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PubMed2004

Determination of resting membrane potential of individual neuroblastoma cells (IMR-32) using a potentiometric dye (TMRM) and confocal microscopy.

The potentiometric dye, Tetramethylrhodamine methyl ester (TMRM) has been extensively used with fluorometry or optical microscopy to evaluate the electric potential across plasma or mitochondrial membranes. We present here a TMRM confocal microscopy-based potential measurement technique. Corrections are introduced to minimize nonspecific dye binding and insensitivity to low background levels. We have used this technique to compare the resting membrane potential of proliferating and differentiated human neuroblastoma cells (IMR-32).

Cell Differentiation↗
PubMed2003

Uptake of chitosan and associated insulin in Caco-2 cell monolayers: a comparison between chitosan molecules and chitosan nanoparticles.

PURPOSE: To evaluate the uptake of chitosan molecules (fCS) and nanoparticles (fNP), and their ability to mediate insulin transport in Caco-2 cell monolayers. METHODS: Cell-associated fCS and fNP were evaluated by fluorometry, trypan blue quenching, and confocal microscopy using FITC-labeled chitosan. Chitosan-mediated transport of FITC-labeled insulin was studied in Caco-2 cell monolayers cultured on permeable inserts. RESULTS: Caco-2 cells showed twofold higher association with fNP than fCS after 2-h incubation with 1 mg/ml samples. fNP uptake was a saturable (Km 1.04 mg/ml; Vmax 74.15 microg/mg/h), concentration- and temperature-dependent process that was inhibited by coadministered chlorpromazine. fCS uptake was temperature dependent, but was less sensitive to concentration and was inhibited by filipin. Postuptake quenching with 100 microg/ml of trypan blue suggests a significant amount of intracellular fNP, although the bulk of fCS was extracellular. Internalized fNP were located by confocal microscopy at 15 microm from the apical membrane, but there was no apparent breaching of the basal membrane. This might explain the failure of the nanoparticles to mediate significant insulin transport across the Caco-2 cell monolayer. CONCLUSIONS: Formulation of chitosan into nanoparticles transforms its extracellular interactions with the Caco-2 cells to one of cellular internalization via clathrin-mediated endocytosis.

Caco-2 Cells↗
PubMed2004

Uptake and cytotoxicity of chitosan molecules and nanoparticles: effects of molecular weight and degree of deacetylation.

PURPOSE: To evaluate the effects of molecular weight (Mw) and degree of deacetylation (DD) on the cellular uptake and in vitro cytotoxicity of chitosan molecules and nanoparticles. METHODS: Chemical depolymerization and reacetylation produced chitosans of Mw 213,000 to 10,000 and DD 88-46%, respectively. Chitosan was labeled with FITC and transformed into nanoparticles by ionotropic gelation. Uptake of chitosan by confluent A549 cells was quantified by fluorometry, and in vitro cytotoxicity was evaluated by the MTT and neutral red uptakeassays. RESULTS: Nanoparticle uptake was a saturable event for all chitosan samples, with the binding affinity and uptake capacity decreasing with decreasing polymer Mw and DD. Uptake fell by 26% when Mw was decreased from 213,000 to 10,000, and by 41% when DD was lowered from 88% to 46%; the uptake data correlated with the zeta potential of the nanoparticles. Uptake of chitosan molecules did not exhibit saturation kinetics and was less dependent on Mw and DD. Postuptake quenching with trypan blue indicated that the cell-associated chitosan nanoparticles were internalized, but not the cell-associated chitosan molecules. Chitosan molecules and nanoparticles exhibited comparable cytotoxicity, yielding similar IC50 and IC20 values when evaluated against the A549 cells. Cytotoxicity of both chitosan entities was attenuated by decreasing polymer DD but was less affected by a lowering in Mw. CONCLUSIONS: Transforming chitosan into nanoparticles modified the mechanism of cellular uptake but did not change the cytotoxicity of the polymer toward A549 cells. Chitosan DD had a greater influence than Mw on the uptake and cytotoxicity of chitosan nanoparticles because of its effect on the zeta potential of the nanoparticles.

Acetylation↗
PubMed2003

In vitro kinetics of allergen- and microbe-induced IL-4 and IFN-gamma mRNA expression in PBMC of pollen-allergic patients.

