Cinnamate uptake by rat small intestine: transport kinetics and transepithelial transfer

Due to their ubiquitous occurrence in the plant kingdom, plant phenolics, including monomeric cinnamic acids, are ingested by man and animals in variable amounts with their natural diets. Recently, Na(+)-dependent saturable transport of cinnamic acid across the brush-border membrane of rat jejunum has been described. It was the aim of the present study to characterize this mechanism in more detail. We therefore determined the transport kinetics of mucosal uptake of radioactively labelled cinnamic acid under various conditions using a short-term mucosal uptake technique. In addition, the transfer of cinnamic acid across the jejunal wall was investigated using everted intestinal sacs. Investigations of the kinetics of cinnamic acid uptake by the mid-jejunal mucosa revealed the involvement of two transport components, a diffusive Na(+)-independent mechanism and a saturable Na(+)-dependent mechanism. The results obtained with everted sacs provided further evidence of the existence of an active Na+ gradient-driven transport of cinnamic acid across the intestinal epithelium. In the presence of Na+, a significant accumulation of cinnamate occurred inside the serosal compartment and this was strongly inhibited by serosal ouabain. A decrease in the extracellular pH stimulated mucosal cinnamate uptake by increasing the apparent affinity (1/km). This may be attributable to the involvement of a transmembrane H+ gradient in Na(+)-dependent cinnamate transport because the protonophore FCCP caused a significant reduction of cinnamate uptake only in the presence of Na+. The kinetics of cinnamate transport in the absence or presence of a surplus of either unlabelled cinnamate or unlabelled butyrate indicates a reduction in the apparent affinity of the Na(+)-dependent mechanism involved in cinnamate uptake. These results may be explained by a modification of the mechanism by the intracellular pH. Additionally, competitive inhibition of cinnamate uptake by substances structurally related to cinnamic acid may also be involved.     

Bidirectional transepithelial water transport: measurement and governing mechanisms

In the search for the mechanisms whereby water is transported across biological membranes, we hypothesized that in the airways, the hydration of the periciliary fluid layer is regulated by luminal-to-basolateral water transport coupled to active transepithelial sodium transport. The luminal-to-basolateral (JWL-->B) and the basolateral-to-luminal (JWB-->L) transepithelial water fluxes across ovine tracheal epithelia were measured simultaneously. The JWL-->B (6.1 microliter/min/cm2) was larger than JWB-->L (4.5 microliter/min/cm2, p < 0.05, n = 30). The corresponding water diffusional permeabilities were PdL-->B = 1.0 x 10(-4) cm/s and PdB-->L = 7.5 x 10(-5) cm/s. The activation energy (Ea) of JWL-->B (11.6 kcal/mol) was larger than the Ea of JWB-->L (6.5 kcal/mol, p < 0.05, n = 5). Acetylstrophanthidin (100 microM basolateral) reduced JWL-->B from 6.1 to 4.4 microliter/min/cm2 (p < 0. 05, n = 5) and abolished the PD. Amiloride (10 microM luminal) reduced JWL-->B from 5.7 to 3.7 microliter/min/cm2 (p < 0.05, n = 5) and reduced PD by 44%. Neither of these agents significantly changed JWB-->L. These data indicate that in tracheal epithelia under homeostatic conditions, JWB-->L was dominated by diffusion (Ea = 4.6 kcal/mol), whereas approximately 30% of JWL-->B was coupled to the active Na+,K+-ATPase pump (Ea = 27 kcal/mol).     

