Intracellular Fluorescent Dye Research Articles

Low Incidence of Acute Rejection after Living-Donor Liver Transplantation: Immunologic Analyses by Mixed Lymphocyte Reaction using a Carboxyfluorescein Diacetate Succinimidyl Ester Labeling Technique

To monitor antidonor alloreactivity for accurate diagnosis of acute rejection after living-donor liver transplantation (LDLT), we used a mixed lymphocyte reaction (MLR) assay using an intracellular fluorescent dye carboxyfluorescein diacetate succimidyl ester (CFSE)-labeling technique (CFSE-MLR) in 29 consecutive patients who underwent adult-to-adult LDLT. For patients who developed moderate or severe disorders in liver function, CFSE-MLR was performed together with needle biopsy of the liver allografts immediately after liver dysfunction had occurred. CFSE-labeled peripheral blood mononuclear cells (PBMC) from recipients and irradiated autologous, donor, or third-party PBMC were cultured, and then proliferation and CD25 expression in each of the CD4+ and CD8+ T cell subsets were analyzed by flow cytometry. Twelve (41.4%) of the 29 patients developed moderate or severe disorders in liver function within 6 months after LDLT. Eight of the 12 patients (overall incidence of 27.6%) suffering from liver function disorder were diagnosed on the basis of liver biopsy results as having mild or moderate acute rejection. However, only 4 of the 12 patients (overall incidence of 13.8%) showed remarkable proliferation of CD8+ T cells in association with CD25 expression on antidonor CFSE-MLR. The other eight patients were eventually diagnosed as having recurrence of original hepatitis, drug-induced hepatotoxicity, or congestion of the anterior segment of the liver allograft by further extensive examinations or in retrospect. The results of CFSE-MLR assays, which could be used for rigorously monitoring rejection, provided evidence of low incidence of acute rejection after LDLT.

Carbonic anhydrase inhibitors that directly inhibit anion transport by the human Cl−/HCO3−exchanger, AE1

Carbonic anhydrases (CA, EC 4.2.1.1.) catalyze reversible hydration of CO2 to HCO3- + H+. Bicarbonate transport proteins, which catalyze the transmembrane movement of membrane-impermeant bicarbonate, function in cooperation with CA. Since CA and bicarbonate transporters share the substrate, bicarbonate, we examined whether novel competitive inhibitors of CA also have direct inhibitory effects on bicarbonate transporters. We expressed the human erythrocyte membrane Cl-/HCO3- exchanger, AE1, in transfected HEK293 cells as a model bicarbonate transporter. AE1 activity was assessed in both Cl-/NO3- exchange assays, which were independent of CA activity, and in Cl-/HCO3- exchange assays. Transport was measured by following changes of intracellular [Cl-] and pH, using the intracellular fluorescent reporter dyes 6-methoxy-N-(3-sulfopropyl)quinolinium and 2',7'-bis-(2-carboxyethyl)-5-(and-6)carboxyfluorescein, respectively. We examined the effect of 16 different carbonic anhydrase inhibitors on AE1 transport activity. Among these 12 were newly-reported compounds; two were clinically used non-steroidal anti-inflammatory drugs (celecoxib and valdecoxib) and two were anti-convulsant drugs (topiramate and zonisamide). Celecoxib and four of the novel compounds significantly inhibited AE1 Cl-/NO3- exchange activity with EC50 values in the range 0.22-2.8 microM. It was evident that bulkier compounds had greater AE1 inhibitory potency. Maximum inhibition using 40 microM of each compound was only 22-53% of AE1 transport activity, possibly because assays were performed in the presence of competing substrate. In Cl-/HCO3- exchange assays, which depend on functional CA to produce transport substrate, 40 microM celecoxib inhibited AE1 by 62+/-4%. We conclude that some carbonic anhydrase inhibitors, including clinically-used celecoxib, will inhibit bicarbonate transport at clinically-significant concentrations.

Effects of fluorescent and nonfluorescent tracing methods on lymphocyte migration in vivo.

