Chloride-activated water permeability in the frog corneal epithelium

We have previously reported that the isolated frog corneal epithelium (a Cl^–-secreting epithelium) has a large diffusional water permeability (P_dw 1.8×10^–4 cm/s). We now report that the presence of Cl^– in the apical-side bathing solution increases the diffusional water flux, J_dw (in both directions) by 63% from 11.3 to 18.4 l min^–1 · cm^–2 with 60 mm [Cl] exerting the maximum effect. The presence of Cl^– in the basolateral-side bathing solution had no effect on the water flux. In Cl^–-free solutions amphotericin B increased J_dw by 29% but only by 3% in Cl^–-rich apical-side bathing solution, suggesting that in Cl^–-rich apical side bathing solution, the apical barrier is no longer rate limiting. Apical Br^– (75 mm) also increased J_dw by 68%. The effect of Cl^– on J_dw was observed within 1 min after its addition to the apicalside bathing solution. HgCl₂ (0.5 mm) reduced the Cl^–-increased P_dw by 31%. The osmotic permeability (P_f) was also measured under an osmotic gradient yielding values of 0.34 and 2.88 (x 10^–3 cm/s) in Cl^–-free and Cl^–-rich apical-side bathing solutions respectively. It seems that apical Cl^–, or Cl^– secretion into the apical bath could activate normally present but inactive water channels. In the absence of Cl^–, water permeability of the apical membrane seems to be limited to the permeability of the lipid bilayer.This work was supported by National Eye Institute grants EY-00160 and EY-01867.     

Immortalized hypothalamic luteinizing hormone-releasing hormone (LHRH) neurons: A new tool for dissecting the molecular and cellular basis of LHRH physiology

Summary 1. Two LHRH neuronal cell lines were developed by targeted tumorigenesis of LHRH neuronsin vivo. These cell lines (GN and GT-1 cells) represent a homogeneous population of neurons. GT-1 cells have been further subcloned to produce the GT1-1, GT1-3, and GT1-7 cell lines. While considerable information is accumulating about GT-1 cells, very little is currently known about the characteristics and responses of GN cells.2. By both morphological and biochemical criteria, GT-1 cells are clearly neurons. All GT-1 cells immunostain for LHRH and the levels of prohormone, peptide intermediates, and LHRH in the cells and medium are relatively high.3. GT-1 cells biosynthesize, process, and secrete LHRH. Processing of pro-LHRH appears to be very similar to that reported for LHRH neuronsin vivo. At least four enzymes may be involved in processing the prohormone to LHRH.4. LHRH neurons are unique among the neurons of the central nervous system because they arise from the olfactory placode and grow back into the preoptic-anterior hypothalamic region of the brain. Once these neurons reach this location, they send their axons to the median eminence. With respect to the immortalized neurons, GN cells were arrested during their transit to the brain. In contrast, GT-1 cells were able to migrate to the preoptic-anterior hypothalamic region but were unable correctly to target their axons to the median eminence. These problems in migration and targeting appear to be due to expression of the simian virus T-antigen.5. While GT-1 cells are a homogeneous population of neurons, they are amenable to coculture with other types of cells. Coculture experiments currently under way should help not only to reveal some of the molecular and cellular cues that are important for neuronal migration and axonal targeting, but they should also highlight the nature of the cellular interactions which normally occurin situ.6. GT-1 cells spontaneously secrete LHRH in a pusatile manner. The interpulse interval for LHRH from these cells is almost identical to that reported for release of LH and LHRHin vivo. GT-1 cells are interconnected by both gap junctions and synapses. The coordination and synchronization of secretion from these cells could occur through these interconnections, by feedback from LHRH itself, and/or by several different compounds that are secreted by these cells. One such compound is nitric oxide.7. GT-1 cells have Na⁺, K⁺, Ca²⁺, and Cl^– channels. Polymerase chain reaction experiments coupled with Southern blotting and electrophysiological recordings reveal that GT-1 cells contain at least five types of Ca²⁺ channels. R-type Ca²⁺ channels appear to be the most common type of channel and this channel is activated by phorbol esters in the GT-1 cells.8. LHRH is secreted from GT-1 cells in response to norepinephrine, dopamine, histamine, GABA (GABA-A agonists), glutamate, nitric oxide, neuropeptide Y, endothelin, prostaglandin E₂, and activin A. Phorbol esters are very potent stimulators of LHRH secretion. Inhibition of LHRH release occurs in response to LHRH, GABA (GABA-B agonists), prolactin, and glucocorticoids.9. Compared to secretion studies, far fewer agents have been tested for their effects on gene expression. All of the agents which have been tested so far have been found either to repress LHRH gene expression or to have no effect. The agents which have been reported to repress LHRH steady-state mRNA levels include LHRH, prolactin, glucocorticoids, nitric oxide, and phorbol esters. While forskolin stimulates LHRH secretion, it does not appear to have any effect on LHRH mRNA levels.     

