Effects of cystic fibrosis serum on the rat parotid gland

Summary Injections of serum from human patients with cystic fibrosis into adult rats caused pronounced structural modifications and increased mitotic rate in the parotid gland. Mitotic rate was increased from a low level of 0.02/1,000 acinar cells in parotid glands of adult rats to 6.5/1,000 acinar cells after 2 or 3 days of serum injection. At the light and electron microscopic levels, significant acinar cell atrophy and degranulation were observed. Cellular necrosis, and increases in quantity of lysosome-like dense bodies, mast cells, and macrophages were also detected. These changes are suggestive of tissue response to injurious foreign protein. Furthermore, the fact that normal sera pronounced the same kind of effects (but greatly reduced in extent) strengthens the view that these effects result from the immunologic response of the host organ to foreign antigen. Since, however, the responses of the rat parotid to cystic fibrosis serum were considerably more marked than those elicited by normal serum, the rat parotid may thus have potential usefulness in assaying for the presence of human cystic fibrosis factor.This work was supported in part by U.S.P.H.S. Grant DE 02110The authors wish to thank Dr. Alexander Spock, Cystic Fibrosis Center, Duke University Medical Center, Durham, North Carolina, and Dr. Ralph Tiller, Children's Hospital, University of Alabama Medical Center, for generously supplying blood from patients with cystic fibrosis. The authors also want to thank Dr. A. Siegel, Department of Pathology, University of Alabama Medical Center, and Mr. R. Siegel, for determinations of serum catecholamine levels     

Morphogenesis and secretory activity of mouse mammary cultures on EHS biomatrix

Summary The nature of the substratum profoundly influences the growth and function of epithelia in tissue culture. Mammospheres, hollow spherical structures, develop when epithelial clusters are plated on a biomatrix derived from the Engelbreth-Holm-Swarm murine tumour (EHS matrix). Morphologic examination of mammosphere development demonstrates that morphogenesis is a two stage process. Over the first 48 h the cells aggregate into spheres, drawing the matrix up and over themselves to become buried within the material. Changes in matrix morphology emphasize the importance of the quasi-fluid nature of the substratum on which the cells are plated. Lumen formation ensues over the next 2 to 5 days as the cells, polarized by basal contact with the matrix, differentiate. They form tight junctions at their apical borders and synthesize milk proteins, secreting caseins into the enlarging interior cavity and transferrin from their basal surfaces into the medium. These experiments demonstrate that the physical properties of the EHS matrix allow epithelial cells to develop the cuboidal shape necessary for secretory activity.Abbreviations EHS Engelbreth-Holm-Swarm biomatrix     

99TcmO4 as an indicator for specialized cell function in organ cultures of the human submaxillary gland

Summary Salivary gland tissue was cultured in vitro and viable cells were present during 14 days. The specific glandular cell function was estimated from the capacity to accumulate ⁹⁹Tc^mO₄ from the culture medium. The morphology of the cultured specimens was examined by light and electron microscopy. Structural changes and loss of the ⁹⁹Tc^mO₄ accumulation capacity increased with increasing culture time in vitro. A correlation was observed between the loss of ⁹⁹Tc^mO₄ accumulation capacity and the disappearance of zymogen granules from the cultured cells. However, the causative link between these two phenomena requires further analysis.Supported by grants from Karolinska Institutet     

Synergistic action of vasopressin and aldosterone on basolateral Na+-K+-ATPase in the cortical collecting duct