The use of fluorescent dyes to monitor in vivo cellular migration and proliferation has greatly expanded, but little is known about their potential influence on cell migration. Adoptive transfer studies of lymphocytes labeled with various dyes were performed, and their in vivo homing was compared with that of coinjected unlabeled control cells. In addition, in vitro migration and binding studies were performed to analyze the various steps of transmigration separately. These data showed that the intracellular fluorescent dyes calcein acetoxymethyl ester, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester, 5-chloromethylfluorescein diacetate, 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester, and fluorescein isothiocyanate affect in vivo homing of especially B lymphocytes to lymphoid organs, without any direct effect on in vitro chemotactic or adhesive activity. The only label that did not affect migration was the extracellular and nonfluorescent molecule biotin, provided that the labeling was performed at room temperature. Interestingly, by using the highly versatile congenic Ly5.1-Ly5.2 system, we also demonstrated intrinsic differences in lymphocyte migration based on allelic differences. Our data showed that fluorescent labeling of lymphocytes has a severe effect on their homing capacity in vivo. Labeling of cells with biotin appeared to be a good alternative for this purpose; however, if direct fluorescence is required, the negative effects on cell migration should be considered.

Vaccination does not exacerbate juvenile idiopathic arthritis disease activity in a cohort of Dutch patients

In 2002, all children in the Netherlands from four to 18 years of age, including the Juvenile Idiopathic Arthritis (JIA) population, were vaccinated with the group-C meningococci conjugate vaccine. The aim of this study is to determine whether the vaccine increases JIA disease activity by monitoring the core set criteria of JIA patients as well as investigates the efficacy of this vaccine in those receiving DMARDs. The core set criteria included Childhood Health Assessment Questionnaire (CHAQ) disability, CHAQ pain, CHAQ well being, number of inflamed joints, range of motion, ESR, CRP, and semi-quantitative physicians global assessment. These were monitored for six months prior to as well as after vaccination for any changes in 86 JIA patients. Blood samples were taken from patients to measure antibody production using ELISA as well as T cell proliferation through intracellular fluorescent dye CFSE. In summary, no significant differences (P>0.05) were found when comparing the core set criteria six months before as well as six months after vaccination. However, there were significant increases of CD4+ T cell proliferation both against the carrier protein of the vaccine (P=0.034) as well as the whole vaccine (P=0.035). Furthermore, anti-Men C IgG antibody production increased significantly (>18µg/ml) irrespective of DMARD therapy. In conclusion, our data suggests that the group-C meningococci conjugated vaccine does not increase JIA disease activity. Moreover, B as well as T lymphocyte activity against group-C meningococci are not significantly diminished in patients undergoing immune suppressive treatments.

Immunosuppressive properties of human amniotic membrane for mixed lymphocyte reaction.

The combination of allograft limbal transplantation (ALT) and amniotic membrane transplantation (AMT) has been applied in the treatment of severe ocular surface diseases. The beneficial effect of this combination has been thought to result from possible immunosuppressive ability of amniotic membrane (AM). However, the mechanisms of any such ability remain unknown. In this study, we investigated whether human AM has the ability to suppress allo-reactive T cell responses in vitro. For mixed lymphocyte reaction (MLR), lymphocytes isolated from lymph nodes of C57BL/6 mice (Mls1b, Vbeta6+) were cultured with irradiated splenocytes from DBA/2 mice (Mls1a, Vbeta6-) with or without human AM. For carboxyfluorescein diacetate succinimidyl ester (CFSE) experiments, responder lymph node cells were labelled with a stable intracellular fluorescent dye and cultured with irradiated stimulator cells. The ratio of responder Vbeta6+ T cells was then determined by FACS analysis, and the division profiles of responder Vbeta6+ T cells were analysed by CFSE content. Furthermore, Th1 and Th2 cytokine synthesis by allo-reactive T cells in MLR culture supernatants was determined by enzyme-linked immunosorbent assay (ELISA). Addition of AM to the MLR culture resulted in the significant inhibition of thymidine incorporation compared with control culture lacking AM. The population of responder CD4+Vbeta6+ T cells was significantly reduced in the AM-treated culture in comparison to control. CFSE analysis revealed less division and lower proliferation of responder CD4+Vbeta6+ T cells in cultures with AM than without. In addition, allo-rective T cell synthesis of both Th1 (IL-2 and IFNgamma) and Th2 (IL-6 and IL-10) type cytokine was significantly decreased in the presence of AM. These results indicate that human AM has the ability to suppress allo-reactive T cells in vitro. This inhibitory effect likely contributes to the success of the ALT-AMT combination.

