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We evaluated the efficacy of an ointment containing bacitracin, polymyxin B, and gramicidin for the eradication of colonization by methicillin-resistant Staphylococcus aureus in 11 medical patients, 10 (91%) of whom had previously failed a 1-week course of topical mupirocin. Mupirocin resistance was documented in 5 (45%) of 11 patients. Successful decolonization was achieved in 9 (82%) of 11 patients (based on 2 months of follow-up).

In this review, we discuss evidence that supports the hypothesis that adrenergic stimulation of transepithelial Na absorption across the alveolar epithelium occurs indirectly by activation of apical Cl channels, resulting in hyperpolarization and an increased driving force for Na uptake through amiloride-sensitive Na channels. This hypothesis differs from the prevailing idea that adrenergic-receptor activation increases the open probability of Na channels, leading to an increase in apical membrane Na permeability and an increase in Na and fluid uptake from the alveolar space. We review results from cultured alveolar epithelial cell monolayer experiments that show increases in apical membrane Cl conductance in the absence of any change in Na conductance after stimulation by selective beta-adrenergic-receptor agonists. We also discuss possible reasons for differences in Na-channel regulation in cells grown in monolayer culture compared with that in dissociated alveolar epithelial cells. Finally, we describe some preliminary in vivo data that suggest a role for Cl-channel activation in the process of amiloride-sensitive alveolar fluid absorption.

Background: Some individual components of complement are synthesized by the kidney. However, it is not known whether these form functional pathways that are able to mediate more fundamental cellular events. We examined the ability of HK-2 tubular cells to produce an intact alternative pathway of complement and to respond to the C3a fragment thus produced through the C3a receptor., Methods: The production of mRNA for alternative pathway components was detected by reverse transcription-polymerase chain reaction, whereas protein synthesis was investigated by probing Western blots of concentrated culture supernatants with polyclonal antisera. Levels of C3a and inositol phosphate produced by HK-2 cells were determined by radioimmunoassay, whereas those of transforming growth factor-beta1 (TGF-beta1) were measured by ELISA. Intracellular tyrosine phosphorylation in response to C3a was evaluated by Western blotting and chemiluminescence., Results: HK-2 cells produce the complement polypeptides C3a, C3, and factors B and H. They also contain mRNA for all components of the alternative pathway and the C3a receptor. mRNA levels were up-regulated by interleukin-1alpha, interleukin-1beta, and tumor necrosis factor-alpha. Incubation of HK-2 cells with C3a led to an increase in intracellular inositol phosphate and to tyrosine phosphorylation of at least two proteins in a pertussis-toxin-sensitive fashion. C3a and C3a desarg also up-regulated the secretion of TGF-beta1 by these cells., Conclusion: HK-2 cells produce an intact alternative pathway of complement. In addition, both locally produced and urinary C3a have the potential to activate these cells, resulting in inflammatory events such as TGF-beta1 production.

The objective of this study was to investigate the effects of insulin and insulin-like growth factor I on transepithelial Na(+) transport across porcine glandular endometrial epithelial cells grown in primary culture. Insulin and insulin-like growth factor I acutely stimulated Na(+) transport two- to threefold by increasing Na(+)-K(+) ATPase transport activity and basolateral membrane K(+) conductance without increasing the apical membrane amiloride-sensitive Na(+) conductance. Long-term exposure to insulin for 4 d resulted in enhanced Na(+) absorption with a further increase in Na(+)-K(+) ATPase transport activity and an increase in apical membrane amiloride-sensitive Na(+) conductance. The effect of insulin on the Na(+)-K(+) ATPase was the result of an increase in V(max) for extracellular K(+) and intracellular Na(+), and an increase in affinity of the pump for Na(+). Immunohistochemical localization along with Western blot analysis of cultured porcine endometrial epithelial cells revealed the presence of alpha-1 and alpha-2 isoforms, but not the alpha-3 isoform of Na(+)-K(+) ATPase, which did not change in the presence of insulin. Insulin-stimulated Na(+) transport was inhibited by hydroxy-2-naphthalenylmethylphosphonic acid tris-acetoxymethyl ester [HNMPA-(AM)(3)], a specific inhibitor of insulin receptor tyrosine kinase activity, suggesting that the regulation of Na(+) transport by insulin involves receptor autophosphorylation. Pretreatment with wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase as well as okadaic acid and calyculin A, inhibitors of protein phosphatase activity, also blocked the insulin-stimulated increase in short circuit and pump currents, suggesting that activation of phosphatidylinositol 3-kinase and subsequent stimulation of a protein phosphatase mediates the action of insulin on Na(+)-K(+) ATPase activation.

