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PubMed2006

Different glibenclamide-sensitivity of ATP-sensitive K+ currents using different patch-clamp recording methods.

Electorophysiological and pharmacological properties of the levcromakalim-induced inward ATP-sensitive K+ currents (K(ATP) currents) in pig proximal urethra were investigated by use of two different whole-cell patch-clamp techniques, namely conventional whole-cell and nystatin-perforated patch recordings. In conventional whole-cell configuration, the levcromakalim (100 microM)-induced K(ATP) current decayed by about 30% in 8 min at a holding potential of -50 mV. In contrast, with the nystatin-perforated patch, 96% of the levcromakalim-induced K(ATP) current still remained even after 8 min application of levcromakalim. The peak amplitude of the levcromakalim-induced inward K(ATP) currents in nystatin-perforated patch was approximately half of those observed in conventional whole-cell configuration. When cytosolic extract of pig urethra was included in the pipette solution, approximately 90% of the levcromakalim (100 microM)-induced K(ATP) current remained at 8 min, even after the establishment of conventional whole-cell configuration. In conventional whole-cell configuration, glibenclamide suppressed the levcromakalim-induced K(ATP) currents in a concentration-dependent manner (Ki=175 nM). Inclusion of 1 mM uridine 5'-diphosphate (UDP) in the pipette solution shifted the glibenclamide-sensitivity (Ki=640 nM) to the right in comparison with that in the absence of UDP (i.e., control). In contrast, using nystatin-perforated patch, glibenclamide inhibited the levcromakalim-induced K(ATP) currents with two affinity sites (high-affinity site, Ki1=10 nM; low-affinity site, Ki2=9 microM). The concentration response curves regarding the inhibitory effects of K(ATP) channel pore blockers (Ba2+ and flecainide) on the levcromakalim-induced K(ATP) currents in conventional whole-cell recording nearly overlapped with those in nystatin-perforated patch recording. These results indicate that the glibenclamide-sensitivity of pig urethral K(ATP) channels in nystatin-perforated patch recording was significantly different from that in a conventional whole-cell configuration, and that the glibenclamide-sensitivity may be modified by some cytosolic factor(s).

ATP-Binding Cassette Transporters↗
PubMed1997

Controversies on the sensitivity of the diabetic heart to ischemic injury: the sensitivity of the diabetic heart to ischemic injury is decreased.

Controversy exists as to whether the diabetic heart is more or less sensitive to ischemic injury. Although a considerable number of experimental studies have directly determined the effects of ischemia on the diabetic heart, there is still no general agreement as to whether metabolic changes within the myocardium contribute to the severity of ischemic injury. This paper reviews the evidence suggesting that the diabetic heart can actually be less sensitive to an episode of severe ischemia. Possible reasons for this decreased sensitivity to injury are discussed, which include a decreased accumulation of glycolytic products during ischemia (lactate and protons), as well as alterations in the regulation of intracellular pH in the diabetic heart. Based on existing studies, we suggest that although impaired glucose metabolism in the diabetic heart contributes to injury in hypoxic hearts or in hearts subjected to low-flow ischemia, diabetes-induced decreases in glycolysis can actually be beneficial to the diabetic heart during and following a severe ischemic episode. A decreased clearance of protons via the Na+/H+ exchanger may also contribute to the decreased sensitivity to ischemic injury in the diabetic heart.

Animals↗
PubMed1997

Monoamine oxidase inhibitor sensitive site implicated in sensitization to quinpirole.

Clorgyline (1.0 mg/kg/day) administered via osmotic minipumps blocked the development of locomotor sensitization to the dopamine receptor agonist quinpirole (0.5 mg/kg every 3 days for 8 injections). In male rats already well sensitized to quinpirole, the continuous infusion of clorgyline (1.0 mg/kg/day for 28 days) produced a progressive decline in locomotor activity, despite a continued regimen of quinpirole injections (0.5 mg/kg every 3 days). It is suggested that the development, as well as the maintenance, of locomotor sensitization to quinpirole is modulated by the activation of an monoamine oxidase inhibitor-sensitive site. This site may be a dopamine D2 receptor-linked monoamine oxidase inhibitor-displaceable quinpirole binding site, the enzyme monoamine oxidase-A, or other clorgyline binding sites.