BACKGROUND: According to a hypothesis allergens induce Th2 responses in allergic patients, and microbes induce Th1 responses. We studied the kinetics of in vitro allergen-, tuberculin (PPD)- and tetanus toxin (TT)-induced IFN-gamma and IL-4 mRNA expression in peripheral blood mononuclear cell (PBMC) cultures of pollen-allergic patients and healthy controls. METHODS: PBMC of 10 birch or timothy pollen-allergic patients and of 13 healthy controls were stimulated in vitro with allergen (birch or timothy), PPD or TT. Pellets and supernatants were collected at 24, 48, 72 and 96 h after stimulation. IFN-gamma and IL-4 production was measured by enzyme linked immunosorbent assay and mRNA expression using RT-PCR and time-resolved fluorometry. RESULTS: Allergen induced IFN-gamma production and mRNA expression in PBMC more in allergic patients than in healthy controls. Also allergen induced IL-4 mRNA expression more in allergic patients than in healthy controls. PPD induced IFN-gamma mRNA expression both in allergic patients and healthy controls, whereas IFN-gamma production was induced only in healthy controls and IL-4 was not induced at all. TT induced IFN-gamma mRNA expression in both groups, IFN-gamma production in allergic patients, and IL-4 mRNA expression in both allergic patients and healthy controls. CONCLUSIONS: In vitro stimulation with allergen induced both IFN-gamma and IL-4 mRNA expression of PBMC in allergic patients. These observations challenge the clearcut division of microbe-specific Th1 and allergen-specific Th2 responses in peripheral blood.

Adult↗
PubMed2002

Melatonin's unique radical scavenging properties - roles of its functional substituents as revealed by a comparison with its structural analogs.

Melatonin's O-methyl and N-acetyl residues are not only the basis of its amphilicity enabling the molecule to enter all organs and all subcellular compartments, but are also decisive for its antioxidant properties. We have compared melatonin's redox chemistry with that of several structural analogs: tryptamine, N-acetyltryptamine, serotonin, N-acetylserotonin, 5-methoxytryptamine, 6-chloromelatonin and 2-iodomelatonin. Scavenging of hydroxyl radicals (*OH) was measured in a scavenger competition assay based on ABTS cation radical (ABTS(*)+) formation. The capability of undergoing single-electron transfer reactions was studied using an ABTS(*)+ reduction assay, reflecting the more general property of scavenging organic cation radicals. Direct scavenging of superoxide anions (O2(*)-), under non-catalyzed conditions, was investigated in a hematoxylin autoxidation assay. Measurements of chemiluminescence were used for studying scavenging of O2(*)- under catalyzed conditions, either by hemin-mediated interaction or by combination with the respective indolyl cation radicals. Light emission was determined in the absence or presence of the *OH scavenger dimethylsulfoxide and the O2(*)- scavenger Tiron. Products formed by oxidation of the respective indoles in a moderately alkaline, hemin-catalyzed H2O2 system were analyzed by thin-layer chromatography and fluorometry. Absence of either the O-methyl or the N-acetyl residue causes marked diminutions in the capacities of scavenging *OH and ABTS(*)+ as well as in chemiluminescence emitted during oxidation. The importance of the N-acetyl group is insofar remarkable as it seems, at first glance, to be isolated from the indolic moiety; interactions between side chain and indolic moiety are therefore decisive for melatonin's redox properties. The 5-hydroxylated compounds are not generally more efficient scavengers, but particularly better reducers of ABTS(*)+; in the alkaline H2O2 system generating *OH and O2(*)-, melatonin was much more rapidly oxidized than the 5-hydroxylated and non-substituted analogs. Oxidative products formed from any of the compounds studied contained much less of substituted kynuramines as in the case of melatonin, indicating that radical chain termination by O2(*)- is considerably more efficient with melatonin. These findings are supported by measurements of chemiluminescence, which largely reflects pyrrole ring cleavage as a result of combination with superoxide anions. In this regard, only 6-chloromelatonin equalled melatonin, whereas the efficiency of 2-iodomelatonin was much lower, another indication for the importance of 2,3-dioxygenation.

5-Methoxytryptamine↗
PubMed2001

Up-converting phosphor reporters for nucleic acid microarrays.