The respective effects of aldosterone and arginine vasopressin (AVP) were examined on the number of active Na⁺-K⁺-ATPase and their pumping activity in nonperfused microdissected mouse cortical collecting tubules (CCD) by measuring specific ³H-ouabain binding and ouabain-sensitive ⁸⁶Rb uptake. In adrenalectomized (ADX) animals, incubation of CCD with AVP (10^–8 m for 5 min) had no effect on the number of pumps. In contrast, in ADX animals replete with aldosterone, AVP induced a 40% increase in the number of pumps. This was accompanied by a 60–65% increase in ouabain-sensitive Rb uptake. AVP effect was dose-dependent (10^–10–10^–8 m) and was reproduced by dDAVP, forskolin and 8-Br cAMP, indicating a V₂ pathway. It was inhibited by amiloride 10^–5 m, and did not occur in CCD incubated in hyperosmotic solution, suggesting that the signal was transmitted via apical sodium entry and cell swelling. Finally, the AVP-dependent increase in the number of pumps was rapid (within 5 min) and transient (<25 min).These results demonstrate that, in the CCD, aldosterone and AVP act synergistically to increase not only the apical sodium entry but also the basolateral Na⁺-K⁺-ATPase transport capacity: AVP allows a rapid recruitment and/or activation of an aldosterone-dependent pool of latent Na⁺-K⁺-ATPase.     

Effects of 20-HETE on Na+ transport and Na+ -K+ -ATPase activity in the thick ascending loop of Henle

Previous studies have indicated that 20-hydroxyeicosatetraenoic acid (20-HETE) inhibits Na+ transport in the medullary thick ascending loop of Henle (mTALH), but the mechanisms involved remain uncertain. The present study compared the effects of 20-HETE with those of ouabain and furosemide on intracellular Na+ concentration ([Na+]i), Na+ -K+ -ATPase activity, and 86Rb+ uptake, an index of Na+ transport, in mTALH isolated from rats. Ouabain (2 mM) increased, whereas furosemide (100 microM) decreased, [Na+]i in the mTALH of rats. Ouabain and furosemide inhibited 86Rb+ uptake by 91 and 30%, respectively. 20-HETE (1 microM) had a similar effect as ouabain and increased [Na+]i from 19 +/- 1 to 30 +/- 1 mM. 20-HETE reduced Na+ -K+ -ATPase activity by 30% and 86Rb+ uptake by 37%, but it had no effect on 86Rb+ uptake or [Na+]i in the mTALH of rats pretreated with ouabain. 20-HETE inhibited 86Rb+ uptake by 12% and increased [Na+]i by 19 mM in mTALH pretreated with furosemide. These findings indicate that 20-HETE secondarily inhibits Na+ transport in the mTALH of the rat, at least, in part by inhibiting the Na+ -K+ -ATPase activity and raising [Na+]i.     

Insulin and glucagon stimulation of (Na+-K+)-ATPase transport activity in isolated rat hepatocytes

The effects of insulin and glucagon on the (Na+-K+)-ATPase transport activity in freshly isolated rat hepatocytes were investigated by measuring the ouabain-sensitive, active uptake of 86Rb+. The active uptake of 86Rb+ was increased by 18% (p less than 0.05) in the presence of 100 nM insulin, and by 28% (p less than 0.005) in the presence of nM glucagon. These effects were detected as early as 2 min after hepatocyte exposure to either hormone. Half-maximal stimulation was observed with about 0.5 nm insulin and 0.3 nM glucagon. The stimulation of 86Rb+ uptake by insulin occurred in direct proportion to the steady state occupancy of a high affinity receptor by the hormone (the predominant insulin-binding species in hepatocytes at 37 degrees C. For glucagon, half-maximal response was obtained with about 5% of the total receptors occupied by the hormone. Amiloride (a specific inhibitor of Na+ influx) abolished the insulin stimulation of 86Rb+ uptake while inhibiting that of glucagon only partially. Accordingly, insulin was found to rapidly enhance the initial rate of 22Na+ uptake, whereas glucagon had no detectable effect on 22Na+ influx. These results indicate that monovalent cation transport is influenced by insulin and glucagon in isolated rat hepatocytes. In contrast to glucagon, which appears to enhance 86Rb+ influx through the (Na+-K+)-ATPase without affecting Na+ influx, insulin stimulates Na+ entry which in turn may increase the pump activity by increasing the availability of Na+ ions to internal Na+ transport sites of the (Na+-K+)-ATPase.     