Animals↗
PubMed2001

Oral irritation by sodium chloride: sensitization, self-desensitization, and cross-sensitization to capsaicin.

Psychophysical methods were used to investigate the irritant sensory properties of concentrated NaCl. The first experiment investigated potential sensitization and desensitization properties. Subjects rated the intensity of the irritation elicited by 10 successive applications of 5 M NaCl on one side of the dorsal surface of the tongue. The mean irritant sensation increased significantly across trials, consistent with sensitization. To test for self- and cross-desensitization effects of unilateral sequential stimulation with NaCl followed by a 10-min rest period, either 5 M NaCl or 10 microM capsaicin was applied bilaterally. In a two-alternative forced-choice (2-AFC) test, subjects indicated which side of the tongue had a stronger irritant sensation. They also rated the intensity of irritation on each side separately. When NaCl was applied bilaterally, the side not previously receiving NaCl was chosen as stronger by a significant majority of subjects and was given significantly higher intensity ratings, consistent with self-desensitization. In contrast, when capsaicin was applied bilaterally, the side that had previously received sequential NaCl was perceived as having a significantly more intense irritation, consistent with cross-sensitization. In a second experiment, the effect of amiloride on NaCl-evoked irritation was studied. One side of the tongue was treated with 1 mM amiloride, after which 5 M NaCl was applied bilaterally and subjects performed the same 2-AFC and rating procedures. Since amiloride significantly reduced the intensity of the irritant sensation, the contribution of amiloride-sensitive ionic currents or the Na+/H+ exchange pump (NHE) are suggested as possible transduction mechanisms in lingual nociceptors mediating NaCl-evoked oral irritation.

Adolescent↗
PubMed2000

Tritanopic color matches and the middle- and long-wavelength-sensitive cone spectral sensitivities.

Tritanopic color matches (i.e. matches that depend on the middle- (M) and long- (L), but not short- (S) wavelength-sensitive cones) were made between two half-fields: one illuminated by either a 405 or a 436 nm Hg spectral line; the other by a light of variable wavelength and radiance. Our purpose was to test between rival M- and L-cone spectral sensitivities, which should predict the tritanopic matches. The observers were tritanopes, in whom functioning S-cones are lacking, or normal trichromats, in whom artificial tritanopia was induced by a strong, violet adapting field. The wavelengths found to match the 405 and 436 nm lights agreed poorly with those predicted by the cone spectral sensitivities of Smith and Pokorny (1975) [Vision Research, 15, 161], while the 405 nm matching wavelength agreed poorly with that predicted by Stockman, MacLeod and Johnson (1993) [Journal of the Optical Society of America, A10, 2491]. Both matching wavelengths agreed well, however, with the predictions of the Stockman and Sharpe (2000) [Vision Research] M- and L-cone spectral sensitivities, which lie within the range of measured matches.

Color Perception↗
PubMed2003

Amphetamine-sensitized rats show sugar-induced hyperactivity (cross-sensitization) and sugar hyperphagia.

The goal was to determine the locomotor and consummatory effects of sugar in amphetamine-sensitized rats. Following a 30-min locomotor activity baseline using a photocell cage, male rats were administered either 3.0 mg/kg amphetamine or saline i.p. daily for 6 days. On the final day of injections, locomotor activity was measured again to affirm amphetamine sensitization. Experiment 1: Seven days later, half of each group was offered 10% sucrose or water for 1 min in the home cages, followed by a 30-min locomotor activity test to determine whether or not the animals had become hyperactive in response to sugar. Results showed that amphetamine-sensitized animals were hyperactive following a taste of sugar, but not water. Experiment 2: All subjects were then given access to 10% sucrose for 1 h daily for five consecutive days. Results showed that the amphetamine-sensitized group consumed more sucrose across the 5-day measurement period. These results suggest that sugar may be acting on the same system as amphetamine to trigger hyperactivity, and that alterations in this system caused by repeated doses of amphetamine can instigate an appetite for sugar that persists for at least a week.

