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H Kanaide

Publications and source records attributed to H Kanaide.

At least 91 records · Page 5Linked to original sources
PubMed1995

Effects of U46619 on intracellular Ca++ concentration and tension in human umbilical artery.

OBJECTIVE: The aim was to examine the effects of U46619, a thromboxane A2 analog, on intracellular Ca++ concentration and tension in human umbilical artery. STUDY DESIGN: By means of front-surface fluorometry and human umbilical arterial strips loaded with fura-2 we simultaneously measured intracellular Ca++ concentration and tension. RESULTS: In the presence of extracellular Ca++ (1.25 mmol/L), U46619 elevated intracellular Ca++ concentration and tension in a concentration-dependent manner. Oscillations of both intracellular Ca++ concentration and tension were evident in many cases. In the absence of extracellular Ca++ U46619 induced a transient elevation of intracellular Ca++ concentration that was associated with a transient increase and a sustained tension. At any given intracellular Ca++ concentration level the tension induced by the cumulative application of extracellular Ca++ during depolarization with high external K+ in the presence of U46619 was greater than that in the absence of this drug; thus the intracellular Ca++ concentration-tension relationship in the presence of U46619 shifted to the left. CONCLUSION: The tension developed by U46619 depends both on an influx of Ca++ from the extracellular space and on release of intracellular Ca++, and U46619 increased Ca++ sensitivity of the contractile apparatus.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1995

Effects of midazolam on intracellular Ca2+ and tension in airway smooth muscles.

BACKGROUND: It has been demonstrated that a group of minor tranquilizers, benzodiazepines, are able to relax airway smooth muscles. To determine the underlying mechanisms of this phenomenon, the effects of midazolam on the intracellular Ca2+ concentration ([Ca2+]i) and tension in airway smooth muscles were investigated. METHODS: Using front-surface fluorometry and fura-2-loaded porcine tracheal smooth muscle strips, both [Ca2+]i and isometric tension developments were simultaneously recorded. RESULTS: When the tracheal strips were exposed to a high external K(+)-solution (40 mM) or 10(-7) M carbachol containing 1.25 mM Ca2+, both [Ca2+]i and tension increased rapidly until they reached a plateau (the steady state). During steady-state contraction induced by K(+)-depolarization or carbachol, the cumulative application of midazolam (10(-7) approximately 10(-4) M) caused decreases in both [Ca2+]i and tension, in a concentration-dependent manner. During 40 mM K(+)-induced depolarization, the stepwise increases in the extracellular Ca2+ concentration induced the stepwise increases in [Ca2+]i and tension. Midazolam (3 x 10(-5) M) inhibited these increases in [Ca2+]i and tension, but had no effect on the [Ca2+]i-tension relationship. In the presence of 3 x 10(-3) M NiCl2 (a nonselective cation channel blocker), midazolam (3 x 10(-5) M) did not cause any additional reduction of [Ca2+]i or tension during the contraction induced by carbachol (10(-7) M). In the absence of extracellular Ca2+, midazolam (3 x 10(-5) M) had no effect on the transient increases in either [Ca2+]i or the tension induced by carbachol (10(-7) M) or caffeine (20 mM). Pretreatment with both 10(-5) M flumazenil (a specific central antagonist of benzodiazepines) and 10(-5) M PK11195 (a specific peripheral antagonist of benzodiazepines) did not influence the effect of 10(-5) M midazolam on [Ca2+]i or tension during the contractions induced by carbachol. CONCLUSIONS: Midazolam directly relaxes airway smooth muscles by decreasing [Ca2+]i; this can be attributed to the inhibition of the influx of extracellular Ca2+. Midazolam has no effect on the release of stored Ca2+. In addition, midazolam has no effect on Ca2+ sensitivity of the contractile apparatus. Finally, benzodiazepine antagonists, flumazenil and PK11195, have no effect on this mechanism of direct action of midazolam on airway smooth muscles.

Animals↗
PubMed1995

Cytosolic calcium concentration-force relation during contractions in the rabbit femoral artery: time-dependency and stimulus specificity.