The role of glutathione in renal cortical tissue. effects of diamide on Na+ and GSSG levels,amino acid transport and Na+-K+-ATPase activity

Summary The effects of diamide were studied in rat kidney cortical tissue. It was found that diamide increased oxidized glutathione levels and inhibited Na⁺-K⁺-ATPase activity. Consistent with this finding was the observation that diamide compromised the sodium gradients maintained in renal cortical slices. Amino acid transport studies with ouabain or a sodium-free buffer indicated that diamide interferes with both Na⁺-dependent and Na⁺-independent transport systems. These results indicate that diamide has a number of different effects on renal cortical tissue and emphasize the important role of glutathione in maintaining control of a number of key metabolic pathways.     

The secretory cells of the submaxillary gland in the perinatal period of development in the rat

Summary Secretory cell differentiation in the submaxillary gland of the rat was investigated electron microscopically within the period of the 19th–22nd embryonic and the 1st–6th postnatal days.Within this period three types of secretory cells were identified which differed mainly in the morphological appearance of their secretory granules. In addition to the previously described cells of the terminal tubules and the glandular cells of the acini, we observed secretory cells with polymorphic granules, characterized by the presence of small osmiophilic bodies in the matrix of the granules, both after simple and double fixation. The chronological occurrence as well as the quantitative representation of these three types of secretory cells changes in the course of the period investigated. The cells of the terminal tubules are differentiated at the 19th embryonic day; the secretory cells with polymorphic granules by the 21st embryonic day; the glandular cells of the acini do not appear until after birth. It is presumed that the cells of the terminal tubules are precursors of the glandular cells of the acini and that the cells containing polymorphic granules represent an intermediate stage between them.

---

Zusammenfassung Die Differenzierung der Drüsenzellen der Glandula submaxillaris bei Ratten in der Periode vom 19.–22. Tage pränatal und vom 1.–6. Tage postnatal wurde elektronenmikroskopisch studiert.In dieser Periode haben wir in dieser Drüse drei Typen von sezernierenden Zellen festgestellt, welche sich morphologisch hauptsächlich bezüglich verschiedenartiger Sekretionsgranula voneinander unterscheiden. Zu den schon früher beschriebenen Zellen der terminalen Tubuli und den Drüsenzellen der Azini haben wir neu die sezernierenden Zellen mit polymorphen Granula, welche sich durch das Vorhandensein osmiophiler Körperchen in der Matrix der Granula nach einfacher sowohl als auch zweifacher Fixation auszeichnen, angereiht. Das zeitliche Erscheinen und die quantitative Verteilung der erwähnten drei Typen von Sekretionszellen änderten sich während der Beobachtungsperiode dahin, daß die Zellen der terminalen Tubuli am 19. Tage, die Sekretionszellen mit polymorphen Granula am 21. Tage der pränatalen Periode sich differenzieren und die Drüsenzellen der Azini erst nach der Geburt erscheinen.Unserer Ansicht nach sind die Zellen der terminalen Tubuli die Mutterzellen der Drüsenzellen der Azini und die sezernierenden Zellen mit polymorphen Granula das Übergangsstadium zwischen ihnen beiden.

     

The effect of harmaline on unidirectional potassium fluxes and ouabain binding in renal cell cultures

Harmaline inhibits K⁺ influx into primary cell cultures of ground squirrel kidneys to a greater extent than either ouabain or furosemide. A concentration of 200 μM harmaline was required to inhibit half of the total K⁺ influx; this effect was also seen at low temperature (5°C), and in another species (hamster). Although kinetic analysis of K⁺ influx indicates that harmaline does not compete with extracellular K⁺, harmaline did reduce the binding of [³H]ouabain to the cells. K⁺ efflux was also reduced. Therefore, harmaline may inhibit the furosemide-sensitive Na⁺/K⁺ cotransport system as well as the ouabain-sensitive Na⁺/K⁺ pump.     