Amphetamine↗
PubMed1999

Increased olfactory sensitivity in first episode psychosis and the effect of neuroleptic treatment on olfactory sensitivity in schizophrenia.

Olfactory sensitivity to two odorants, isoamyl acetate and androstenone, was assessed in 19 male schizophrenic patients and 10 control subjects. Tests were performed during a drug-free period and 2-3 weeks after initiation of neuroleptic drug therapy. Olfactory sensitivity in schizophrenic patients was significantly impaired during the drug-free period and neuroleptic treatment further reduced olfactory sensitivity in these patients. The same olfactory tests were administered to 22 first-episode-psychosis patients, 12 first-episode-schizophrenia and 10 brief-psychotic-disorder patients, as well as to 20 age-matched control subjects. The first-episode-psychosis patients had significantly higher sensitivity to isoamyl acetate and to androstenone, but the incidence of anosmia to androstenone was not higher in the first episode patient group as compared to the control group. We conclude that olfactory dysfunction in schizophrenic patients, and possibly other forms of psychosis, is mainly due to long-term effects of commonly used neuroleptic drugs.

Acute Disease↗
PubMedDate not supplied

Use of hexyl isocyanate antigen to detect antibodies to hexamethylene diisocyanate (HDI) in sensitized guinea pigs and in a sensitized worker.

Hypersensitivity to hexamethylene diisocyanate (HDI) has been reported following occupational exposure. Diagnosis of sensitivity is usually made from clinical evaluation of symptomatology. An in vitro serologic assay for HDI sensitivity was developed by immunizing guinea pigs with HDI and with hexyl isocyanate (HMI). Animals injected intradermally with HMI produced hapten-specific antibodies whereas guinea pigs injected with HDI produced antibodies specific for larger determinants which included the HDI hapten. The larger determinants were assumed to be composed of portions of "self" molecules which reacted in vivo with HDI. Serum albumin appeared to be one such molecule. No cross reactions were noted between antibodies to HDI and another widely used industrial isocyanate, toluene diisocyanate (TDI). Antigens effective in detecting antibodies to HDI or HMI were tested for ability to detect reaginic antibodies in a worker with clinical "HDI" asthma. Using a radioimmunoassay (RAST), antibodies reacted with conjugates containing either HDI or HMI as haptens. In addition, the prevalance of HDI polyisocyanates (Desmodur N) in spray paints prompted its use as a hapten. Antibodies reacted with Desmodur N antigen conjugates in RAST. RAST inhibition further indicated that Desmodur N antigen reacted more readily with the patient's antibodies than did HDI or HMI antigens. These results suggest that the patient may have been exposed to HDI polyisocyanates in spray paint application. Use of Rast inhibition for diagnosis of sensitivity may indicate the precise sensitizing agent within a mixture.

Animals↗
PubMed1998

The guinea-pig skin sensitization test revisited: an evaluation formula to predict possible sensitization levels for eight chemicals used in household products.

In predicting human skin sensitization due to possible risky chemicals, it is not sufficient to evaluate solely the minimum induction dose (MID) or the standard challenge dose (SCD) in the Guinea Pig Maximization Test (GPMT). Nakamura et al. (1994) (Nakamura, A., Momma, J., Sekiguchi, H., Noda, T., Yamano, T., Kaniwa, M., Kojima, S., Tsuda, M., Kurokawa, Y., 1994. A new protocol and criteria for quantitative determination of sensitization potencies of chemicals by guinea pig maximization test. Contact Dermatitis 31, 72-85) previously measured the residual dose of chemicals in the products implicated in human allergic accidents, and stated that '... the level of chemical in the products (direct exposure-dose = DED) was similar to or higher than value of sensitization potency.' However, several of the chemicals listed in their article, show an even lower value of sensitization potency than the DED, although a potential correlation between results of the GPMT and the DED was seemed to be evident; a key question about the essential rule of those parameters therefore remains open. Using the data of Nakamura et al. (1994), we analyzed the functional rules of the three independent parameters, the MID, the SCD, and the DED on which the GPMT is based. Calculations of the degree of allergic reactions elicited in humans provided a range of discrimination constants (D) using the formula; D = DED/(MID*SCD). Possible human allergic accidents may be predicted when the dose of a candidate chemical in a chemical product (equal to DED) exceeds the value; D*(MID*SCD), following the correct evaluation of the MID as well as the SCD.