Alternative paths of zinc transepithelial transport in the small intestine

PURPOSE: Neutrophil invasion is a primary event in the development of herpetic keratitis. It has been reported that HSV-1 infection of keratocytes induces the synthesis of IL-8, a potent neutrophil chemoattractant, while corneal epithelium does not. Nevertheless, little is known about the correlation between neutrophil migration and the production of chemotactic factors by HSV-1-infected corneal cells, especially in epithelial cells which form an initial barrier of the ocular surface. We examined whether human corneal epithelial cells as well as keratocytes could induce neutrophil chemotaxis in response to HSV-1 infection. METHODS: Human corneal epithelial cells immortalized with SV40 (HCE) and human keratocytes were infected with HSV-1. The culture fluids collected at 4, 12, 24 h after infection were assayed for human neutrophil chemotaxis using a modified Boyden chamber method. IL-8 levels in these supernatants were measured using enzyme-linked immunosorbent assay (ELISA). RESULTS: The chemotactic activity induced by HCE and keratocytes after MP strain of HSV-1 infection peaked as early as 4 h postinfection, then declined. Chemotactic activity induced by HSV-1-infected HCE and IL-8 levels on these supernatants paralleled with the infectious virus titer. It was inhibited by monoclonal anti-IL-8 antibody. UV-inactivation of MP strain abrogated neither the induction of chemotactic activity nor IL-8 secretion of infected HCE. CONCLUSIONS: At the early phase of HSV-1 infection, corneal epithelial cells play an important role in inducing neutrophil chemotaxis, which was mediated by IL-8.     

A pH-dependent, carrier-mediated system for transport of 5-methyltetrahydrofolate in rat jejunum

The intestinal transport of the naturally occurring folate coenzyme, 5-methyltetrahydrofolate, was studied using everted sacs of rat jejunum. The study provides evidence that intestinal transport of 5-methyltetrahydrofolate is composed of two systems: 1) an active, carrier-mediated system which is demonstrable at low concentrations; and 2) a diffusion system which is demonstrable at high concentrations. The active system is characterized by: 1) saturation kinetics with Km congruent to 0.3 microM; 2) accumulation against a concentration gradient with a serosal-to-mucosal ratio of 1.8; 3) inhibition by metabolic poisons; 4) inhibition by oxidized and reduced folate analogs; 5) temperature dependence; 6) sodium dependence; 7) glucose dependence; and 8) specificity for the jejunum. These features are strongly pH-dependent, and demonstration of active transport of 5-methyltetrahydrofolate requires a buffer pH of 6, glucose in the incubation medium and a substrate concentration of less than 10(-6) M. The diffusion process is characterized by: 1) linear increase in the mucosal-to-serosal transport of 5-methyltetrahydrofolate with increasing mucosal concentration to 10(-6) M and above; 2) energy independence; 3) pH independence; and 4) temperature independence. These studies clarify the mechanism of intestinal transport of 5-methyltetrahydrofolate, show the similarities to transport of other folate compounds and provide a unified concept of intestinal folate transport.     

The absence of stereoselective P-glycoprotein-mediated transport of R/S-verapamil across the rat jejunum

We have studied the potential stereoselective transport and metabolism of R/S-verapamil in rat jejunum, in-situ. A regional single-pass perfusion of the rat jejunum was performed on 24 rats in six separate groups. The effective permeability (Peff) was assessed for three different concentrations of verapamil, 4, 40 and 400 mg L(-1). The Peff of each enantiomer was also determined at 400 mg L(-1) when chlorpromazine (10 mM) was added to the perfusion solution. Two other groups of rats received R/S-verapamil as an intravenous infusion and the intestinal secretion and metabolism were studied by simultaneously perfusing the jejunum with a control or with chlorpromazine (10 mM) added. The concentrations in the outlet perfusate of each enantiomer of verapamil and norverapamil were assayed with HPLC. R/S-Verapamil is a high permeability drug in the proximal rat small intestine throughout the luminal concentration range studied and complete intestinal absorption was expected. There was an increase of Peff from 0.42 x 10(-4) cm s(-1) to 0.80 x 10(-4) cm s(-1) (P < 0.05) at concentrations from 4 to 400 mg L(-1), respectively. The observed concentration-dependent jejunal Peff and fraction absorbed (P < 0.05) of R/S-verapamil is consistent with the saturation of an efflux mechanism. When chlorpromazine (a P-glycoprotein inhibitor/substrate) was added the jejunal Peff increased to 1.47 x 10(-4) cm s(-1). There was no difference between the Peff of the two enantiomers in any of these experiments. The efflux of R/S-norverapamil into the rat jejunum was high after intravenous administration of R/S-verapamil, suggesting extensive metabolism in the enterocyte. In conclusion, both R/S-verapamil enantiomers are P-glycoprotein substrates, but there is no stereoselective transport of R/S-verapamil in the rat jejunum. The results also suggests that R/S-norverapamil is formed inside the enterocytes.     