Alveolar epithelial cells were isolated from adult Sprague-Dawley rats and grown to confluence on membrane filters. Most of the basal short-circuit current (Isc; 60%) was inhibited by amiloride (IC50 0. 96 microM) or benzamil (IC50 0.5 microM). Basolateral addition of terbutaline (2 microM) produced a rapid decrease in Isc, followed by a slow recovery back to its initial amplitude. When Cl- was replaced with methanesulfonic acid, the basal Isc was reduced and the response to terbutaline was inhibited. In permeabilized monolayer experiments, both terbutaline and amiloride produced sustained decreases in current. The current-voltage relationship of the terbutaline-sensitive current had a reversal potential of -28 mV. Increasing Cl- concentration in the basolateral solution shifted the reversal potential to more depolarized voltages. These results were consistent with the existence of a terbutaline-activated Cl- conductance in the apical membrane. Terbutaline did not increase the amiloride-sensitive Na+ conductance. We conclude that beta-adrenergic stimulation of adult alveolar epithelial cells results in an increase in apical Cl- permeability and that amiloride-sensitive Na+ channels are not directly affected by this stimulation.

Objectives: To examine the hypothesis that a sustained rise in plasma acylation stimulating protein (ASP, C3a desarg) accompanies the elevation in triacylglycerol that follows the ingestion of an oral fat load., Design: Following an overnight fast, blood samples were obtained from healthy volunteers while fasting and 15 min, 1, 2, 4, 6 and 8 h following ingestion of: (i) a liquid meal, rich in dairy fat (eight subjects) and (ii) a semi-liquid meal, with higher total fat content and rich in polyunsaturated fat (six subjects)., Subjects and Methods: Four male and four female volunteers (age range: 22-51 y; body mass index (BMI): 17.9-26.9 kg/m2) received the first meal. Six subjects (age range: 32-60 y; BMI: 18.0-28.4 kg/m2), including three from the first study, received the second meal using the same protocol. ASP and C5a were measured by radioimmunoassay (RIA) and the complement proteins C3, factor B and C5 by radial immunodiffusion or nephelometry. Tumour necrosis factor (TNF)-alpha was measured by enhanced ELISA, and plasma cholesterol and triacylglycerol by an automated enzymatic method. The presence of chylomicrons was assessed in post-prandial plasma samples taken after the second meal., Results: There was no significant change in mean ASP concentration in either group at any time point, following ingestion of either meal. However, there was a significant positive linear trend in ASP following the second fat challenge (ANOVA; P < 0.05). There was also no change in complement proteins, plasma cholesterol or TNF-alpha. Plasma triacylglycerol rose significantly after the first and second meals (P < 0.05 and P < 0.001 at 2 h post-prandially); the mean maximum rise above the fasting level was 58 +/- 41% and 89 +/- 38% respectively (mean +/- s.d.). Chylomicrons were detected in samples taken from each subject after the second meal. Analysis of individual ASP data showed a sustained rise in one subject after the first meal and two subjects after the second meal. Substantial variation in ASP concentration was observed in samples taken in the first 2 h post-prandially., Conclusion: There was no significant change in ASP nor other complement proteins for either group of subjects following ingestion of the lipid loads. Individual data showed substantial variation in post-prandial ASP, but multiple plasma sampling did not define the basis for this variation.