Use of the Intracellular Fluorescent DyeCFSEto Monitor Lymphocyte Migration and Proliferation

The stable incorporation of the intracellular dye CSFE into lymphocytes is a powerful tool to monitor lymphocyte migration in vivo and to quantitatively analyze cell division both in vivo and in vitro. This unit describes methods for labeling high or low numbers of lymphocytes with CSFE. Protocols are provided to use CSFE-labeled cells in cell transfer studies or as cells to be cultured in vitro. Detailed guidelines for positioning of CSFE-labeled lymphocytes in lymphoid organs or other tissues are included for those wishing to use this approach to study lymphocyte migration.

Effects of glucocorticoids on generation of reactive oxygen species in platelets.

Since prednisolone and dexamethasone are known as potent anti-inflammatory agents, the effects of prednisolone and dexamethasone on production of intracellular reactive oxygen species (ROS) were investigated in human platelets. Platelet ROS were measured using the intracellular fluorescent dye dichlorofluorescein diacetate after activation of protein kinase C by phorbol-12-myristate-13-acetate (PMA) or 1-oleoyl-2-acetyl-sn-glycerol (OAG). NAD(P)H oxidase activity was measured photometrically. PMA and OAG significantly increased ROS in platelets (P<0.001). Prednisolone or dexamethasone concentration-dependently reduced the PMA-induced ROS production. The PMA-induced ROS increase was significantly reduced in the presence of 10 micromol/l prednisolone to 9+/-1% (n=31; P<0.001) or in the presence of 10 micromol/l dexamethasone to 9+/-1% (n=24; P<0.001). The inhibitory effect of prednisolone or dexamethasone could also be observed in the presence of the glucocorticoid receptor inhibitor, mifepristone (RU486). Administration of testosterone or aldosterone did not significantly reduce PMA-induced ROS increase. Prednisolone had no effect on platelet NAD(P)H oxidase activity. The inhibition of oxidative phosphorylation by sodium azide reduced platelets ROS to 8+/-1% (n=35). It is concluded that glucocorticoids, prednisolone and dexamethasone, directly inhibit production of intracellular ROS. This effect may contribute to the anti-inflammatory actions of these agents.

Generation of reactive oxygen species by xanthine derivatives in MDA-MB-231 human breast cancer cells.

Theophylline reduces cell number in MDA-MB-231 cells through mechanisms over and above phosphodiesterase inhibition. In the current study, we used an intracellular fluorescent dye to show that theophylline and, to a much greater extent, 3-isobutyl-1-methylxanthine, evoke the generation of reactive oxygen species and also sensitize the cells to insult by other oxidants. Xanthine derivatives may therefore offer novel strategies for antitumor therapeutics.

Antioxidant effect of estrogen on cytomegalovirus-induced gene expression in coronary artery smooth muscle cells.

Pathogens infecting the arterial wall with resultant inflammation may contribute to atherogenesis. Human coronary artery smooth muscle cells (SMCs) infected with human cytomegalovirus (CMV) demonstrate a rapid increase in reactive oxygen species (ROSs), with activation of genes involved in viral replication and inflammation. Because estrogen appears to have antioxidant properties, we wished to determine whether this hormone attenuates SMC responses to CMV infection. Using confocal microscopy and an intracellular fluorescent dye activated by ROSs, we found that 17beta-estradiol (0.1 to 10 nmol/L) and its stereoisomer 17alpha-estradiol (which has low affinity for the estrogen receptor) dose-dependently inhibited ROS generation in CMV-infected SMCs. These effects were not blocked by the estrogen receptor inhibitor ICI 182,780. 3-Methoxyestrone, which lacks the phenolic hydroxyl group, did not interfere with ROS generation. We found that 17beta-estradiol and 17alpha-estradiol, but not 3-methoxyestrone, prevented binding of nuclear factor (NF)-kappaB to DNA. Furthermore, in SMCs transfected with the reporter constructs 3XkappaB-CAT, MIEP-CAT, or ICAM-CAT, cotransfection with a CMV-IE72 expression plasmid caused promoter and CAT activation. Treatment with 17beta-estradiol and 17alpha-estradiol, but not 3-methoxyestrone, inhibited CAT activity and, in CMV-infected SMCs, prevented IE72 and ICAM-1 protein expression and cytopathic effects. These findings indicate that estrogen molecules with an A-ring hydroxyl group have estrogen receptor-independent anti-CMV effects at physiological concentrations by inhibiting ROS generation, NF-kappaB activation, NF-kappaB-dependent transcription, and viral replication. To the extent that chronic infection of the vascular wall with CMV contributes to atherogenesis, these antioxidant actions of estrogen may be of therapeutic importance.