An important application of robotically spotted DNA microarrays is the monitoring of RNA expression levels. A clear limitation of this technology is the relatively large amount of RNA that is required per hybridization as a result of low hybridization efficiency and limiting detection sensitivity provided by conventional fluorescent reporters. We have used a recently introduced luminescent reporter technology, called UPT (up-converting phosphor technology). Down-converting phosphors have been applied before to detect nucleic acids on filters using time-resolved fluorometry. The unique feature of the phosphor particles (size 0.4 microm) used here is that they emit visible light when illuminated with infrared (IR) light (980 nm) as a result of a phenomenon called up-conversion. Because neither support material of microarrays nor biomolecules possess up-conversion properties, an enhanced image contrast is expected when these nonfading phosphor particles are applied to detect nucleic acid hybrids on microarrays. Comparison of the UPT reporter to cyanin 5 (Cy5) in a low-complexity model system showed a two order of maginitude linear relationship between phosphor luminescence and target concentration and resulted in an excellent correlation between the two reporter systems for variable target concentrations (R2 = 0.95). However, UPT proved to be superior in sensitivity, even though a wide-field microscope equipped with a xenon lamp was used. This higher sensitivity was demonstrated by complementary DNA (cDNA) microarray hybridizations using cDNAs for housekeeping genes as probes and complex cDNA as target. These results suggest that a UPT reporter technology in combination with a dedicated IR laser array-scanner holds significant promise for various microarray applications.

Biotinylation↗
PubMed1981

ICRF 159-induced cell-cycle perturbation in vitro: its relationship to inhibition of colony-forming ability.

The effects of single doses of ICRF 159 (Razoxane) on the cell-cycle kinetics of lines of BKH 21S cells in vitro were studied by means of flow micro-fluorometry (FMF). A characteristic accumulation of cells in the 4n DNA region after 6 h in the presence of ICRF 159 was evident in cells which were sensitive to the cytotoxic effects of the agents, as judged by colony-forming assays. The subsequent accumulation of 8n cells as the major population within 24 h indicates that this 6 h effect is associated with the induction of tetraploidy through abnormal mitosis, rather than reflecting a G2 block of potential value in combination therapy. In ICRF 159-resistant sublines, the cell-cycle distributions were similarly affected, but only the doses of the drug high enough to reduce their surviving fraction. Somatic cell hybrids from crosses between sensitive and resistant cells demonstrated intermediate responses in ICRF 159, both in terms of cell-cycle kinetics and impairment of reproductive integrity. These data suggest a relationship between the 2 manifestations of the cellular action of ICRF 159, and also a possible predictive role for FMF in the assessment of response to this particular agent.

Animals↗
PubMed1996

Creatine kinase BB isoenzyme levels in tumour cytosols and survival of breast cancer patients.

Creatinine kinase BB (CK-BB) is elevated in many tumours including those of the breast. We have recently described a new, highly sensitive and specific method for measuring CK-BB, based on monoclonal antibodies and time-resolved fluorometry. Using this method, we quantitated CK-BB in 172 breast tumour cytosols and examined the associations between CK-BB and other clinicopathological variables and patient survival. High CK-BB levels were seen more frequently in tumours from patients who were younger (age < 50 years), patients who qualified for chemotherapy and patients with oestrogen receptor-positive tumours. No association was seen between CK-BB and tumour stage, grade, size, histological type or the progesterone receptor. In univariate analysis, the risk of relapse or death was higher in the group with tumours containing high CK-BB levels but the difference did not reach statistical significance. In multivariate analysis, the risk of death was statistically significantly higher in the high-CK-BB group. Analysis of subsets of patients revealed that patients with oestrogen receptor-negative cancer have higher risk of death if their tumours contain high levels of CK-BB. Our data suggest that, in general, CK-BB is associated with more aggressive tumours but its value as a prognostic indicator is limited. CK-BB content of breast tumours may be more useful as an aid in selecting therapy directed at inhibiting this enzyme activity and thus depriving tumour cells of their energy source.

Adult↗
PubMed1990

Metabolic and hemodynamic activation of postischemic rat brain by cortical spreading depression.