Contribution of dermal macrophage trafficking in the sensitization phase of contact hypersensitivity

We investigated cellular trafficking of dermal macrophages that express a macrophage calcium-type lectin (MMGL) during the sensitization of delayed-type hypersensitivity. In skin, dermal macrophages, but not epidermal Langerhans cells, have been shown to express MMGL. Epicutaneous sensitization by FITC produced a transient increase in MMGL-positive cells in regional lymph nodes. To directly investigate whether the increase was due to cell migration from dermis, MMGL-positive cells purified from skin were intradermally injected into syngeneic mice after labeling with a fluorescent cell tracer, followed by epicutaneous sensitization over the site of injection. MMGL-positive cells containing the tracer were found in the regional lymph nodes after sensitization. The majority of the MMGL-positive cell migrants were negative for FITC fluorescence despite the presence of FITC-labeled cells that included Langerhans cell migrants. Because the extent of MMGL-positive cell migration was greatly influenced by the selection of vehicles to dissolve FITC, the efficiency of sensitization was compared using the ear swelling test. Migration of both Langerhans cells (FITC-labeled cells) and MMGL-positive cells contributed positively to the efficiency of sensitization. Interestingly, MMGL-positive cell migration was induced by vehicle alone, even in the absence of FITC. These results suggest that migration of dermal MMGL-positive cells accounts for the adjuvant effects of vehicles at least in part.     

Functional characterisation of tachykinin receptors mediating ion transport in porcine jejunum

Reconstructed human skin was prepared from human keratinoblasts. After 1 week of cultivation at the air-liquid interface a stratified layer developed, similar to native human epidermis. Liposomes with an average diameter of 50 nm, made of phosphatidylcholine (PC), phosphatidylserine (PS) and human stratum corneum lipids (hSCL) were applied on top of this culture system. The rate of penetration through the reconstructed human epidermis was 1.38, 0.55 and 0.013 ng lipidh-1cm-2 for PC, hSCL and PS liposomes, respectively. Electron microscopy and confocal laser scanning microscopy showed that PS and hSCL liposomes aggregated at the skin surface, while PC liposomes remained homogeneously dispersed. Fluorescence measurements demonstrated that vesicles, made of native human stratum corneum lipids rapidly mixed with PS liposomes, weakly with hSCL liposomes and did not mix with PC liposomes.     

Catecholamine-induced mechanical sensitization of cutaneous nociceptors in the rat

C-Fiber mechanoheat (C-MH) nociceptors from the saphenous nerve were studied, in control rats and in rats that underwent surgical sympathectomy. Intradermal injection, alone, of either norepinephrine (NE) or the calcium ionophore, A23187, did not affect mechanical threshold. The combination of A23187 and NE, however, significantly decreased mechanical threshold. In the presence of the alpha 2-adrenergic antagonist, yohimbine, or the cyclooxygenase inhibitor, indomethacin, C-MHs were not sensitized by the combination of NE + A23187. One week after surgical sympathectomy, the number of C-MHs sensitized by NE + A23187 was significantly reduced. In summary, NE appears to sensitize nociceptors indirectly. These data are compatible with the suggestion that a sympathetic postganglionic neuron-dependent release of prostaglandins mediates the sensitization. NE appears to act at an alpha 2-adrenergic receptor, only in the presence of an increased intracellular Ca2+.     