Previous studies in our laboratory have shown that Na absorption across the porcine endometrium is stimulated by PGF2alpha and cAMP-dependent activation of a barium-sensitive K channel located in the basolateral membrane of surface epithelial cells. In this study, we identify and characterize this basolateral, barium-sensitive K conductance. Porcine uterine tissues were mounted in Ussing chambers and bathed with KMeSO4 Ringer solution. Amphotericin B (70 microM) was added to the luminal solution to permeabilize the apical membrane and determine the current-voltage relationship of the basolateral K conductance after activation by 100 microM CPT-cAMP. An inwardly rectifying current was identified which possessed a reversal potential of -53 mV when standard Ringer solution was used to bathe the serosal surface. The K:Na selectivity ratio was calculated to be 12:1. Administration of 5 mM barium to the serosal solution completely inhibited the current activated by cAMP under these conditions. In addition to these experiments, amphotericin-perforated whole cell patch clamp recordings were obtained from primary cultures of porcine surface endometrial cells. The isolated cells displayed an inwardly rectifying current under basal conditions. This current was significantly stimulated by CPT-cAMP and blocked by barium. These results together with our previous studies demonstrate that cAMP increases Na absorption in porcine endometrial epithelial cells by activating an inwardly rectifying K channel present in the basolateral membrane. Similar patch clamp experiments were conducted using cells from a human endometrial epithelial cell line, RL95-2. An inwardly rectifying current was also identified in these cells which possessed a reversal potential of -56 mV when the cells were bathed in standard Ringer solution. This current was blocked by barium as well as cesium. However, the current from the human cells did not appear to be activated by cAMP, indicating that distinct subtypes of inwardly rectifying K channels are present in endometrial epithelial cells from different species.

In the present paper, the mechanisms underlying the neural and hormonal regulation of mucosal ion transport in the pig intestinal tract are reviewed. The active transport of NaCl by isolated sheets of porcine intestinal mucosa is modulated by cholinergic and non-cholinergic neurons of undetermined neurochemical identity that lie in the submucosa. The application of electrical field stimulation to mucosa-submucosa preparations from porcine jejunum, ileum, or colon produces rapid elevations in short-circuit current which are inhibited by tetrodotoxin or omega-conotoxin GVIA, blockers of neuronal Na+ and Ca2+ channels, respectively. In porcine ileum, these elevations in current are mimicked in large part by cholinergic agonists and have been attributed to anion secretion. The majority of classical neurotransmitters and gut peptides that have been examined to date increase active transepithelial anion secretion through interactions with G protein-coupled receptors associated with submucosal neurons or situated on the basolateral membranes of epithelial cells. A small number of neuropeptides interact with neuronal receptors to augment NaCl absorption or decrease anion secretion. Noradrenergic control of intestinal transport differs in the porcine small and large intestines, and displays considerable inter-species variability in its cellular underpinnings. Transport regulation by bombesin-like peptides may be mediated by receptors distributed in both the apical and basolateral membrane domains of epithelial cells in porcine colon. The transport process affected by these peptides may be linked to epithelial growth and differentiation. The pig intestinal tract appears to be a useful biological model for resolving the cellular mechanisms by which gut neurotransmitters and hormones act in regulating transepithelial ion fluxes. Its general relevance to human intestinal function is discussed.

Of particular interest for voltage-gated K+ channels are the effects of membrane voltage and pharmacologic agents on channel kinetics. We have characterized in detail properties of Kv1.2 channel expressed in oocytes as the basis for investigation of its structure-function relationships. This channel exhibited a voltage-dependent rate of activation with a V1/2 of -21 mV. Voltage-dependent steady-state inactivation overlapped the activation curve with half-maximal inactivation occurring at -22 mV. Dendrotoxin inhibited channel activation with an IC50 of 8.6 nM at + 35 mV. Charybdotoxin also blocked this K+ channel (IC50 = 5.6 nM). While dendrotoxin block was not affected by channel activation, charybdotoxin exhibited additional accumulation of block following activation, which was relieved with a time constant of 0.5 s upon repolarization of the membrane. The deactivation of this channel was accelerated in the presence of charybdotoxin while not significantly affected by dendrotoxin.

A Quick Response Program (QRP) was developed and implemented at St George Hospital during 1995 and 1996. The program sought to improve the service provided to elderly people presenting to the emergency department by offering a new rapid response service pathway to community-based care. Emergency department discharge planning and crisis intervention evolved as important QRP functions during the program's life. Evaluation findings indicated that QRP penetration into the elderly sub-acute emergency department patient population was high, and that hospital admissions were avoided without affecting emergency department process times. Health outcomes were not compromised by the program, and patient and general practitioner satisfaction were high. The program grappled with the inherent conflict of interest between the aims of the hospital (acute care services) and those of the community service (support and maintenance). The program sought to bridge the gap between these service parameters in the name of meeting patient needs.