Interplay between the Efflux Pump and the Outer Membrane Permeability Barrier in Fluorescent Dye Accumulation in Pseudomonas aeruginosa

Pseudomonas aeruginosa encodes three types of xenobiotic efflux pumps, MexAB-OprM, MexCD-OprJ, and MexEF-OprN, which are regulated by the nalB, nfxB, and nfxC genes, respectively, and their high expression renders the cells resistant to multiple species of antibiotics. We evaluated the role of the outer membrane permeability barrier and the efflux pump in lowering the intracellular concentration of fluorescent probes. The wild-type, nalB, nfxB, and nfxC strains with an intact outer membrane showed equally high capability in draining out intracellular fluorescent dye, 2-(4-dimethylaminostyryl)-1-ethylpyridinium and ethidium bromide. When the outer membrane barrier was dismantled by the EDTA treatment, wild-type, nfxC, nfxB, and nalB strains showed significantly different levels of dye accumulation. The polymyxin B-treated cells showed an even more pronounced difference in dye accumulation among the nfxC, nfxB, and nalB mutants. We concluded from these results that the xenobiotic extrusion pumps interplay with the outer membrane permeability barrier in lowering the intracellular substrate concentration. Among three extrusion pumps in P. aeruginosa, MexAB-OprM was the most efficient, followed by MexCD-OprJ and MexEF-OprN pumps for the fluorescent dye extrusion.

Functional reconnection of severed mammalian spinal cord axons with polyethylene glycol.

We describe a technique using the water-soluble polymer polyethylene glycol (PEG) to reconnect the two segments of completely transected mammalian spinal axons within minutes. This was accomplished by fusing completely severed strips of isolated guinea pig thoracic white matter maintained in vitro in a double sucrose gap recording chamber. The faces of the severed segments were pressed together, and PEG (MW 1,400-3,500 d; approximately 50% by weight in distilled water) was applied directly to this region through a micropipette and removed by aspiration within 2 min. Successful fusion was documented by the immediate restored conduction of compound action potentials through the original transection and by the variable numbers of fused axons in which anatomical continuity was shown to be restored by high-resolution light microscopy and by the diffusion of intracellular fluorescent dyes through fused axons. These data support the conclusion that some severed and subsequently PEG-fused spinal axons both demonstrate restored anatomical continuity and also are physiologically competent to conduct action potentials. This work adds to our previous demonstration that PEG application can immediately repair severely crushed, rather than cut, spinal cord white matter, and may lead to novel treatments for acute trauma to the central and peripheral nervous systems.

Intracellular alkalinisation in Vero cells parasitised by Trypanosoma cruzi

We studied the intracellular pH of Vero cells parasitised by Trypanosoma cruzi, using different methods: fluorimetric measurement after labelling the cells with the pH-sensitive intracellular fluorescent dye 2′,7′,-bis- (2-carboxyethyl)-5- (and-6)-carboxyfluorescein, acetoxymethyl ester; flow cytometry; and image analysis after staining the cells with neutral-red vital stain. The results show that the intracellular pH of the parasitised cells rose in comparison with that of the uninfected control cells. A study of the population of parasitised cells made by flow cytometry allowed us to subdivide the cells from the infected cultures into two populations according to their pH as obtained by fluorimetric measurements. Image analysis showed that the cell cytoplasm was more alkaline in the vicinity of the sites containing parasites. Treatment of the parasitised cells with amiloride, ouabain, or with 4,4′-diisothiocyano-2,2′-stilbene disulphate consistently lowered the pH values of the parasitised cells, but not sufficiently to return to the values of the non-parasitised control cells. When the control cells were subject to similar treatments with the inhibitors, only amiloride acidified the cytoplasm to any extent. The basification undergone by the parasitised cells was independent of the transport systems and may be a consequence of the release of NH + 4 by the intracellular amastigotes. © 1998 Australian Society for Parasitology.