Following transient ischemia of the brain, the coupling between somatosensory activation and the hemodynamic-metabolic response is abolished for a certain period despite the partial recovery of somatosensory evoked responses. To determine whether this disturbance is due to alterations of the stimulus-induced neuronal excitation or to a breakdown of the coupling mechanisms, cortical spreading depression was used as a metabolic stimulus in rats before and after ischemia. Adult rats were subjected to 30 min of global forebrain ischemia and 3-6 h of recirculation. EEG, cortical direct current (DC) potential, and laser-Doppler flow were continuously recorded. Local CBF (LCBF), local CMRglc (LCMRglc), regional tissue contents of ATP, glucose, and lactate, and regional pH were determined by quantitative autoradiography, substrate-induced bioluminescence, and fluorometry. Amplitude and frequency of the DC shifts did not differ between groups. In control animals, spreading depression induced a 77% rise in cortical glucose consumption, a 66% rise in lactate content, and a drop in tissue pH of 0.3 unit. ATP and glucose contents were not depleted. During the passage of DC shifts, transient increases (less than 2 min) in laser-Doppler flow were observed, followed by a post-spreading depression hypoperfusion. A comparable although less expressed pattern of hemodynamic and metabolic changes was observed in the postischemic rats. Although baseline LCMRglc was depressed after ischemia, it was activated 47% during spreading depression. Lactate increased by 26%, pH decreased by 0.3 unit, and ATP and glucose remained unchanged. The extent of the transient increase in laser-Doppler flow did not differ from that of the control group, and a post-spreading depression hypoperfusion was also found.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Cerebral blood flow and oxygen consumption in cortical spreading depression.

We determined the effects of spreading depression on local cerebral O2 supply, oxygenation, and O2 consumption in the anesthetized rat. Spreading depression was induced by application of 0.5 M KCl to the frontal cortex. Regional cerebral blood flow was determined with [14C]iodoantipyrine and regional O2 extraction was determined microspectrophotometrically. The passage of the spreading depression wave was determined with a multiprobe assembly that recorded NADH redox state (surface fluorometry), extracellular K+ activity, and DC steady potential (surface minielectrodes). As the wave of spreading depression passed, there was an increase in extracellular K+ and a decrease in NADH. Cerebral blood flow was significantly increased (120 +/- 51 ml/min/100 g, mean +/- SD) during the wave as compared with other regions. In the affected cortex, blood flow was not different from that in the contralateral cortex (69 +/- 28 ml/min/100 g) either before or after the wave of spreading depression passed. Arterial and venous O2 saturation were unaffected by the wave and the histogram of O2 saturations of examined veins followed a similar normal distribution in all regions. Oxygen extraction was not altered by the wave of spreading depression. Oxygen consumption was significantly increased during the wave to 7.4 +/- 3.7 ml O2/min/100 g compared with the contralateral cortex (5.1 +/- 2.6 ml/min/100 g) and other regions. It can be concluded that spreading depression caused an increase in cerebral O2 consumption that was adequately matched by an increase in local blood flow. Oxygen delivery was not limited during spreading depression and its effects were quickly over as evidenced by the lack of alteration in oxygenation after the wave of spreading depression passed.

Animals↗
PubMed1992

Direct evidence for increased continuous histamine release in the striatum of conscious freely moving rats produced by middle cerebral artery occlusion.

Extracellular histamine in the stratum of conscious freely moving rats collected by intracerebral microdialysis 1 day after implantation of a U-shaped dialysis probe was measured by HPLC coupled with postcolumn o-phthalaldehyde derivatization fluorometry. The basal fractional histamine outputs were almost constant from 1 to 7 h after the start of perfusion (5.9-8.4 pg/30 min). Depolarization by perfusion with a high K+ (100 mM)-containing medium produced a significant (124%) increase and neuronal blockade by perfusion with a tetrodotoxin (1 microM)-containing medium resulted in a 68% reduction in the histamine output. The histamine output was markedly reduced by intraperitoneal injection of alpha-fluoromethylhistidine (100 mg/kg), an irreversible inhibitor of histidine decarboxylase, or (R)-alpha-methylhistamine (5 mg/kg), a potent and specific H3-receptor agonist. After middle cerebral artery (MCA) occlusion, the histamine output gradually increased, and reached four times the control value 8 h later. When rats were pretreated with metoprine (10 mg/kg), a histamine N-methyltransferase inhibitor, there was no significant difference in the histamine output between the MCA-occluded and the sham-operated groups during the first 3.5 h after the operation, but the histamine output gradually increased thereafter in the MCA-occluded group. In rats treated with alpha-fluoromethylhistidine, MCA occlusion failed to cause an increase in the histamine output. These results demonstrate that MCA occlusion induces a long-lasting increase in neuronal histamine release in the rat striatum.