1. By use of front-surface fluorometry with fura-2-loaded rabbit femoral arterial strips, both the cytosolic Ca2+ concentration ([Ca2+]i) and force were simultaneously monitored. By utilizing the [Ca2+]i-force curves, we were thus able to examine the temporal changes in the relationships between [Ca2+]i and force ([Ca2+]i-force relationship) during contractions induced by a high external K+ solution, noradrenaline (NA) and 5-hydroxytryptamine (5-HT). 2. The 'basic' [Ca2+]i-force relationship of the Ca(2+)-induced contractions was obtained by the cumulative applications of extracellular Ca2+ (0-10 mM) during 118 mM K(+)-depolarization (Ca(2+)-contractions). 3. When each vascular strip was exposed to high external K+ (30 mM K(+)-118 mM K+) solutions, the [Ca2+]i abruptly increased until it reached a peak, and then slightly decreased and eventually reached a steady-state level. The force also rapidly rose to reach a maximum plateau level. The changes in [Ca2+]i were more rapid than those in the force. Thus, the [Ca2+]i-force curves observed during the contractions induced by high+ (30 mM-118 mM) solutions showed a counter-clockwise rotation, over time. The entire curve shifted to the right, in a concentration-dependent manner, as compared with the line of the 'basic' [Ca2+]i-force relationship of the Ca(2+)-contraction. However, the [Ca2+]i-force relationship of the steady-state of contractions induced by the single dose applications of high K+ (30 mM-118 mM) overlapped with the line of the 'basic' [Ca2+]i-force relationship of Ca(2+)-contractions. 4. As references, the levels of [Ca2+]i and the force at rest (without stimulation) and at the steady-state of the contractions induced by a single dose application of 118 mM K+ solution were designated as 0% and 100%, respectively. When the vascular strips were exposed to NA (10(-5) M) and to 5-HT (10(-4) M), the [Ca2+]i abruptly rose, and reached a peak (107.1 +/- 5.8%) and 101.3 +/- 2.8%, respectively) after 1 min and 2 min, respectively (the [Ca2+]i-rising phase), and thereafter declined with a similar time course (the [Ca2+]i-declining phase) until reaching a low steady level (the steady-state phase). The force induced by 10(-5) M NA and 10(-4) M 5-HT reached a peak at 4 min (129%) and at 2 min (115%), respectively, and thereafter gradually declined. In contrast to the similarity in the [Ca2+]i transient between NA and 5-HT, the force induced by NA declined more slowly and reached higher steady levels than that seen with 5-HT. The level of force 20 min after the application of NA and 5-HT was 112% and 72%, respectively.5. In the entire time course of the 5-HT-induced contraction, i.e., in [Ca2+]i-rising, [Ca2+]i-declining and the steady-state phases, the [Ca2+]i-force relation was almost the same as that of the Ca2+-contractions.In the [Ca2+]i-rising phase of NA-induced contraction, the [Ca2+]i-force relation was similar to that of the Ca2+-contractions. However, in the [Ca2+] -declining and the steady-state phases, NA produced a greater force than that expected from a given change in the [Ca2+]i of the Ca2+-contractions, which resulted in a leftward shift of the [Ca2+]i-force relation. The extent of the leftward shift depended on the concentration of NA.6. These results suggest that (1) changes in [Ca2+]i precede changes in the force during the high K+-induced contraction, (2) in the initial [Ca2+]i-rising phase of the contractions induced by NA or by 5-HT, the [Ca2+]i-force relation is similar to that of Ca2+-contractions, and (3) in the subsequent[Ca2+]i-declining and the steady-state phases of the contractions, 5-HT demonstrated little enhancement in force for the given levels of [Ca2+]i, while NA induced a greater force for the given levels of [Ca2+],, in the rabbit femoral artery. Based on the above findings we suggest the presence of a time-dependent and stimulus-specific modulation of the Ca2+ sensitivity in the contractile apparatus of arterial smooth muscles.

Animals↗
PubMed1995

Endothelin-1 and endothelin-3 regulate differently vasoconstrictor responses of smooth muscle of the porcine coronary artery.