Measurement ofCandida-specific blastogenesis: Comparison of carboxyfluorescein succinimidyl ester labelling of T cells, thymidine incorporation, and CD69 expression

Measurement of the T cell blastogenic response to Candida may be useful in the evaluation of patients with suspected immunodeficiency. The classic blastogenesis assay is based on uptake of [3H]thymidine by peripheral blood lymphocytes stimulated with Candida antigens for 5 days. An alternative approach involves staining peripheral blood lymphocytes with the intracellular fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) and measuring mitotic activity by the successive twofold reductions in fluorescent intensity using flow cytometry (FCM). The two approaches were compared in 16 subjects who demonstrated various proliferative responses to Candida. FCM-derived indices all involved initial gating on CD3+ T cells and included 1) blastic transformation as measured by changes in light scatter, 2) cell division, measured by CFSE fluorescence, and 3) CD69 expression. A good correlation was found between [3H]thymidine uptake and CFSE-derived indices, irrespective of the analysis algorithm used to interpret CFSE division profiles. Furthermore, significant T cell proliferation occurred only in subjects who had had one or more symptomatic episodes of vaginal candidiasis whereas controls with no such history, and patients with chronic vaginal infection, showed minimal proliferation. The increase in proportion of CD69+ T cells in culture also correlated with the blastogenic response to Candida, but less well than mitotic indices. CFSE-derived indices of T cell blastogenesis to Candida are equivalent to [3H]thymidine-based assays and may allow useful laboratory distinction between subjects who have been exposed to and recovered from vaginal Candida infection, who have a strong proliferative response, from those with no exposure or chronic infection who demonstrate a poor response.

Differential sensitivity to nickel and SK&F96365 of second messengerm operated and receptormoperated calcium channels in rat submandibular ductal cells

The intracellular concentration of calcium ([Ca 2+] i) of rat submandibular ductal cells was measured with the intracellular fluorescent dye Fura-2. Carbachol (100 μM) and ATP (1 mM) both increased the [Ca 2+] j. The late response to ATP was blocked by 0.5 mM Ni 2+. This concentration of Ni 2+ also blocked the increase of the [Ca 2+] i and the uptake of manganese and calcium in response to 2′- and 3′-O-(4-benzoylbenzoyl) adenosine 5′-triphosphate (BzATP, 100 μM), a specific agonist of P2X receptors from salivary glands. The increase of the [Ca 2+] i in response to 2-methylthioadenosine 5′-triphosphate (2-McSATP, 100 μM) a specific P2Y agonist in salivary glands or to a muscarinic agonist (carbachol) was not affected by 0.5 mM Ni 2+. Only higher concentrations of Ni 2+ (in the millimolar range) inhibited the uptake of extracellular calcium in response to carbachol. SK&F 96365, a blocker of store-operated calcium channels, inhibited the uptake of extracellular calcium in response to carbachol without affecting the response to BzATP. It is concluded that at low concentrations (below 0.5 mM), Ni 2+ inhibits the non-specific cation channel coupled to P2X receptors. The uptake of extracellular calcium by store-operated calcium channels is inhibited by higher concentrations of Ni 2+ and by SK&F96365.

Acidosis impairs rabbit trabecular smooth muscle contractility.

The aim of our study was to define the effects of acidosis on the contractility of trabecular smooth muscle. Rabbit corpus cavernosal strips were mounted in organ chambers to measure isometric tension. Additionally, intracellular free Ca2+ concentration ([Ca2+]i) and tension were measured simultaneously utilising the intracellular fluorescent dye, FURA-2, and isometric tension recordings. Contraction of corpus cavernosum smooth muscle following transmural electrical stimulation (TES) of constrictor nerves or exposure to norepinephrine was depressed under acidic (pH 6.9) vs. control (pH 7.4) conditions. Twenty mM K(+)-induced contractions were also inhibited by acidosis, however 40, 80, and 120 mM K+ contractions were unaffected. Relaxation responses to acetylcholine and electrical stimulation, in phenylephrine contracted tissues, were unaffected by acidosis. Tissues contracted with 20 mM K+ under control conditions, relaxed approximately 50% when exposed to an acidic environment. This relaxation was blocked by exposing the tissue to 80 mM K+. Acidic conditions inhibited basal tone and [Ca2+]i as well as normal increases in both intracellular free Ca2+ and tension upon exposure to 20 mM K+, while 80 mM K(+)-induced increases in Ca2+ and tension were comparable under both neutral and acidic conditions. Acidosis impairs trabecular smooth muscle contractility. This alteration is probably secondary to the interference of [H+] with the intra and extracellular mechanisms that regulate homeostasis of [Ca2+]i. Since acidosis is an early complication of ischemic priapism, we propose that the reduced contractility of trabecular smooth muscle may be a significant factor in the perpetuation of the ischemic state.