Angiotensin II Receptor Type I-Regulated Anion Secretion in Cystic Fibrosis Pancreatic Duct Cells

The β-adrenergic (cAMP-dependent) regulation of Cl⁻ conductance is defective in cystic fibrosis (CF). The present study explored alternative regulation of anion secretion in CF pancreatic ductal cells (CFPAC-1) by angiotensin II (AII) using the short-circuit current (I _SC ) technique. An increase in I _SC could be induced in CFPAC-1 cells by basolateral or apical application of AII in a concentration-dependent manner (EC₅₀ at 3 μm and 100 nm, respectively). Angiotensin receptor subtypes were identified using specific antagonists, losartan and PD123177, for AT₁ and AT₂ receptors, respectively. It was found that losartan (1 μm) could completely inhibit the AII-induced I _SC , whereas, PD123177 exerted insignificant effect on the I _SC , indicating predominant involvement of AT₁ receptors. The presence of AT₁ receptors in CFPAC-1 cells was also demonstrated by immunohistochemical studies using specific antibodies against AT₁ receptors. Confocal microscopic study demonstrated a rise in intracellular Ca²⁺ upon stimulation by AII indicating a role of intracellular Ca²⁺ in mediating the AII response. Depletion of intracellular but not extracellular pool of Ca²⁺ diminished the AII-induced I _SC . Treatment of the monolayers with a Cl⁻ channel blocker, DIDS, markedly reduced the I _SC , indicating that a large portion of the AII-activated I _SC was Cl⁻-dependent. AII-induced I _SC was also observed in monolayers whose basolateral membranes had been permeabilized by nystatin, suggesting that the I _SC was mediated by apical Cl⁻ channels. Our study indicates an AT₁-mediated Ca²⁺-dependent regulatory mechanism for anion secretion in CF pancreatic duct cells which may be important for the physiology and pathophysiology of the pancreas. Received: 17 June 1996/Revised: 14 November 1996     

Fluid Recirculation in Necturus Intestine and the Effect of Alanine

Previous studies in our laboratory have shown that Na absorption across the porcine endometrium is stimulated by PGF_2α 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 μm) 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 μm 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. Received: 6 February 1997/Revised: 10 July 1997     

Fish Gill Respiratory Cells in Culture: A New Model for Cl−-secreting Epithelia

Primary cultures of sea bass gill cells grown on permeable membranes form a confluent, polarized, functional tight epithelium as characterized by electron microscopy and electrophysiological and ion transport studies. Cultured with normal fetal bovine serum (FBS) and mounted in an Ussing chamber, the epithelium presents a small short-circuit current (I _sc : 1.4 ± 0.3 μA/cm²), a transepithelial voltage (V _t ) of 12.7 ± 2.7 mV (serosal positive) and a high transepithelial resistance (R _t : 12302 ± 2477 Ω× cm²). A higher degree of differentiation and increased ion transport capacities are observed with cells cultured with sea bass serum: numerous, organized microridges characteristic of respiratory cells are present on the apical cell surface and there are increased I _sc (11.9 ± 2.5 μA/cm²) and V _t (25.9 ± 1.7 mV) and reduced R _t (4271 ± 568 Ω× cm²) as compared with FBS-treated cells. Apical amiloride addition (up to 100 μm) had no effect on I _sc . The I _sc , correlated with an active Cl⁻ secretion measured as the difference between ³⁶Cl⁻ unidirectional fluxes, was partly blocked by serosal ouabain, bumetanide, DIDS or apical DPC or NPPB and stimulated by serosal dB-cAMP. It is concluded that the chloride secretion is mediated by a Na⁺/K⁺/2Cl⁻ cotransport and a Cl⁻/HCO₃ ⁻ exchanger both responsible for Cl⁻ entry through the basolateral membrane and by apical cAMP-sensitive Cl⁻ channels. This study gives evidence of a functional, highly differentiated epithelium in cultures composed of fish gill respiratorylike cells, which could provide a useful preparation for studies on ion transport and their regulation. Furthermore, the chloride secretion through these cultures of respiratorylike cells makes it necessary to reconsider the previously accepted sea water model in which the chloride cells are given the unique role of ion transport through fish gills. Received: 12 July 1996/Revised: 5 November 1996     

中华大蟾蜍输卵管促受精因子的实验分析

在孕酮作用厂,切除卵巢的中华大蟾蜍输卵管上皮细胞不仅能够使外源移植的卵母细胞在输卵管中正常运行,还保证卵母细胞正常受精。根据输卵管卷曲部分泌物的ＳＤＳ－ＰＡＧＥ分析,存在依赖孕酮的３３ｋＤ蛋白质。经兔抗３３ｋＤ蛋白抗体处理具输卵管分泌物的卵母细胞,受精率受到显著抑制·提示该蛋白有促受精活性。输卵管分泌物介入受精涉及复杂的多种因子。     