The finding of a massive unilateral segmental defect with normal ventilation upon lung scanning does not always secure the diagnosis of acute pulmonary embolus. We present a patient whose ventilation-perfusion lung scan suggested a significant embolic phenomenon, but who was subsequently found to have compression of her right pulmonary artery by a dissecting aortic aneurysm. Conditions that present with a unilateral perfusion defect merit further evaluation with pulmonary arteriography.

Mycobacterial diseases are common in people infected with human immunodeficiency virus. Mycobacterium tuberculosis (MTB) and Mycobacterium avium intracellulare (MAI), the specific pathogens most frequently involved, cause pulmonary tuberculosis and disseminated MAI infections. Pulmonary tuberculosis incidence was on the decline from 1950 to 1985, but since 1985 has been on the rise worldwide. Prior to the onset of AIDS, MAI infections were rare in humans. However, disseminated MAI seems to be associated with the terminal stage of AIDS. The symptomatology of MTB and MAI infections is similar, yet diagnosis and treatment vary. Pulmonary TB can be treated effectively with chemotherapy and isolation to prevent transmission. Because MAI infection is not a communicable disease, isolation is not necessary. Effective treatment for disseminated MAI remains under investigation; currently, a regimen of four to five drugs is recommended. There are however, significant side effects associated with this therapy. Because the number of AIDS patients is increasing, it is imperative for clinicians to understand the mycobacterial diseases and how best to manage them.

Porcine distal colon epithelium was mounted in Ussing chambers and bathed in plasma-like Ringer solution. Tissue conductances ranged from 10 to 15 mS and the short-circuit current (Isc) ranged from -15 to 220 microA.cm-2. Variations in basal Isc resulted from differences in the amount of amiloride (10 microM mucosal addition)-sensitive Na+ absorption. Ion substitution and transepithelial flux experiments showed that 10 microM amiloride produced a decrease in the mucosal-to-serosal (M-S) and net Na flux, and that this effect on Isc was independent of Cl- and HCO3- replacement. When the concentration of mucosal amiloride was increased from 10 to 100 microM, little change in Isc was observed. However, increasing the concentration to 1 mM produced a further inhibition, which often reversed the polarity of the Isc. The decrease in Isc due to 1 mM amiloride was dependent on both Cl- and HCO3-, and was attributed to reductions in the M-S and net Na+ fluxes as well as the M-S unidirectional Cl- flux. Ion replacement experiments demonstrated that Cl- substitution reduced the M-S and net Na fluxes, while replacement of HCO3- with HEPES abolished net Cl- absorption by reducing the M-S unidirectional Cl- flux. From these data it can be concluded that: (1) Na+ absorption is mediated by two distinct amiloride-sensitive transport pathways, and (2) Cl- absorption is completely HCO3- dependent (presumably mediated by Cl-/HCO3- exchange) and occurs independently of Na+ absorption.

The production of corticotropin or corticotropin-releasing factor by non-pituitary, non-adrenal tumors may rarely be associated with an overt clinical expression of hypercortisolism. Recent studies have emphasized the importance of careful thoracic evaluation when such ectopic hormone secretion is suspected.

In 1985 the U.S. Department of Defense took action to establish regulations for the protection of national defense personnel against the AIDS epidemic. Since that time specific procedures have been carried out in each military unit to effectively handle soldiers infected with the AIDS virus. A unique six-stage classification system for monitoring disease progression of AIDS is currently used in military medical facilities around the world, and military medicine remains active in the fight against AIDS.

