junctions: vesicular transport: or channel formation across cerebral blood vessels. The role of vesicular transport has been questioned, in part, because many cytoplasmic vesicles are induced by aldehyde fixation. Cryofixation reduces this artefact and was used to see structural changes in frog cerebral endothelium made permeable to plasma solutes after perivascular exposure to hyperosmotic (3 M) urea, or injury with a cold probe (−50 °C). Some control and experimental frogs were made hypothermic so as to inhibit endocytosis and autolytic changes. The brains of some untreated controls were immerse-fixed in aldehydes. Other controls and all other brains of normothermic or hypothermic animals were rapidly frozen, then substituted with acetone-fixative. The interendothelial tight junctions separate partially or completely, after hyperosmotic exposure, in one third of the junctions. Blood-borne ferritin and Evans blue pass through some of the patent junctions. Junctional opening is caused by cell shrinkage, because the perimeter/area ratio of individual endothelial cells in the hyperosmotic group is significantly greater than in the control, due to a decreased area. Large 0.08–0.32-μm-wide invaginations or pits of the endothelial cell membrane characterize both cryofixed and aldehyde-fixed vessels. The pits often appear as isolated vesicles in the cytoplasm, but serial sections reveal that many communicate with either the capillary lumen or subendothelial space. No series of pits opened onto both lumen and space to form a transendothelial channel. The number of vesicles in aldehyde-fixed specimens is about 4 times greater ( P < 0.01) and in the cold injured, cryofixed brain capillary, about two times greater ( P < 0.01), than in the cryofixed control. Hyperosmotic exposure does not increase the number of pits. The permeabilization of anuran cerebral endothelium by hyperosmotic treatment or cold injury is thus by means of an intercellular rather than a transcellular route.