Allergens↗
PubMed2000

Interleukin-1beta reduces temperature sensitivity but elevates thermal thresholds in different populations of warm-sensitive hypothalamic neurons in rat brain slices.

Extracellularly recorded firing rates of neurons in slices of the preoptic area and anterior hypothalamus (PO/AH) of the rat were determined during thermal stimulation. Human recombinant interleukin-1beta (20 ng/ml) did not influence temperature-insensitive neurons, but reduced the firing rate and thermosensitivity in linear warm-sensitive neurons, and shifted the thermal thresholds of activation in threshold warm- and cold-sensitive neurons by 1.1-2.3 degrees C to hyperthermic temperatures. The data support the suggestion that endogenous pyrogens may act on different populations of thermosensitive PO/AH neurons to induce fever. The shift of the thermal thresholds of activation of threshold warm- and cold-sensitive neurons in combination with the otherwise maintained temperature sensitivity of these neurons appears to play a major part for the controlled shift of body temperature and the maintenance of the elevated body temperature during cytokine-induced fever.

Animals↗
PubMed2002

Ondansetron given in the acute withdrawal from a repeated cocaine sensitization dosing regimen reverses the expression of sensitization and inhibits self-administration.

Male Sprague-Dawley rats were given two separate sensitizing regimens of cocaine (7 days on, 7 days off, 7 days on; 40 mg/kg/day s.c.) along with saline controls. Furthermore, animals also received the 5-HT(3) antagonist ondansetron (0.2 mg/kg s.c.) either during the second dosing regimen (3.5 h after each cocaine/saline injection) or during the first five days of the second withdrawal period. Animals were then challenged, on day 10 of withdrawal, with cocaine (7.5 mg/kg i.p.) and assessed by a behavioral rating scale and locomotor activity monitoring. The cocaine regimen induced behavioral and locomotor sensitization on day 10 of withdrawal, further, ondansetron inhibited sensitization regardless of whether given after each second cocaine regimen dose or during the second withdrawal period, although treatment 3.5 h after each cocaine injection appeared more effective. Ondansetron did not inhibit behavior in control animals. In a second experiment animals were trained to self-administer cocaine via an indwelling jugular catheter. After stable fixed-ratio responding (FR1 then FR2) they were given a progressive ratio (PR) schedule until PR each day was stable. During the first five days of withdrawal they were given either ondansetron (0.2 mg/kg s.c.) or saline injections. On day 10 of withdrawal the cocaine PR schedule was reinstated. The ondansetron treated rats showed only a non-significant decrease in break point. After day 2 of the PR session rats were again injected with either ondansetron (0.2 mg/kg s.c.) or saline, 3.5 h after each PR session for five days. Ondansetron inhibited cocaine self-administration on each of the following days. Ondansetron may be a useful treatment for cocaine addicts who have undergone previous sensitization periods.

Acute Disease↗
PubMed1993

Sensitivity to nitrate and other oxyanions further distinguishes the vanadate-sensitive osteoclast proton pump from other vacuolar H(+)-ATPases.

The osteoclast proton pump (OC H(+)-ATPase) differs from other vacuolar H(+)-ATPases (V-ATPases) in its sensitivity to vanadate and in the subunit composition of its catalytic domain, where isoforms of subunits A and B are expressed [Chatterjee et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6257-6261]. In the present study, the sensitivity of the osteoclast H(+)-ATPase to various oxyanions was tested. The results indicate that H+ transport by microsomal preparations isolated from chicken osteoclasts is 20-100-fold more sensitive to nitrate that any other animal and fungal V-ATPases and 10-20-fold more sensitive than plant V-ATPases, as is the ATPase activity of the affinity-purified enzyme. This inhibition by nitrate is not due to a chaotropic effect of the oxyanion and is complete at 1 mM concentrations with an IC50 of 100 microM. In contrast, proton transport by the OC H(+)-ATPase was insensitive to other oxyanions (phosphate, sulfate, and acetate) which inhibit other V-ATPases. These results further demonstrate that the proton pump present in osteoclast membranes differs from other vacuolar ATPases. It is speculated that, since cells of the macrophage lineage can generate high intracellular concentrations of nitrate, it may be possible to physiologically or therapeutically regulate the activity of the OC H(+)-ATPase in the osteoclast without affecting the other V-ATPases in the same or in other cells.