Rat hypothalamic extract inhibits vasopressin-stimulated Na+-K+-ATPase activity in the rat renal medulla

Arginine vasopressin stimulates Na⁺-K⁺-ATPase activity located in the rat thick ascending limb of s'Henle loop. Mammalian hypothalamus appears to produce a factor capable of inhibiting Na⁺-K⁺-ATPase activity in a variety of tissues. The effect of a purified rat hypothalamic extract with and without AVP on rat renal Na⁺-K⁺-ATPase activity was evaluated by a cytochemical technique. The hypothalamic extract alone failed to affect basal Na⁺-K⁺-ATPase activity throughout renal segments after 10 min exposure. Na⁺-K⁺-ATPase activity stimulated by AVP (1–10 fmol l^?1) for 10 min was inhibited by rat hypothalamic extract over the concentration range 10^?7–10^?3 U ml^?1 in a dose-dependent manner. Complete inhibition of AVP-stimulated Na⁺-K⁺-ATPase activity occurred at a hypothalamic extract concentration of 10^?3 U ml^?1. Only Na⁺-K⁺-ATPase activity located in the renal medullary thick ascending limb was influenced by the rat hypothalamic extract.     

Opposing effects of coupled and uncoupled NOS activity on the Na+-K+ pump in cardiac myocytes

Pharmacological delivery of nitric oxide (NO) stimulates the cardiac Na(+)-K(+) pump. However, effects of NO synthesized by NO synthase (NOS) often differ from the effects of NO delivered pharmacologically. In addition, NOS can become "uncoupled" and preferentially synthesize O(2)(.-), which often has opposing effects to NO. We tested the hypothesis that NOS-synthesized NO stimulates Na(+)-K(+) pump activity, and uncoupling of NOS inhibits it. To image NO, we loaded isolated rabbit cardiac myocytes with 4,5-diaminofluorescein-2 diacetate (DAF-2 DA) and measured fluorescence with confocal microscopy. L-arginine (L-arg; 500 micromol/l) increased DAF-2 DA fluorescence by 51% compared with control (n = 8; P < 0.05). We used the whole cell patch-clamp technique to measure electrogenic Na(+)-K(+) pump current (I(p)). Mean I(p) of 0.35 +/- 0.03 pA/pF (n = 44) was increased to 0.48 +/- 0.03 pA/pF (n = 7, P < 0.05) by 10 micromol/l L-Arg in pipette solutions. This increase was abolished by NOS inhibition with radicicol or by NO-activated guanylyl cyclase inhibition with 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one. We next examined the effect of uncoupling NOS using paraquat. Paraquat (1 mmol/l) induced a 51% increase in the fluorescence intensity of O(2)(.-)-sensitive dye dihydroethidium compared with control (n = 9; P < 0.05). To examine the functional effects of uncoupling, we measured I(p) with 100 micromol/l paraquat included in patch pipette solutions. This decreased I(p) to 0.28 +/- 0.03 pA/pF (n = 12; P < 0.001). The paraquat-induced pump inhibition was abolished by superoxide dismutase (in pipette solutions). We conclude that NOS-mediated NO synthesis stimulates the Na(+)-K(+) pump, whereas uncoupling of NOS causes O(2)(.-)-mediated pump inhibition.     

番茄碱对人红细胞膜Na+-K+-ATPase活性影响的研究

以低渗法从新鲜健康人红细胞中制得膜Na^ -K^ -ATPase,研究了番茄碱(tomatine)X~人红细胞膜Na^ -K^ -ATPase活性的影响。实验结果表明,反应体系中的tomatine浓度低于1mmol／L时,对不依赖钙调蛋白(CAM)激活的Na^ -K^ -ATPase(称之为基本酶活)影响不大,浓度达1mmol／L时,该酶活性仍保持在95％左右;而在此浓度范围内,tomatine对依赖CaM的Na^ -K^ -ATPase有明显的抑制作用,其IC50值为0．16mmol／L．说明tomatine对膜酶Na^ -K^ -ATPase活性的影响可能是通过阻断CaM激活的途径而起作用,从而为进一步研究番茄碱的作用机制奠定了基础。     

Dexamethasone prevents transport inhibition by hypoxia in rat lung and alveolar epithelial cells by stimulating activity and expression of Na+-K+-ATPase and epithelial Na+ channels