Animals↗
PubMed1990

Identification and characteristics of a Na+/Ca2+ exchanger in cultured human mesangial cells.

Excitable cells express Na+/Ca2+ exchange activity among other mechanisms modulating rapid fluctuations of cytosolic free Ca2+ ([Ca2+]i). We studied functions and regulation of a Na+/Ca2+ exchanger in cultured human glomerular mesangial cells. Fura-2-loaded confluent monolayers reacted to removal of ambient Na+ with an immediate, transient elevation of [Ca2+]i, assessed by single/dual wavelength fluorometry. Peak [Ca2+]i was inversely correlated with the extracellular Na+ concentration. Ca2+ influx was the sole mechanism implicated, as the [Ca2+]i rise was prevented by EGTA. The process was inhibited by 1 mM amiloride, but not by blockers of voltage-operated Ca2+ channels. Re-addition of Na+ resulted in a rapid decrease of [Ca2+]i, indicating bimodal operation of the exchanger. Na(+)-loading the cells with monensin and ouabain enhanced Ca2+ uptake. Prior stimulation of [Ca2+]i with the thromboxane A2 mimetic, U-46619, or angiotensin II also increased Ca2+ uptake upon subsequent Na+ removal, suggesting induction of the exchanger by vasoconstrictors. Moreover, the magnitude of agonist-induced [Ca2+]i transients was amplified by Na+ removal, indicating that the exchanger modulates the effects of vasoconstrictors. These results demonstrate that an inducible Na+/Ca2+ antiporter is operative in resting and stimulated human mesangial cells, further confirming their smooth muscle origin and potential regulatory role on glomerular hemodynamics.

Calcium↗
PubMed1996

Adhesion of U-937 monocytes induces cytotoxic damage and subsequent proliferation of cultured human mesangial cells.

In an in vitro model of monocyte adhesion to glomerular cells, U-937 myelomonocytic leukemia cells irreversibly bind to human mesangial cell monolayers. Adhesion is enhanced in mesangial cells proliferating in response to fetal bovine serum, and in the presence of several cytokines and vasoactive agents. In the present study, co-culture with U-937 followed by removal of non-adherent cells time-dependently decreased viability of mesangial cells, measured either by fluorometry after dual labeling with calcein acetoxymethylester and ethidium homodimer, or by the release of lactate dehydrogenase. The cytotoxic effects of co-culture with U-937 cells were significantly reduced by a combination of free radical scavengers, indicating involvement of reactive oxygen species. U-937 cells also stimulated subsequent proliferation of mesangial cells, assessed by [3H]-TdR incorporation and direct cell counts 24 hours later (from 1,034 +/- 83 to 14,611 +/- 959 and from 2,931 +/- 201 to 19,400 +/- 2,124 cpm/well, quiescent/cycling mesangial cells, respectively, P < 0.01). Controls to rule out TdR incorporation by adherent U-937 cells included selective [3H]-TdR labeling and demecolcine pretreatment. Cell counts at 24 hours confirmed U-937-induced proliferation of quiescent HMC, from 50,575 +/- 3,596 to 143,012 +/- 10,039 cells/cm2 (P < 0.01). Agents that promote U-937 cell adhesion, such as the TxA2 mimetic, U-46619, or angiotensin II, enhanced cytotoxicity while inhibiting the proliferation of both quiescent and cycling mesangial cells, when added during co-culture and the subsequent 24 hours (+1 microM U-46619, 1,875 +/- 131 and 2,546 +/- 125 cpm/well, respectively, 79,793 +/- 5,744 cells/cm2, P < 0.01 vs. U-937 only; +1 microM Ang II, 5066 +/- 560 and 5,784 +/- 306 cpm/well, respectively, 81,068 +/- 4,671 cells/cm2, P < 0.05). Blocking antibodies against the adhesion molecule ICAM-1 and leukocyte counterreceptors (LFA-1, VLA-4) prevented the proliferative response, which could not be duplicated with the conditioned media of U-937 alone or co-cultured with mesangial cells. These findings may reflect the interactions occurring in vivo between infiltrating leukocytes and resident cells during glomerular inflammation.