Nitric oxide signaling in pain and nociceptor sensitization in the rat

We investigated the role of nitric oxide (NO) in inflammatory hyperalgesia. Coinjection of prostaglandin E2 (PGE2) with the nitric oxide synthase (NOS) inhibitor NG-methyl-L-arginine (L-NMA) inhibited PGE2-induced hyperalgesia. L-NMA was also able to reverse that hyperalgesia. This suggests that NO contributes to the maintenance of, as well as to the induction of, PGE2-induced hyperalgesia. Consistent with the hypothesis that the NO that contributes to PGE2-induced sensitization of primary afferents is generated in the dorsal root ganglion (DRG) neurons themselves, L-NMA also inhibited the PGE2-induced increase in tetrodotoxin-resistant sodium current in patch-clamp electrophysiological studies of small diameter DRG neurons in vitro. Although NO, the product of NOS, often activates guanylyl cyclase, we found that PGE2-induced hyperalgesia was not inhibited by coinjection of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a guanylyl cyclase inhibitor. We then tested whether the effect of NO depended on interaction with the adenylyl cyclase-protein kinase A (PKA) pathway, which is known to mediate PGE2-induced hyperalgesia. L-NMA inhibited hyperalgesia produced by 8-bromo-cAMP (a stable membrane permeable analog of cAMP) or by forskolin (an adenylyl cyclase activator). However, L-NMA did not inhibit hyperalgesia produced by injection of the catalytic subunit of PKA. Therefore, the contribution of NO to PGE2-induced hyperalgesia may occur in the cAMP second messenger pathway at a point before the action of PKA. We next performed experiments to test whether administration of exogenous NO precursor or donor could mimic the hyperalgesic effect of endogenous NO. Intradermal injection of either the NOS substrate L-arginine or the NO donor 3-(4-morphinolinyl)-sydnonimine hydrochloride (SIN-1) produced hyperalgesia. However, this hyperalgesia differed from PGE2-induced hyperalgesia, because it was independent of the cAMP second messenger system and blocked by the guanylyl cyclase inhibitor ODQ. Therefore, although exogenous NO induces hyperalgesia, it acts by a mechanism different from that by which endogenous NO facilitates PGE2-induced hyperalgesia. Consistent with the hypothesis that these mechanisms are distinct, we found that inhibition of PGE2-induced hyperalgesia caused by L-NMA could be reversed by a low dose of the NO donor SIN-1. The following facts suggest that this dose of SIN-1 mimics a permissive effect of basal levels of NO with regard to PGE2-induced hyperalgesia: (1) this dose of SIN-1 does not produce hyperalgesia when administered alone, and (2) the effect was not blocked by ODQ. In conclusion, we have shown that low levels of NO facilitate cAMP-dependent PGE2-induced hyperalgesia, whereas higher levels of NO produce a cGMP-dependent hyperalgesia.     

cAMP mediates transepithelial K+ and Na+ transport in a strial marginal cell line

The recently described gastrointestinal glutathione peroxidase (GI-GPx) is the fourth member of the family of the selenoenzymes glutathione peroxidases (GPx). In contrast to the more uniform distribution of, for example, the classical glutathione peroxidase (cGPx), it is expressed exclusively in the gastrointestinal tract and has, therefore, been suggested to function as a primary barrier against alimentary hydroperoxides. In order to get an idea of its relative importance we investigated its position in the hierarchy of selenoprotein expression. The selenium-dependent expression of GI-GPx was analyzed in comparison with that of other GPx types at the level of mRNA and protein in HepG2 and CaCo-2 cells. Furthermore, the selenocysteine insertion sequence (SECIS) efficiencies of GI-GPx, phospholipid hydroperoxide glutathione peroxidase (PHGPx) and cGPx in response to selenium were determined by a reporter-gene assay in human hepatoma cells and baby hamster kidney cells. GI-GPx mRNA levels increased during selenium deficiency, whereas cGPx mRNA levels decreased and PHGPx mRNA levels remained almost unaffected. In cells grown in selenium-poor media, all GPx-types were low in both activity and immunochemical reactivity. Upon selenium repletion immunoreactive GI-GPx protein reached a plateau after 10 h, whereas cGPx started to be expressed at 24 h and did not reach its maximum level before 3 days. SECIS efficiencies decreased in the order PHGPx > cGPx > GI-GPx. The augmentation of SECIS efficiencies by selenium was highest for cGPx and intermediate for PHGPx, whereas it was marginal for GI-GPx. The high mRNA stability under selenium restriction, the speed of biosynthesis upon selenium repletion and the marginal effect of selenium on the SECIS efficiency indicate that of the GPx isotypes, GI-GPx ranks highest in the hierarchy of selenoproteins and point to a vital role of GI-GPx in the gastrointestinal tract.     