Acidosis Impairs Rabbit Trabecular Smooth Muscle Contractility

The aim of our study was to define the effects of acidosis on the contractility of trabecular smooth muscle.Rabbit corpus cavernosal strips were mounted in organ chambers to measure isometric tension. Additionally, intracellular free Ca2+ concentration ([Ca2+]i) and tension were measured simultaneously utilising the intracellular fluorescent dye, FURA-2, and isometric tension recordings.Contraction of corpus cavernosum smooth muscle following transmural electrical stimulation (TES) of constrictor nerves or exposure to norepinephrine was depressed under acidic (pH 6.9) vs. control (pH 7.4) conditions. Twenty mM K(+)-induced contractions were also inhibited by acidosis, however 40, 80, and 120 mM K+ contractions were unaffected. Relaxation responses to acetylcholine and electrical stimulation, in phenylephrine contracted tissues, were unaffected by acidosis. Tissues contracted with 20 mM K+ under control conditions, relaxed approximately 50% when exposed to an acidic environment. This relaxation was blocked by exposing the tissue to 80 mM K+. Acidic conditions inhibited basal tone and [Ca2+]i as well as normal increases in both intracellular free Ca2+ and tension upon exposure to 20 mM K+, while 80 mM K(+)-induced increases in Ca2+ and tension were comparable under both neutral and acidic conditions.Acidosis impairs trabecular smooth muscle contractility. This alteration is probably secondary to the interference of [H+] with the intra and extracellular mechanisms that regulate homeostasis of [Ca2+]i. Since acidosis is an early complication of ischemic priapism, we propose that the reduced contractility of trabecular smooth muscle may be a significant factor in the perpetuation of the ischemic state.

Intracellular calcium signaling by Jurkat T-lymphocytes exposed to a 60 Hz magnetic field.

To explore possible biochemical mechanisms whereby electromagnetic fields of around 0.1 mT might affect immune cells or developing cancer cells, we studied intracellular calcium signaling in the model system Jurkat E6-1 human T-leukemia cells during and following exposure to a 60 Hz magnetic field. Cells were labeled with the intracellular calcium-sensitive fluorescent dye Fluo-3, stimulated with a monoclonal antibody against the cell surface structure CD3 (associated with ligand-stimulated T-cell activation), and analyzed on a FACScan flow-cytometer for increases in intensity of emissions in the range of 515-545 nm. Cells were exposed during or before calcium signal-stimulation to 0.15 mTrms 60 Hz magnetic field. The total DC magnetic field of 78.2 microT was aligned 17.5 degrees off the vertical axis. Experiments used both cells cultured at optimal conditions at 37 degrees C and cells grown under suboptimal conditions of 24 degrees C, lowered external calcium, or lowered anti-CD3 concentration. These experiments demonstrate that intracellular signaling in Jurkat E6-1 was not affected by a 60 Hz magnetic field when culture and calcium signal-stimulation were optimal or suboptimal. These results do not exclude field-induced calcium-related effects further down the calcium signaling pathway, such as on calmodulin or other calcium-sensitive enzymes.

New methods for maintaining human renal epithelial cells and analyzing their ion transport functions: potential analysis of genetic disease.

New methods are available to immortalize parenchymal cells from exocrine glands and kidney with retention of differentiation. Adaptation of this technology to small, single-donor biopsy material or surgical specimens could provide genetically homogeneous cells for functional analyses and correlation with genetic background and underlying biochemistry. To develop a methodology useful for renal sodium metabolism, epithelial cell line generation was tested in a hypertensive rat model with features similar to salt-sensitive hypertension in humans. This form of hypertension has a large genetic component and is prevalent in African Americans. Protocols were designed to immortalize primary cultures of microdissected proximal tubule epithelial cells from spontaneously hypertensive (SHR) and control, normotensive Wistar-Kyoto (WKY) rats. Immortalization was based on a replication-defective retrovirus coding for SV40 large T-antigen as positive cell cycle regulator. Transport competent cells that grow on porous filters to form confluent monolayers were selected. Several proximal tubule cell lines have been developed from SHR and WKY rats. The cells retain important differentiated features, such as epithelial polarity, low monolayer conductance, and sodium-succinate cotransport. They are suitable for analyses of electrolyte transport by electrophysiology or imaging of intracellular fluorescent indicator dyes, such as sodium-binding benzofuran isophthalate. Feasibility of generating epithelial cell lines from defined renal segments was demonstrated. The cells retain important transport function so that analyses of sodium metabolism and the influence of genetic background on it are possible. The methodology is applicable to human specimens.