Animals↗
PubMed1997

Mechanism of contraction induced by bradykinin in the rabbit saphenous vein.

1. By using fura-PE3 fluorometry and receptor-coupled permeabilization by alpha-toxin, the mechanism of the bradykinin (BK)-induced contraction was determined in the rabbit saphenous vein (RSV). The receptor subtype responsible for the BK-induced contraction of RSV was determined by means of a pharmacological blocker study and reverse transcription polymerase chain reaction (RT-PCR). 2. In the presence of extracellular Ca2+ (1.25 mM), BK (10(-11)-3 x 10(-7) M) induced increases in both the cytosolic Ca2+ concentration ([Ca2+]i) and force, in a concentration-dependent manner. Both the release of Ca2+ from the store site and the influx of extracellular Ca2+ contribute to an increase in [Ca2+]i induced by BK. 3. In the absence of extracellular Ca2+, the application of 10(-7) M BK induced transient elevations of [Ca2+]i and force, both of which thereafter declined to the levels observed before the application of BK. When extracellular Ca2+ was replenished (1.25 mM), [Ca2+]i and force increased to form a peak, followed by a sustained elevation in the presence of BK. When an RSV strip was pretreated with 10(-5) M thapsigargin for 20 min, the BK-induced transient increases in both [Ca2+]i and force were markedly inhibited. 4. These responses induced by BK were inhibited by Hoe 140 (D-Arg-[Hyp3, Thi5, D-Tic7, Oic8] bradykinin), a highly specific bradykinin B2 receptor antagonist, in a concentration-dependent manner. In RT-PCR, B2-receptor mRNA was expressed in the smooth muscle of RSV. 5. The [Ca2+]i-force relationships, which were determined by cumulative applications of extracellular Ca2+ (0-5 mM) during 118 mM K(+)-depolarization, shifted to the upper left in the presence of BK, thus indicating that BK induced a greater force than 118 mM K(+)-depolarization for a given level of [Ca2+]i. 6. In alpha-toxin-permeabilized preparations of RSV, application of 10(-7) M BK after a steady state contraction had been induced by a mixture of 5 x 10(-7) M Ca2+, 10(-6) M GTP and 10(-6) M captopril caused an additional force development at a constant [Ca2+]i. However, treatment with 1 mM guanosine-5'-O-(beta-thiodiphosphate) (GDP beta S) for 5 min before and during the application of BK (10(-7) M), abolished this BK-induced additional contraction. 7. These results indicated that in RSV: (1) BK elicits vasoconstriction by increasing the Ca2+ influx from the extracellular space, Ca2+ release from intracellular thapsigargin-sensitive storage sites and increasing the Ca2+ sensitivity of the contractile apparatus, (2) the BK-induced increase in Ca2+ sensitivity is mediated by G-protein, (3) the BK-induced contractions are mediated via B2-receptors and (4) the smooth muscle cells express B2-receptor mRNA.

Animals↗
PubMed1997

The mechanisms of the relaxation induced by vasoactive intestinal peptide in the porcine coronary artery.