1. Using front-surface fluorometry of fura-2 and medial strips of the porcine coronary artery, we investigated mechanisms by which endothelin-1 (ET-1) and ET-3 function as vasoconstrictors. 2. In the presence of extracellular Ca2+(1.25 mM), ET-1 (10(-10)-10(-7) M) increased cytosolic Ca2+ concentrations ([Ca2+]i) and tension, in a concentration-dependent manner. ET-1, at concentrations greater than 10(-8) M, induced an abrupt elevation of [Ca2+]i which reached a transient peak (the first component, [Ca2+]i-rising phase) and subsequently declined ([Ca2+]i-declining phase) to reach a lower sustained phase (the second component, steady-state phase), while the tension rose monotonically to reach a peak and then slightly and gradually declined. ET-1, at concentrations lower than 10(-8) M, induced slowly developing and sustained increases in [Ca2+]i and tension ([Ca2+]i-rising phase followed by steady-state phase). All concentrations of ET-1 increased tension more slowly than [Ca2+]i. 3. In the presence of extracellular Ca2+, ET-3 (10(-8)-10(-5) M) induced concentration-dependent increases in [Ca2+]i and tension. However, the maximal elevations of [Ca2+]i and tension induced by ET-3 were substantially smaller than those induced by ET-1, indicating the involvement of an ETA receptor subtype. ET-3, at concentrations greater than 6 x 10(-7) M, caused biphasic slowly developing increases in [Ca2+]i and tension. At concentrations lower than 10(-6) M, ET-3 caused monophasic increases in [Ca2+]i and tension. At all concentrations of ET-3, the time courses of increases in [Ca2+]i and tension were similar. 4. The biphasic increases in [Ca2+]i and tension induced by 10-5 M ET-3 and by 1O-7M ET-1 were significantly inhibited by pretreatment with 10-5 M of the Ca2+ entry blocker, diltiazem, although the inhibition of the first component of ET-l-induced [Ca2+]i increase was partial.5. In the absence of extracellular Ca2+, ET-1 induced a concentration-dependent transient increase in[Ca2+]i, possibly due to release of Ca2+ from intracellular stores, and a sustained contraction. In contrast, ET-3 ( 10-6 M) caused little, if any, transient increase in [Ca2+]i and a small sustained contraction.6. Temporal changes in the relationships between [Ca2+]i and tension ([Ca2+]1-tension relationship)during contractions induced by ET-1 and ET-3 were compared with the [Ca2+]i-tension relationship of Ca2+-induced contractions (Ca2+-contractions) obtained by cumulative applications of extracellular Ca2+(0-7.5 mM) to tissues depolarized in the presence of 118 mMK+. In the [Ca2+]i-rising phase, ET-1 increased tension more slowly than [Ca2+]i, thereby shifting the [Ca2+]i-tension relation to the right from that for Ca2+-contractions. In the [Ca2+I-declining and the steady-state phases, ET-1, at concentrations higher than 10-9 M, produced greater tension development than that expected from a given change in[Ca2+ji, resulting in a leftward shift of the [Ca2+]i-tension relation. During ET-3-induced contractions,([Ca2+]i-rising, [Ca2+]i-declining and steady-state phases), the [Ca2+]i-tension relation was similar to that of Ca2+-contractions.7. BQ-123, a selective ETA receptor antagonist, completely inhibited the increases in [Ca2+1]i and tension induced by ET-1 and ET-3.8. These results suggest: (1) That ET-1 elicits vasoconstriction by increasing [Ca2+]i through the activation of Ca2+ influx from the extracellular space and Ca2+ release from intracellular storage sites,and by increasing the Ca2+ sensitivity of the contractile apparatus, whereas ET-3 induces vasoconstriction by increasing [Ca2+1] mainly through Ca2+ influx from the extracellular space. (2) Distinct mechanisms of time-dependent modulation of the Ca2+ sensitivity function in the vasoconstrictor responses to ET-1 and ET-3. (3) That both ET-1- and ET-3-induced contractions seem to be mediated via ETA-receptors in porcine coronary artery, and that the ETA-receptor-mediated effects of ET-1 and ET-3 can be dissociated at the sub-receptor levels of the signal transduction pathway.

Animals↗
PubMed1995

Mechanism of endothelium-dependent relaxation induced by substance P in the coronary artery of the pig.