Electrophysiological properties of neurones with CGRP‐like immunoreactivity in rat dorsal root ganglia

Intracellular voltage recordings and fluorescent dye injections were made in vitro in 107 neurons in lumbar dorsal root ganglia (DRGs) of 6- to 8-week-old rats. Calcitonin gene-related, peptide-like immunoreactivity (CGRP-LI) was examined in these neurones, which were divided into C-, Aδ-, and Aα/β-fibre neurones on the basis of their conduction velocities (CVs). A-fibre neurones with CGRP-LI had significantly longer mean action potential (AP) and afterhyperpolarisation (AHP) durations than those without CGRP-LI. Aδ neurones with CGRP-LI had significantly longer AHP durations, slower CVs, and slower maximal fibre following frequencies than those without CGRP-LI. They also had longer AP durations (not significant). The largest Aδ neurones were CGRP-LI negative, whereas the smaller cells were either positive or negative. Aα/β neurones with CGRP-LI also had longer mean APs (not significant) and AHPs (significant) than those without CGRP-LI, and the cell size distributions were similar for positive and negative neurones. Most A-fibre neurones with CGRP-LI had inflections on the falling phase of the somatic AP. Of the A-fibre neurones with such inflections (Ai neurones), those with CGRP-LI had longer AP durations (not significant) and longer AHP durations (significant) than Ai neurones without CGRP-LI, pointing to a functionally distinct subgroup of Ai neurones. There were no significant differences in electrophysiological properties or cell size measurements between C-fibre neurones with and without detectable CGRP-LI. The patterns of electrophysiological properties of Aδ neuronal somata with CGRP-LI and of most, but not all, Aα/β neuronal somata with CGRP-LI are similar to those reported for cutaneous nociceptors with A fibres in rat (Ritter and Mendell [1992] J. Neurophysiol. 68:2033–2041). Because rat DRG neurones that express CGRP normally also express trkA (Averill et al. [1995] Eur. J. Neurosci. 7:1484–1494), the properties described here of neurones with CGRP-LI are probably the same as those of DRG neurones with trkA. © 1996 Wiley-Liss, Inc.

Electrophysiological properties of neurones with CGRP-like immunoreactivity in rat dorsal root ganglia.

Intracellular voltage recordings and fluorescent dye injections were made in vitro in 107 neurons in lumbar dorsal root ganglia (DRGs) of 6- to 8-week-old rats. Calcitonin gene-related, peptide-like immunoreactivity (CGRP-LI) was examined in these neurones, which were divided into C-, A delta-, and A alpha/beta-fibre neurones on the basis of their conduction velocities (CVs). A-fibre neurones with CGRP-LI had significantly longer mean action potential (AP) and afterhyperpolarisation (AHP) durations than those without CGRP-LI. A delta neurones with CGRP-LI had significantly longer AHP durations, slower CVs and slower maximal fibre following frequencies than those without CGRP-LI. They also had longer AP durations (not significant). The largest A delta neurones were CGRP-LI negative, whereas the smaller cells were either positive or negative. A alpha/beta neurones with CGRP-LI also had longer mean APs (not significant) and AHPs (significant) than those without CGRP-LI, and the cell size distributions were similar for positive and negative neurones. Most A-fibre neurones with CGRP-LI had inflections on the falling phase of the somatic AP. Of the A-fibre neurones with such inflections (Ai neurones), those with CGRP-LI had longer AP durations (not significant) and longer AHP durations (significant) than Ai neurones without CGRP-LI, pointing to a functionally distinct subgroup of Ai neurones. There were no significant differences in electrophysiological properties or cell size measurements between C-fibre neurones with and without detectable CGRP-LI. The patterns of electrophysiological properties of A delta neuronal somata with CGRP-LI and of most, but not all, A alpha/beta neuronal somata with CGRP-LI are similar to those reported for cutaneous nociceptors with A fibres in rat (Ritter and Mendell [1992] J. Neurophysiol. 68:2033-2041). Because rat DRG neurones that express CGRP normally also express trkA (Averill et al. [1995] Eur. J. Neurosci. 7:1484-1494), the properties described here of neurones with CGRP-LI are probably the same as those of DRG neurones with trkA.