1. This study was designed to investigate the mechanism of the relaxation induced by vasoactive intestinal peptide (VIP) in medial strips of the porcine coronary artery, by determining the effect on the cytosolic Ca2+ concentration ([Ca2+]i), the [Ca2+]i-force relation and the involvement of G-protein. 2. Front-surface fluorometry of fura-2 revealed that U46619, a thromboxane A2 analogue, and the high K(+)-depolarization induced increases in both the [Ca2+]i and force of the medial strips. At a steady state of contraction, the extent of an increase in [Ca2+]i induced by 100 nM U46619 was similar to that induced by 30 mM K(+)-depolarization. VIP concentration-dependently (1 nM-1 microM) induced transient decreases in both the [Ca2+]i and force of the medial strips precontracted with 100 nM U46619. The decreases in the [Ca2+]i and force induced by VIP during the contraction with U46619 were much greater than those with 30 mM K(+)-depolarization. 3. The VIP-induced decreases in the [Ca2+]i and force were attenuated by K+ channel blockers such as tetrabutylammonium (TBA: non-selective K+ channel blocker), charybdotoxin (large conductance Ca(2+)-activated K+ channel blocker), and 4-aminopyridine (4-AP: voltage-dependent K+ channel blocker). However, neither glibenclamide (ATP-sensitive K+ channel blocker) nor apamin (small conductance Ca(2+)-activated K+ channel blocker) had any significant inhibitory effect. 4. In the 30 mM K(+)-depolarized strips, pretreatment with thapsigargin, a specific Ca(2+)-ATPase inhibitor of the Ca2+ store sites, completely abolished the VIP-induced decrease in [Ca2+]i, but partially attenuated the VIP-induced decrease in force. 5. VIP shifted the [Ca2+]i-force relation of the U46619-induced contractions to the right in a concentration-dependent manner. In the alpha-toxin-permeabilized strips, VIP decreased the force development at a constant [Ca2+]i level (pCa = 6.5) in a GTP-dependent manner, which was antagonized by guanosine-5'-O-(beta-thiodiphosphate) (GDP beta S). 6. We thus conclude that VIP relaxes the coronary artery via three mechanisms: (1) a decrease in [Ca2+]i by inhibiting the Ca2+ influx presumably through the membrane hyperpolarization mediated by the activation of the large conductance Ca(2+)-activated (charybdotoxin-sensitive) K+ channels and voltage-dependent (4-AP-sensitive) K+ channels; (2) a decrease in [Ca2+]i by sequestrating cytosolic Ca2+ into thapsigargin-sensitive Ca2+ store sites; and (3) a decrease in the Ca(2+)-sensitivity of the contractile apparatus through the activation of G-protein.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1998

Direct activation of endothelial NO pathway by Ba2+ in canine coronary artery.

1. We have reported that Ba2+ causes endothelium-dependent relaxation of canine coronary arteries through NO synthesis in Ca2+-free and depolarizing solution. To determine the cellular mechanisms by which the endothelium-dependent relaxation occurs, we used fura-2 fluorometry (F350 and F390; excitation wavelengths, 350 and 390 nm, respectively) and estimated the intracellular Ba2+ concentration in endothelial and vascular smooth muscle cells. 2. Ba2+ (10(-3) M) increased the fura-2 ratio (F350/F390) recorded from a combined preparation of smooth muscle and endothelium (0.445+/-0.073, n = 4) and contracted the arteries in the presence of 80 mM K+ (0.22+/-0.06 g, n = 4). 3. Diltiazem (3 x l0(-6) M) blocks Ba2+ entry into vascular smooth muscle cells via L-type Ca2+ channels. In this condition, Ba2+ increased the fura-2 ratio in endothelial cells (0.141+/-0.014, n = 5) and relaxed the underlying smooth muscle (0.08+/-0.01 g, n = 5) by a mechanism which was sensitive to 10(-4) M NG-methyl-L-arginine (L-NMMA). 4. Ba2+-induced relaxation was not attenuated with repeated application and was elicited even after endothelium-dependent relaxations in response to 10(-6) M bradykinin were abolished due to tachyphylaxis. Neither 10(-2) M caffeine nor 10(-6) M thapsigargin had effect upon Ba2+-induced relaxation. 5. To further rule out changes in intracellular Ca2+ as a mechanism of Ba2+-induced relaxation, fura-2 fluorescence was measured at the isosbestic wavelengths for Ca2+ (360 nm) and Ba2+ (370 nm) in endothelium-intact arteries. Ba2+ altered F360, but not F370, suggesting little or no contribution of intracellular Ca2+ to the phenomenon of Ba2+-induced relaxation. 6. These results suggest that the Ba2+-induced relaxation is due to its direct activation of endothelial NO synthesis without mobilization of intracellular Ca2+.

Animals↗
PubMed1998

Mechanisms of galanin-induced contraction in the rat myometrium.