The role of epithelium in the modulation of contractile responses to electrical field stimulation (EFS), acetylcholine (ACh), and KCl were studied in vitro in strips of equine tracheal smooth muscle (TSM). EFS with 0.5 ms pulses of voltage (70 V) resulted in frequency dependent contractions of equine TSM that were sensitive to tetrodotoxin (TTX) and atropine. In TSM without epithelium, preincubation with indomethacin significantly potentiated contractile responses to EFS. The potentiating effect of indomethacin on EFS contractions was abolished by the addition of 3 nM prostaglandin E2 (PGE2). ACh and KCl cumulative concentration-response curves were shifted to the left by removal of epithelium from equine TSM strips with a significant decrease in the 50% effective concentration (EC50) for both ACh and KCl. The mean EC50 (+/- SE) for ACh in TSM without epithelium was 0.51 +/- 0.09 microM vs 4.30 +/- 1.03 microM in TSM with epithelium. Similarly, the mean EC50 (+/- SE) for KCl in TSM without epithelium was 22.20 +/- 2.61 mM vs 32.35 +/- 2.66 mM in TSM with epithelium. The addition of indomethacin (3 microM) had no effect on the ACh concentration-response curves in TSM strips with or without epithelium. Our results suggest that in the equine airway there is (1) an epithelial-derived relaxant factor that modulates tracheal smooth muscle contractility postsynaptically, and (2) a nonepithelial-derived inhibitory factor, possibly PGE2, that modulates ACh release from nerves presynaptically.

Porcine distal colon epithelium was mounted in Ussing chambers and bathed with porcine Ringer's solution. The effects of vasoactive intestinal polypeptide, norepinephrine, and carbamylcholine on Na and Cl fluxes and transepithelial electrical parameters were determined after their serosal administration. Vasoactive intestinal peptide increased the Cl-dependent component of the short-circuit current with a half-maximal effect at 115 nmol/L. Transepithelial Na and Cl flux studies demonstrated that the increase in current was caused by stimulation of Cl secretion. Norepinephrine also stimulated Cl secretion and increased the serosal-to-mucosal Na flux, producing a half-maximal effect at 1.6 mumol/L. Selective blockade of alpha 1 adrenoceptors by 0.5 mumol/L prazosin produced an eightfold decrease in norepinephrine potency. Carbamylcholine produced a significant increase in Cl secretion and decreased absorption of both Na and Cl with a concentration of 10 mumol/L producing a half-maximal effect. The muscarinic cholinoceptor blocker atropine (0.1 mumol/L) produced a 22-fold decrease in carbamylcholine potency. The effects of all three transmitter substances were unaffected after pretreatment of tissues with the neuronal conduction-blocker tetrodotoxin or an inhibitor of arachidonic acid metabolism. These results indicate that (a) vasoactive intestinal polypeptide stimulates Cl secretion without affecting Na absorption; (b) norepinephrine acting through alpha 1 adrenoceptors stimulates net Cl secretion and activates a serosal-to-mucosal Na transport mechanism; and (c) carbamylcholine acting through muscarinic receptors stimulates Cl secretion and inhibits Na and Cl absorption.

Acetylcholine and cholinomimetics such as carbachol are potent stimulants of epithelial Cl- secretion in the small and large intestines of several mammalian species. In this study, the effects of carbachol were characterized in vitro on active ion transport in sheets of submucosa-mucosa from the distal jejunum of swine. Carbachol (10 microM) produced an increase in serosa-positive short-circuit current (Isc) in this tissue after its serosal, but not luminal administration. The Na(+)-K(+)-Cl- transport blocker bumetanide (10 microM) produced a 50% decrease in the carbachol-induced Isc elevation after its serosal administration. Peak increases in Isc evoked by carbachol were significantly reduced by 60-85% in tissues bathed in media lacking Cl-, HCO3-, or both anions. The initial drug-induced increase in net charge transfer from serosa to lumen was dependent upon both HCO3- and Cl-, whereas sustained elevations in charge transfer were dependent upon extracellular Cl- only. Radiotracer flux analyses revealed that the drug decreased net Na+ absorption and increased Cl- secretion. In the absence of HCO3-, carbachol decreased Cl- absorption. The effects of carbachol on HCO3- transport were examined by pH-stat titration. The drug rapidly alkalinized the luminal medium immediately after its serosal administration. These results suggest that carbachol stimulates electrogenic anion secretion in the mucosa of the porcine distal jejunum. Furthermore, the ability of carbachol to inhibit spontaneous Na+ absorption is dependent upon extracellular HCO3-.