Hypoxia inhibits Na and lung fluid reabsorption, which contributes to the formation of pulmonary edema. We tested whether dexamethasone prevents hypoxia-induced inhibition of reabsorption by stimulation of alveolar Na transport. Fluid reabsorption, transport activity, and expression of Na transporters were measured in hypoxia-exposed rats and in primary alveolar type II (ATII) cells. Rats were treated with dexamethasone (DEX; 2 mg/kg) on 3 consecutive days and exposed to 10% O(2) on the 2nd and 3rd day of treatment to measure hypoxia effects on reabsorption of fluid instilled into lungs. ATII cells were treated with DEX (1 muM) for 3 days before exposure to hypoxia (1.5% O(2)). In normoxic rats, DEX induced a twofold increase in alveolar fluid clearance. Hypoxia decreased reabsorption (-30%) by decreasing its amiloride-sensitive component; pretreatment with DEX prevented the hypoxia-induced inhibition. DEX increased short-circuit currents (ISC) of ATII monolayers in normoxia and blunted hypoxic transport inhibition by increasing the capacity of Na(+)-K(+)-ATPase and epithelial Na(+) channels (ENaC) and amiloride-sensitive ISC. DEX slightly increased the mRNA of alpha- and gamma-ENaC in whole rat lung. In ATII cells from DEX-treated rats, mRNA of alpha(1)-Na(+)-K(+)-ATPase and alpha-ENaC increased in normoxia and hypoxia, and gamma-ENaC was increased in normoxia only. DEX stimulated the mRNA expression of alpha(1)-Na(+)-K(+)-ATPase and alpha-, beta-, and gamma-ENaC of A549 cells in normoxia and hypoxia (1.5% O(2)) when DEX treatment was begun before or during hypoxic exposure. These results indicate that DEX prevents inhibition of alveolar reabsorption by hypoxia and stimulates the expression of Na transporters even when it is applied in hypoxia.     

Acetylcholine-Induced Na+ influx in the mouse lacrimal gland acinar cells: Demonstration of multiple Na+ transport mechanisms by intracellular Na+ activity measurements

Summary In the isolated, superfused mouse lacrimal gland, intracellular Na⁺ activities (aNa _i ) of the acinar cells were directly measured with double-barreled Na⁺-selective microelectrodes. In the nonstimulated conditionaNa _i was 6.5±0.5 mM and membrane potential (V _m ) was –38.9±0.4 mV. Addition of 1 mM ouabain or superfusion with a K⁺-free solution slightly depolarized the membrane and caused a gradual increase inaNa _i . Stimulation with acetylcholine (ACh, 1 M) caused a membrane hyperpolarization by about 20 mV and an increase inaNa _i by about 9 mM in 5 min. The presence of amiloride (0.1 mM) reduced the ACh-induced increase inaNa _i by approximately 50%, without affectingV _m and input resistance in both nonstimulated and ACh-stimulated conditions. Acid loading the acinar cells by an addition/withdrawal of 20 mM NH₄Cl or by replacement of Tris⁺-buffer saline solution with HCO ₃ ^– /CO₂-buffered solution increasedaNa _i by a few mM. Superfusion with a Cl^–-free NO ₃ ^– solution or 1 mM furosemide or 0.5 mM bumetanide-containing solution had little effect on the restingaNa _i levels, however, it reduced the ACh-induced increase inaNa _i by about 30%. Elimination of metabolite anions (glutamate, fumarate and pyruvate) from the superfusate reduced both the restingaNa _i and the ACh-induced increase inaNa _i .The present results suggest the presence of multiple Na⁺ entry mechanisms activated by ACh, namely, Na⁺/H⁺ exchange, Na-K-Cl cotransport and organic substrate-coupled Na⁺ transport mechanisms.     