A neuropeptide, galanin, regulates the reproductive process and directly induces myometrial contraction. The aim of this study was to determine the mechanism of galanin-induced myometrial contraction. For this purpose, we simultaneously measured intracellular Ca2+ concentration ([Ca2+]i) and tension using fura-PE3-fluorometry and the rat longitudinal myometrium. The effect of galanin on the Ca2+ sensitivity of the contractile apparatus was examined in beta-escin permeabilized strips. The expression of galanin and the galanin receptors mRNAs in the rat myometrium were determined by reverse transcription polymerase chain reaction (RT-PCR). Galanin (10-300 nM) induced phasic contraction with or without oscillation in the pregnant rat myometrium in a concentration-dependent manner. The maximal response was obtained at 100 nM. There was no significant difference either in the maximal responses or EC50 values for galanin-induced myometrial contractions among myometriums from non-pregnant and pregnant (day 4, day 11, day 20, day 22) rats. In the day 20 and 22 pregnant myometriums, assigning the levels of [Ca2+]i and tension at 40 mM K+-depolarization to be 100%, galanin increased the [Ca2+]i and tension to 126.9+/-2.9% and 116.3+/-2.7%, respectively. Diltiazem (10 microM) inhibited the galanin-induced elevation of [Ca2+]i and tension to 71.9+/-2.4% and 16.2+/-0.7%, respectively. Ni2+, by itself, decreased the basal [Ca2+]i to -50.2+/-3.9% without affecting resting tension. After Ni2+ treatment, galanin-induced increases in [Ca2+]i and tension were -19.6+/-3.4% and 0.9+/-0.1%, respectively. In myometrium treated with diltiazem, no oscillation in [Ca2+]i and tension was observed. In Ca2+-free solution with 0.1 mM EGTA, galanin increased [Ca2+]i from -40.2+/-2.7% to -18.0+/-2.6% and induced transient contraction (3.6+/-0.8%). In beta-escin permeabilized myometrium, galanin enhanced the contraction induced by 0.3 microM Ca2+ in the presence of GTP. In the presence of GDPbetaS (1 mM) instead of GTP, galanin failed to increase the Ca2+ sensitivity of the contractile apparatus. RT-PCR revealed that galanin mRNA was hardly expressed in the non-pregnant rat myometrium and increased to reach a maximal level at mid pregnancy (day 11), but decreased to the same level as in the non-pregnant myometrium at term (day 22). Type 2 galanin receptor (GALR2) mRNA was found to be expressed in the rat myometrium whereas type 1 galanin receptor (GALR1) mRNA expression was not detected. In conclusion, galanin induces contraction of the rat myometrium by increasing [Ca2+]i as well as by increasing Ca2+ sensitivity of the contractile apparatus. Galanin-induced increases in [Ca2+]i are caused by both intracellular Ca2+ release and Ca2+ influx from extracellular space. The responsiveness of the rat myometrium to galanin does not change during pregnancy. The galanin mRNA is expressed in the rat myometrium and it is upregulated during mid-pregnancy. Rat myometrium expresses GALR2 but not GALR1 mRNA. By changing mRNA expression in the myometrium during pregnancy, galanin may act as a paracrine or autocrine mediator in the regulation of myometrial contractility.

Animals↗
PubMed1999

Vasorelaxation and inhibition of the voltage-operated Ca2+ channels by FK506 in the porcine coronary artery.

Using fura-2 fluorometry, the effects of FK506, an immunosuppressant, on changes in cytosolic Ca2+ concentrations ([Ca2+]i) and tension were investigated in porcine coronary arterial strips. The effects of FK506 on the activity of voltage-operated Ca2+ channels were examined by applying a whole cell patch clamp to the isolated smooth muscle cells of porcine coronary artery. FK506 inhibited the sustained increases in both [Ca2+]i and tension induced by 118 mM K+ depolarization and 100 nM U46619 in a concentration-dependent manner (1-30 microM). The extent of inhibition of the K+-induced contraction was greater than that of the U46619-induced contraction. The increases in [Ca2+]i and tension induced by histamine and endothelin- in the presence of extracellular Ca2+ were also inhibited by 10 microM FK506. FK506 (10 microM) had no effect on Ca2+ release induced by caffeine or by histamine in the Ca2+-free solution. FK506 (10 microM) had no effect on the [Ca2+]i-tension relationships of the contractions induced by cumulative increases of extracellular Ca2+ during K+ depolarization or stimulation with U46619. In the patch clamp experiments, FK506 (30 microM) partially inhibited the inward current induced by depolarization pulse from -80 mV to 0 mV. In conclusion, FK506 induces arterial relaxation by decreasing [Ca2+]i mainly due to the inhibition of the L-type Ca2+ channels, with no effect on the Ca2+ sensitivity of the contractile apparatus.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