Equine tracheal epithelium, stripped of serosal muscle, mounted in Ussing chambers, and bathed in plasmalike Ringer solution generates a serosa-positive transepithelial potential of 10-22 mV and a short-circuit current (Isc) of 70-200 microA/cm2. Mucosal amiloride (10 microM) causes a 40-60% decrease in Isc and inhibits the net transepithelial Na flux by 95%. Substitution of Cl with gluconate resulted in a 30% decrease in basal Isc. Bicarbonate substitution with 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid decreased the Isc by 21%. The Cl-dependent Isc was inhibited by serosal addition of 1 mM amiloride. Bicarbonate replacement or serosal amiloride (1 mM) inhibits the net Cl flux by 72 and 69%, respectively. Bicarbonate replacement significantly reduces the effects of serosal amiloride (1 mM) on Isc, indicating its effect is HCO3 dependent. Addition of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP; 100 microM) causes a 40% increase in Isc. This effect is inhibited by subsequent addition of 10 microM serosal bumetanide. Bumetanide (10 microM) reduces net Cl secretion following stimulation with 8-BrcAMP (100 microM). Serosal addition of BaCl2 (1 mM) causes a reduction in Isc equal to that following Cl replacement in the presence or absence of 100 microM cAMP. These results suggest that 1) Na absorption depends on amiloride-inhibitable Na channels in the apical membrane, 2) Cl influx across the basolateral membrane occurs by both a Na-H/Cl-HCO3 parallel exchange mechanism under basal conditions and by a bumetanide-sensitive Na-(K?)-Cl cotransport system under cAMP-stimulated conditions, and 3) basal and cAMP-stimulated Cl secretion depends on Ba-sensitive K channels in the basolateral membrane.

Incorporation of 14C-leucine and 3H-alanine into TCA-precipitable protein. TCA-soluble protein, and antifreeze glycoproteins (AFGP) was measured in isolated hepatocytes from Pagothenia borchgrevinki Boulenger following acclimation to -1.5 degrees C and +4 degrees C. the rate of 3H-alanine incorporation into AFGP followed Michaelis-Menten kinetics with a Vmax of 4.8 nM X mg protein-1 X h-1 at -1.5 degrees C and 7.5 nM X mg protein-1 X h-1 at +4 degrees C. Km values were 27.9 microM and 41.7 microM at -1.5 degrees C and +4 degrees C, respectively. Incorporation of 14C-leucine into TCA-precipitable protein also showed Michaelis-Menten kinetics with a Vmax of 20 nM X mg protein-1 X hr-1 at 1.5 degrees C and 32.3 nM X mg protein-1 X hr-1 at +4 degrees C. Km values were 83.3 microM at -1.5 degrees C and 125 microM at +4 degrees C. AFGP synthesis was monitored over a 120-h period by radioimmunoassay in cultures of hepatocytes from cold acclimated fish (-1.5 degrees C) incubated at both -1.5 degrees C and +4 degrees C. The estimated Q10 for AFGP from these data is 3.23. Polyacrylamide gel electrophoresis of antifreeze glycoproteins produced by isolated hepatocytes showed that all four antifreeze fractions normally present in the serum of P. borchgrevinki are also synthesized by isolated hepatocytes. The two major conclusions from these experiments were that 1) P. brochgrevinki, unlike many northern fishes, does not show thermal acclimation, and 2) environmental factors responsible for modification of peptide antifreeze synthesis in northern fishes do not elicit changes in AFGP synthesis in P. borchgrevinki.

Many polar fishes synthesize a group of eight glycopeptides that exhibit a non-colligative lowering of the freezing point of water. These glycopeptides range in molecular weight between 2600 and 33 700. The largest glycopeptides [1-5] lower the freezing point more than the small ones on a weight basis and contain only two amino acids, alanine and threonine, with the disaccharide galactose-N-acetyl-galactosamine attached to threonine. The small glycopeptides, 6, 7, and 8, also lower the freezing point and contain proline, which periodically substitutes for alanine. Glycopeptides with similar antifreeze properties isolated from the saffron cod and the Atlantic tomcod contain an additional amino acid, arginine, which substitutes for threonine in glycopeptide 6. In this study we address the question of whether differences in amino acid composition or molecular weight between large and small glycopeptides are responsible for the reduced freezing point depressing capability of the low molecular weight glycopeptides. The results indicate that the degree of amino acid substitutions that occur in glycopeptides 6-8 do not have a significant effect on the unusual freezing point lowering and that the observed decrease in freezing point depression with smaller glycopeptides can be accounted for on the basis of molecular weight.