Preliminary studies on rat olfactory tissue: effects of odorants on NA+-K+ atpase activity

Nerve ending particle (B) fractions were prepared from three different rat olfactory endoturbinals. This fraction was tested for Na⁺K⁺ ATPase activity and its response $\text{in vitro}$to low levels of odorants. Quite different differential responses of the enzyme activity were noted between the three tissue preparations for a given odorant and between the three odorants for a given tissue. A possible relationship between enzyme perturbation and initiation of odor sensing mechanism is discussed.     

Moderate-to-severe ischemic conditions increase activity and phosphorylation of the cerebral microvascular endothelial cell Na+-K+-Cl- cotransporter

Brain edema that forms during the early stages of stroke involves increased transport of Na⁺ and Cl^– across an intact blood-brain barrier (BBB). Our previous studies have shown that a luminal BBB Na⁺-K⁺-Cl^– cotransporter is stimulated by conditions present during ischemia and that inhibition of the cotransporter by intravenous bumetanide greatly reduces edema formation in the rat middle cerebral artery occlusion model of stroke. The present study focused on investigating the effects of hypoxia, which develops rapidly in the brain during ischemia, on the activity and expression of the BBB Na⁺-K⁺-Cl^– cotransporter, as well as on Na⁺-K⁺-ATPase activity, cell ATP content, and intracellular volume. Cerebral microvascular endothelial cells (CMECs) were assessed for Na⁺-K⁺-Cl^– cotransporter and Na⁺-K⁺-ATPase activities as bumetanide-sensitive and ouabain-sensitive ⁸⁶Rb influxes, respectively. ATP content was assessed by luciferase assay and intracellular volume by [³H]-3-O-methyl-D-glucose and [¹⁴C]-sucrose equilibration. We found that 30-min exposure of CMECs to hypoxia ranging from 7.5% to 0.5% O₂ (vs. 19% normoxic O₂) significantly increased cotransporter activity as did 7.5% or 2% O₂ for up to 2 h. This was not associated with reduction in Na⁺-K⁺-ATPase activity or ATP content. CMEC intracellular volume increased only after 4 to 5 h of hypoxia. Furthermore, glucose and pyruvate deprivation increased cotransporter activity under both normoxic and hypoxic conditions. Finally, we found that hypoxia increased phosphorylation but not abundance of the cotransporter protein. These findings support the hypothesis that hypoxia stimulation of the BBB Na⁺-K⁺-Cl^– cotransporter contributes to ischemia-induced brain edema formation. edema; blood-brain barrier; bumetanide; cell volume     

The structural unit of the secretory Na+-K+-2Cl- cotransporter (NKCC1) is a homodimer

The oligomeric state of the secretory Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) in rat parotid plasma membranes was studied using the reversible chemical cross-linker DTSSP [3, 3'-dithiobis(sulfosuccinimidyl propionate)]. The monomeric apparent molecular mass of NKCC1 is approximately 170 kDa. However, we show here that this protein migrates as a approximately 355 kDa complex on SDS-PAGE gels after membrane treatment with DTSSP, indicating that NKCC1 exists as an oligomer in the plasma membrane. The stability of this oligomer is such that it is not disrupted by solubilization of the membrane by low concentrations of the nonionic detergent Triton X-100 (0.3%) or the mild ionic detergent deoxycholate (20 mM); however, higher concentrations of Triton X-100 or treatment with the denaturing detergent SDS do result in destabilization of the NKCC1 complex. In additional experiments, we immunoprecipitated the 355 kDa cross-linked complex from biotinylated membranes, then cleaved the cross-linking bonds and analyzed the resulting components of the NKCC1 oligomer by avidin blotting, silver staining, and 2D electrophoresis. In these studies, we were unable to detect the presence of any proteins other than NKCC1 itself in the 355 kDa oligomer, suggesting that this complex is an NKCC1 dimer. Strong evidence for this conclusion was provided by a quantitative analysis of the molecular sizes of oligomers formed by full-length NKCC1 and an N-terminally truncated version of NKCC1 expressed in HEK293 cells. Taken together, our data provide convincing evidence that the dominant structural unit of NKCC1 in the plasma membrane is a homodimer.