The role of physical cues in the regulation of host recognition and acceptance behavior ofAphidius ervi Haliday (Hymenoptera: Braconidae)

The role of color and shape in the host recognition and acceptance behavior ofAphidius ervi Haliday was studied. A quantitative analysis of the oviposition behavior ofA. ervi was carried out with a computer-aided analysis of 150 video-recorded oviposition sequences on its natural host,Acyrthosiphon pisum (Harris). The importance of visual stimuli was assessed in a choice condition bioassay, observing the behavioral reaction of female parasitoids to various test materials flame-sealed into glass capillaries. Glass beads 2 and 6 mm in diameter and a flat arena were coated with cornicle secretion ofA. pisum, and their acceptance rates by both naive and experienced female parasitoids were assessed under no-choice conditions. In most cases,A. ervi females switched from random searching to attack position when the host was within a range of 1 cm, suggesting that host recognition is regulated in part by cues acting before physical contact. The glass capillary bioassay indicated that visual cues are important factors in the host recognition and acceptance phases. Pea aphid color alone can elicit the oviposition response of naiveA. ervi females, and this response is enhanced when color is combined with aphid shape. The cornicle secretion ofA. pisum stimulated an oviposition response which was stronger in naive females ofA. ervi than in experienced ones and was not significantly affected by the glass bead size or flat surface. These results, along with those from previous studies, suggest that manipulation of the oviposition behavior ofA. ervi is feasible under laboratory conditions.     

Changes in metabolism and hormone trafficking during exposure of endocrine cells to elevated ammonium

Endocrine cell cultures have potential in bioprocessing, for the production of biologically active hormones, and in tissue engineering, for the development of implantable artificial tissues for long-term restoration of endocrine function. To optimize such systems, it is necessary to develop a thorough understanding of how inherently present environmental stresses, such as nutrient depletion and metabolite accumulation, affect the cells. This work focuses on the effects of the metabolite ammonium on indicators of endocrine cell metabolism and on the processing, storage and secretion of regulated secretory proteins. Experiments were conducted on recombinant insulin-producing mouse pituitary AtT-20 cells and mouse insulinoma TC3 cells. Exposure for 24–48 hours to 6 mM of exogenous ammonium resulted in higher rates of glucose consumption by both AtT-20 and TC3 cells, while the formation of additional ammonium generally decreased relative to ammonium-free controls. When TC3 cells were discharged of their intracellular insulin stores, the presence of ammonium during a subsequent recharge completely inhibited addition of new insulin-related peptides to the stores, as we had observed previously for both cell lines. There was a correlation between insulin-related peptides stored in TC3 cells during recharging and the amount that could be released upon secretagogue stimulation. Using a combination of radioimmunoassay and high performance liquid chromatography, we found that intracellular insulin and insulin-related peptides changed in the same fashion. Intracellular mechanisms that may be producing the observed results are discussed.Abbreviations IRP insulin-related peptides - HPLC high performance liquid chromatography - DAMP 3-(2,4-dinitroanilino)-3 amino-N-methyldipropylamine     

Phosphorylation of 3 particulate proteins in rat pancreatic acini in response to vasoactive intestinal peptide (VIP), secretin and cholecystokinin (CCK-8)

Rat pancreatic acini were preincubated with 0.4 mM 32Pi for 45 min at 37 degrees C, then exposed for 15 min to VIP, secretin or CCK-8. The incubation was terminated with a stop solution and a fraction rich in mitochondria and zymogen granules was separated from a microsome-rich fraction by differential centrifugation. After heating in the presence of SDS, beta-mercaptoethanol was added and the pattern of equivalent amounts of 32P-labelled proteins was examined by autoradiography of SDS-PAGE gels. VIP, secretin, and CCK-8 stimulated the phosphorylation of a Mr=33 K microsomal protein and that of two proteins of Mr=21 K and Mr=25 K mostly present in a fraction rich in mitochondria and zymogen granules. Stimulations were dose-dependent, the highest stimulant concentrations tested allowing 2- to 3-fold increases of phosphorylation over basal. When 1 nM CCK-8 was used simultaneously with 1 microM VIP, the cyclic AMP levels attained and the pattern of protein phosphorylation were similar to those obtained with VIP alone, and there was a potentiation of amylase secretion; when a supra-maximal 0.1 microM CCK-8 concentration was added, the VIP-induced elevation in cyclic AMP levels and the phosphorylation of the Mr=21 K and Mr=25 K proteins were partially antagonized, and no potentiation any more of secretion occurred. To conclude the in vitro phosphorylation of three particulate proteins (Mr=33 K, 25 K, and 21 K) was similarly increased in rat pancreatic acini in response to secretin and VIP (acting through cyclic AMP) and to CCK-8 (acting mostly through Ca2+).