Measurements of ion concentrations, freezing points and melting points of intestinal fluid were made for several Antarctic fishes and two North Atlantic species. These measurements indicated that plasma and intestinal fluid are nearly isosmotic. Freezing points of intestinal fluid were approximately 0.9 degrees C below the melting points, suggesting the presence of glyco-protein antifreeze within the intestinal fluid of the Antarctic fishes. Polyacrylamide gel electrophoresis and specific immunoprecipitation with glyco-protein antifreeze antibody confirmed the presence of appreciable quantities of antifreeze and showed that the major antifreeze fractions present in the intestinal fluid are low molecular weight glycopeptides.

The effects of several sulfamoyl benzoic acid derivatives on Na-K-Cl cotransport were investigated in winter flounder intestine. The relative efficacy (IC50 values) and order of potency of these derivatives were benzmetanide, 5 X 10(-8) M greater than bumetanide 3 X 10(-7) M greater than piretanide 3 X 10(-6) M greater than furosemide 7 X 10(-6) M greater than amino piretanide 1 X 10(-5) 3-amino-4-penoxy-5-sulfamoyl benzoic acid. Binding of [3H] bumetanide was studied in microsomal membranes from winter flounder intestine and compared to that in bovine kidney outer medulla. Binding was also studied in brush-border membranes from winter flounder intestine. The estimated values for Kd and number of binding sites (n) were: bovine kidney, Kd = 1.6 X 10(-7), n = 10.5 pmol/mg protein; winter flounder intestine, Kd 1.2 X 10(-7), n = 7.3 pmol/mg protein, and brush-border membranes from winter flounder, Kd = 5.3 X 10(-7), n = 20.4 pmol/mg protein. The estimated Kd for bumetanide binding to winter flounder brush-border membranes derived from association and dissociation kinetics was 6.8 X 10(-7) M. The similarity in magnitudes of IC50 and Kd for bumetanide suggests that the brush-border cotransporter is ordinarily rate-limiting for transmural salt absorption and that bumetanide specifically binds to the cotransporter. Measurement of bumetanide binding at various concentrations of Na, K and Cl showed that optimal binding required all three ions to be present at about 5 mM concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Na-K-Cl cotransport stoichiometry and affinities for Na, K and Cl were determined in flounder intestine. Measurement of simultaneous NaCl and RbCl influxes resulted in ratios of 2.2 for Cl/Na and 1.8 for Cl/Rb. The effect of Na and Rb on Rb influx showed first order kinetics with K1/2 values of 5 and 4.5 mM and Hill coefficients of 0.9 and 1.2, respectively. The effect of Cl on rubidium influx showed a sigmoidal relationship with K1/2 of 20 mM and a Hill coefficient of 2.0. The effects of variations in Na and Cl concentration on short-circuit current (Isc) were also determined. The K1/2 for Na was 7 mM with a Hill coefficient of 0.9 and the K1/2 for Cl was 46 mM with a Hill coefficient of 1.9. Based on the simultaneous influx measurements, a cotransport stoichiometry of 1Na:1K:2Cl is concluded. The Hill coefficients for Cl suggest a high degree of cooperativity between Cl binding sites. Measurements of the ratio of net Na and Cl transepithelial fluxes under short-circuit conditions (using a low Na Ringer solution to minimize the passive Na flux) indicate that the Cl/Na flux ratio is approximately 2:1. Therefore, Na recycling from serosa to mucosa does not significantly contribute to the Isc. Addition of serosal ouabain (100 microM) inhibited Rb influx, indicating that Na-K-Cl cotransport is inhibited by ouabain. This finding suggests that a feedback mechanism exists between the Na-K-ATPase on the basolateral membrane and the apical Na-K-2Cl cotransporter.