Animals↗
PubMedDate not supplied

Structure-activity relationships for contact allergenic potential of gamma,gamma-dimethyl-gamma-butyrolactone derivatives. 1. Synthesis and electrophilic reactivity studies of alpha-(omega-substituted-alkyl)-gamma,gamma-dimethyl-gamma-butyrolacton es and correlation of skin sensitization potential and cross-sensitization patterns with structure.

A series of alpha-(X-substituted-methyl)-gamma,gamma-dimethyl-gamma-butyrolactones (series 1), a series of alpha-(2-X-substituted-ethyl)-gamma,gamma-dimethyl-gamma-butyrolactones (series 2), where X is a leaving group, and the compound alpha-(3-bromopropyl)-gamma,gamma-butyrolactone (3) were synthesized. Their reactions as electrophiles toward n-butylamine, used as a model for nucleophilic groups on skin proteins whose in vivo chemical modification leads to skin sensitization, were investigated. The compounds of series 1 were shown to react via a two-stage elimination--Michael addition sequence whereby the elements of HX are eliminated to form alpha-methylene-gamma,gamma-dimethyl-gamma-butyrolactone, which reacts more slowly with n-butylamine to give alpha-[(N-butylamino)methyl]-gamma,gamma-dimethyl-gamma-butyrolactone. The compounds of series 2 and compound 3 were shown to react with n-butylamine via a single-stage substitution reaction to give respectively alpha-[2-(N-butylamino)ethyl]- and alpha-[3-(N-butylamino)propyl]-gamma,gamma-dimethyl-gamma-butyrolactones . Rate constants for these reactions have been determined, and it is found that substitution reactions of series 2 and compound 3 are slower than Michael addition of alpha-methylene-gamma,gamma-dimethyl-gamma-butyrolactone, which in turn is slower than the elimination reactions of series 1. The results of guinea pig skin sensitization tests on these compounds were found to be consistent with the above findings in that the compounds of series 1 were found to be in general much stronger sensitizers than those of series 2 and compound 3. The results of cross-challenge tests indicate that sensitizing compounds from series 2 were cross-reactive with both series 1 and compound 3 but that compound 3 is only weakly cross-reactive with series 1. These observations indicated that for these compounds a difference of two carbon atoms between the determinant groups transferred to protein had a markedly greater effect than a difference of one carbon atom on antigenic specificity.

4-Butyrolactone↗
PubMed2000

Two mouse lines selected for differential sensitivities to beta-carboline-induced seizures are also differentially sensitive to various pharmacological effects of other GABA(A) receptor ligands.

Two mouse lines were selectively bred according to their sensitivity (BS line) or resistance (BR line) to seizures induced by a single i.p. injection of methyl beta-carboline-3-carboxylate (beta-CCM), an inverse agonist of the GABA(A) receptor benzodiazepine site. Our aim was to characterize both lines' sensitivities to various physiological effects of other ligands of the GABA(A) receptor. We measured diazepam-induced anxiolysis with the elevated plus-maze test, diazepam-induced sedation by recording the vigilance states, and picrotoxin- and pentylenetetrazol-induced seizures after i.p. injections. Results presented here show that the differential sensitivities of BS and BR lines to beta-CCM can be extended to diazepam, picrotoxin, and pentylenetetrazol, suggesting a genetic selection of a general sensitivity and resistance to several ligands of the GABA(A) receptor.

Animals↗
PubMed1983

Polyamines inhibit phospholipid-sensitive and calmodulin-sensitive Ca2+-dependent protein kinases.