Relations among transepithelial sodium transport, potassium exchange, and cell volume in rabbit ileum

The relation between active transepithelial Na transport across rabbit ileum and 42K exchange from the serosal solution across the basolateral membranes has been explored. Although 42K influx across the basolateral membranes is inhibited by ouabain and by complete depletion of cell Na, it is not affected when transepithelial Na transport is abolished (i.e. in the presence of an Na-free mucosal solution) or stimulated (i.e. when glucose or alanine is added to the mucosal solution). We are unable to detect any relation between the ouabain-sensitive Na-K exchange mechanism responsible for the maintenance of intracellular Na and K concentrations and active transcellular Na transport. In addition, the maintenance of cell volume (water content) does not appear to be dependent upon transepithelial Na transport or the ouabain-sensitive Na-K exchange pump. Although the results of these studies cannot be considered conclusive, they raise serious questions regarding the role of the Na-K exchange pump, located at the basolateral membranes, in active transepithelial Na transport and the maintenance of cell volume.     

Effects of aging on the regulation of intracellular pH in the rat jejunum

Jejunal villus cells from young-adult (6 months) and senescent (24 months) male Wistar rats were studied to evaluate the effect of aging on intracellular pH (pHi) regulation. pHi was measured by quantitative fluorescence microscopy by using BCECF-AM [2',7'-bis(carboxyethyl)-5(6)-carboxy-fluorescein acetoxy methylester] under basal conditions and after inducing cytoplasmic acidification with pulsed NH4Cl. In the senescent rats, the recovery rate from the acidified levels was significantly lower than that in the young-adult rats (.208 +/- .005 vs .255 +/- .004 pH units/min). The relationship between pHi recovery and external Na+ concentration followed Michaelis-Menten type kinetics, the maximum velocity (Vmax) of alkalinization being significantly lower in the senescent rats than in the young-adult rats (.227 +/- .033 vs .297 +/- .024 pH units/min). These results indicate that the recovery of pHi from an acidic level was slower in the senescent rats, due to the reduced activity of Na+/H+ exchange as revealed by the decreased Vmax value.     

Increased sensitization of the myofilaments in rat neonatal portal vein: a potential mechanism

The purpose of this study was to evaluate the signal processing and analysis methods currently in use for the P wave signal-averaged electrocardiogram and to define optimal parameters for its use. P wave signal-averaged electrocardiograms using the QRS as a trigger for alignment of the analysis window were obtained in 15 subjects with prior atrial fibrillation and 15 controls. Five methods of signal filtering (unidirectional, bidirectional, finite impulse response, least squares fit, and spectral fast-Fourier transform) and three filter frequencies (14, 29, and 60 Hz) were compared with logistic regression analysis. Analysis techniques, including P wave vector duration, individual orthogonal lead duration, and terminal root mean square voltage were also evaluated for the strength of their association with the occurrence of atrial fibrillation. The least-squares fit filter with bandwidth filtering of 29 to 250 Hz produced the strongest association with atrial fibrillation (odds ratio 26). A high correlation (r > 0.92) was noted among the individual orthogonal leads; however, neither individual leads nor total atrial activation determined from individual leads demonstrated a superior association with atrial fibrillation when compared with total vector P wave duration. Terminal P wave RMS volt ages were not significantly different between patients with prior AF and controls.     

Differential retention of sensitization and habituation of the startle response in the rat.

Exposed 27 male albino Sprague-Dawley rats to 50 120-db tones at a 30-sec interstimulus interval on 2 successive days for 4 wk. During the 1st session startle amplitude decreased over tones, while in each of the last sessions startle amplitude increased over tones. Startle amplitude at the beginning of each of the last 7 sessions was actually lower than it had been at the end of each previous session. Results indicate that (a) repetitive stimulus exposure produced habituation, which was relatively permanent, and sensitization, which was relatively transient; and (b) by virtue of their different rates of decay and redevelopment, these processes may be independent. (PsycINFO Database Record (c) 2010 APA, all rights reserved)