Effects of polyamines on various protein kinases were investigated. It was found that both phospholipid-sensitive Ca2+-dependent protein kinase and myosin light-chain kinase (a calmodulin-sensitive species of Ca2+-dependent protein kinase) were inhibited to different degrees by polyamines, with an approximate order of inhibitory potency of spermine = 1, 12-diaminododecane greater than spermidine = 1, 10-diaminodecane much greater than cadaverine = putrescine. Kinetic analysis revealed that spermine inhibited the phospholipid-sensitive enzyme non-competitively with respect to Ca2+ (Ki = 0.84 mM) and phosphatidylserine (Ki = 0.90 mM); it also inhibited myosin light-chain kinase non-competitively with respect to Ca2+ (Ki = 1.82 mM) and calmodulin (Ki = 2.73 mM). 1, 12-Diaminododecane, in comparison, inhibited the phospholipid-sensitive enzyme competitively with respect to Ca2+ (Ki = 0.45 mM) and phosphatidylserine (Ki = 0.50 mM); it also inhibited myosin light-chain kinase competitively with respect to calmodulin (Ki = 0.63 mM) but non-competitively with respect to Ca2+ (Ki = 1.49 mM). Moreover, spermine (0.5 mM) was found to inhibit markedly phosphatidylserine/Ca2+- and calmodulin/Ca2+-stimulated phosphorylation of endogenous proteins in rat brain particulate fraction. All the polyamines tested were practically without effect on cyclic AMP-dependent and cyclic GMP-dependent protein kinases. Polyarginine, like spermine, was found to be a more selective inhibitor of Ca2+-dependent protein kinases, whereas polyglutamate preferentially inhibited the cyclic nucleotide-dependent enzymes. The present results indicated that, in addition to certain lipophilic compounds (such as trifluoperazine, palmitoylcarnitine, adriamycin and naphthalenesulphonamide) and polypeptides with hydrophobic regions (such as melittin and polymyxin B) previously reported, polycationic compounds (exemplified by polyamines) could also inhibit the two classes of Ca2+-dependent protein kinases requiring either phospholipid or calmodulin as a cofactor. Because of the high cellular concentration (up to 10 mM) and the differential effects of polyamines, it is suggested that spermine, and to smaller extents spermidine and putrescine, may be involved in the regulation of certain Ca2+-dependent protein-phosphorylation systems in vivo.

Animals↗
PubMed1985

Insulin sensitivity and responsiveness of epitrochlearis and soleus muscles from fed and starved rats. Recognition of differential changes in insulin sensitivities of protein synthesis and glucose incorporation into glycogen.

The insulin sensitivity of protein synthesis and glucose incorporation into glycogen by the soleus and epitrochlearis muscles from fed rats and 24 h-starved rats was determined in vitro during the first and second hours of incubation after isolation of the muscles. Rates of protein synthesis by both muscles from fed rats in the first hour of incubation were 2-fold higher than in the second hour and were not increased by insulin. Rates of protein synthesis during the first hour in the presence of 6000 microunits of insulin/ml were increased in soleus, but not in epitrochlearis, muscles from starved rats. Rates of protein synthesis in both muscles from fed and starved rats were increased significantly by insulin during the second hour. High concentrations of insulin caused a marked stimulation of the rates of glucose incorporation by both muscles from fed and starved rats in both the first and second hours of incubation. The insulin sensitivity of glucose incorporation during the second hour, defined as the concentration of insulin causing half-maximal stimulation, was increased 10-fold for both muscle types from starved rats (soleus, 65 microunits/ml; epitrochlearis, 45 microunits/ml) relative to muscles from fed rats (soleus, 600 microunits/ml; epitrochlearis, 500 microunits/m). The insulin sensitivity of protein synthesis in the second hour was greater for soleus muscles from starved rats (65 microunits/ml) than from fed rats (500 microunits/ml). In contrast, the insulin sensitivity of protein synthesis in epitrochlearis muscles from starved rats was significantly decreased (225 microunits/ml) compared with fed rats (25 microunits/ml Maximal rates achieved by high concentrations of insulin were not different from those in the same muscle from fed rats. It is suggested that protein synthesis, in distinction to glucose utilization, may be resistant to insulin stimulation during periods of acute starvation in muscles with fibre compositions similar to the epitrochlearis, but not in muscles with fibre compositions similar to the soleus. Partial reversal of the resistance observed in vitro for epitrochlearis muscles from starved rats may be due to the loss of factors which suppress the effect of insulin in vivo.

Animals↗
PubMed1992

Depletion of the inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ store in vascular endothelial cells activates the agonist-sensitive Ca(2+)-influx pathway.

Previous studies in non-excitable cells have suggested that depletion of internal Ca2+ stores activates Ca2+ influx from the extracellular space via a mechanism that does not require stimulation of phosphoinositide hydrolysis. To test this hypothesis in vascular endothelial cells, the effect of the Ca(2+)-ATPase/pump inhibitor 2,5-di-t-butylhydroquinone (BHQ) on cytosolic free Ca2+ concentration ([Ca2+]i) was examined. BHQ produced a dose-dependent increase in [Ca2+]i, which remained elevated over basal values for several minutes and was substantially inhibited in the absence of extracellular Ca2+. Application of bradykinin after BHQ demonstrated that the BHQ-sensitive compartment partially overlapped the bradykinin-sensitive store. Similar results were obtained with thapsigargin and cyclopiazonic acid, two other Ca(2+)-ATPase inhibitors. Although BHQ had no effect on phosphoinositide hydrolysis, both 45Ca2+ influx and efflux were stimulated by this agent. These results suggest that depletion of the agonist-sensitive Ca2+ store is sufficient for activation of Ca2+ influx. Several characteristics of the Ca(2+)-influx pathway activated by internal store depletion were compared with those of the agonist-activated pathway. Bradykinin-stimulated Ca2+ influx was increased at alkaline extracellular pH (pHo), and was inhibited by extracellular La3+, by depolarization of the membrane, and by the novel Ca(2+)-influx blocker 1-(beta-[3-(4-methoxyphenyl)propoxy]-4- methoxyphenethyl)-1H-imidazole hydrochloride (SKF 96365). Additionally, bradykinin stimulated influx of both 45Ca2+ and 133Ba2+, consistent with the hypothesis that the agonist-activated influx pathway is permeable to both of these bivalent cations. Likewise, activation of Ca2+ influx by BHQ, thapsigargin and cyclopiazonic acid was blocked by La3+, membrane depolarization and SKF 96365, but was unaffected by nitrendipine or BAY K 8644. Furthermore, Ca2+ influx stimulated by BHQ was increased at alkaline pHo and BHQ stimulated the influx of both 45Ca2+ and 133Ba2+ to the same extent. These results demonstrate that the agonist-activated Ca(2+)-influx pathway and the pathway activated by depletion of the agonist-sensitive internal Ca2+ store are indistinguishable.

Animals↗
PubMed2003

Induction of cutaneous delayed hypersensitivity reactions in mice sensitized with intragastrically administered hapten: activation of Langerhans cells in the sensitization and elicitation phases.

BACKGROUND: As seen in atopic dermatitis, allergic diseases often produce lesions both in the gastrointestinal tract and the skin, suggesting the involvement of an immunological relationship between the two organs in the pathogenesis. OBJECTIVES: To study the role of gastric and epidermal Langerhans cells (LCs) in the sensitization and elicitation phases, respectively, of cutaneous delayed-type hypersensitivity (DTH) reactions to intragastrically administered hapten. METHODS: BALB/c mice, which were subjected to intragastric administration of trinitrochlorobenzene 5 days previously, received an elicitative challenge of the same hapten to the ear skin. Sections of the ear were immunostained for CD4 and CD8. Epidermal sheets of the ear and epithelial sheets of the forestomach were immunostained for I-A and observed under a confocal laser scanning microscope. RESULTS: Cutaneous DTH reactions were induced in mice, as demonstrated by an increase in ear thickness and a prominent infiltration of CD4+ and CD8+ lymphocytes at 24-36 h after the elicitative challenge. In the elicitation phase, epidermal LCs showed a significant increase in size, indicating in vivo activation, at 24 h. In the sensitization phase, gastric LCs increased in size at 2 h, became round at 6 h, and decreased in number at 24 h, possibly representing the sequential events of LC activation and migration from the epithelium. CONCLUSIONS: The present study demonstrated that gastric LCs and epidermal LCs were activated in vivo in the sensitization and elicitation phases, respectively, of cutaneous DTH reactions in orally sensitized mice.

Animals↗