1. Using front-surface fluorometry of fura-2-loaded porcine coronary arterial strips with the endothelium intact, we investigated the mechanisms of vasorelaxation induced by substance P (SP). Fura-2 fluorescence signals which indicated the cytosolic Ca2+-concentration ([Ca2+]i), were observed to arise exclusively from teh smooth muscle cells in these strips. 2. During the contractions induced by U46619 (100 nM), a thromboxane A2 analogue, an SP-induced endothelium-dependent, biphasic vasorelaxation was observed, which consisted of an initial rapid relaxation phase followed by a sustained phase, with a transient decrease in [Ca2+]i. Pretreatment with indomethacin (Ind) had no effect on the SP-induced relaxation; however, pretreatment with NG-nitro-L-arginine (L-NOARG) partially, but significantly inhibited the decrease in both the [Ca2+]i and tension abolished. Thus, part of the relaxation was considered to be mediated by L-NOARG-sensitive relaxing factor (endothelium-derived relaxing factor: EDRF). 3. During the 40 mM K+-depolarization-induced contraction which may eliminate the effects of endothelium-derived hyperpolarizing factor (EDRF), the vasorelaxation reduced by SP was completely inhibited by L-NOARG. 4. During the vasorelaxation induced SP, the [Ca2+]i-tension relationships shifted to the right of the contractions induced by either U46619 or high K+-depolarization. 5. Using front-surface fluorometry of fura-2 loaded porcine aortic valvular strips, we examined the effects of SP on [Ca2+]i in endothelial cells in situ. SP induced a rapid increase in [Ca2+]i of endothelial cells in situ followed by a small sustained phase in normal PSS (5.9 mM K+). The increase in extracellular K+ had no apparent effect on the SP-induced [Ca2+]i elevation of endothelial cells.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1995

Evidence for the presence of endothelin ETA receptors in endothelial cells in situ on the aortic side of porcine aortic valve.

1. In the present study, we determined whether ETA receptors are present on endothelial cells in situ, by use of front-surface fluorometry of fura-2-loaded porcine aortic valvular strips and reverse transcription polymerase chain reaction (RT-PCR). 2. Although endothelin-1 (ET-1) and endothelin-3 (ET-3) induced maximum elevations of cytosolic Ca2+ concentrations ([Ca2+]i) at 10(-7) M, the peak elevations of [Ca2+]i induced by ET-1 were much greater than those induced by ET-3. 3. The application of ET-1 after ET-3 induced an additional increase in [Ca2+]i, while the application of ET-3 after ET-1 had no effect. A selective ETA receptor antagonist, BQ-123, partially inhibited the ET-1-induced Ca2+ transient but had no effect on ET-3-induced Ca2+ transients. These experiments indicated the presence of functioning ETA receptors in addition to ETB receptors in endothelial cells in situ. 4. The sequence of pig lung ETA receptor complimentary DNA (cDNA) was determined by PCR. RT-PCR, using specific primers for pig ETA receptor sequence and total RNA from endothelial cells on the aortic side of the aortic valve, gave the expected size of band. This PCR product was sequenced and was found to be identical to the sequence of the pig lung ETA receptor. 5. The partial sequence of the pig lung ETB receptor was also determined. RT-PCR for the pig ETB receptor revealed that endothelial cells of the aortic valve express ETB receptor messenger RNA (mRNA). 6. We confirmed that functioning ETA receptors and expression of ETA receptor mRNA exist in the endothelial cells on the aortic side of porcine aortic valves.

Amino Acid Sequence↗
PubMed1995

Resting load regulates vascular sensitivity by a cytosolic Ca(2+)-insensitive mechanism.

The cellular mechanism underlying the regulation of the contraction of vascular smooth muscles by resting load is unknown. To determine the effects of changes in the resting load on vascular sensitivity to high K+ and to 9,11-dideoxy-11 alpha, 9 alpha-epoxy-methanoprostaglandin F2 alpha (U-46619), the force and cytosolic calcium concentration ([Ca2+]i) of arterial strips were recorded at resting loads of 200 (optimal load), 50, and 10 mg. A decrease in the resting load elicited a small decrease in the basal [Ca2+]i level without affecting the extent of maximal [Ca2+]i elevation induced by either stimulus. Through a decrease in the resting load, the concentration-response curves for the force development of high K+ or of U-46619 shifted to the right, whereas those for [Ca2+]i did not. We conclude that the basal [Ca2+]i level and the force development, but not the agonist-induced [Ca2+]i signals, of vascular smooth muscles depend on the resting load. We response that the resting load regulates the sensitivity of vascular smooth muscles, irrespective of types of stimuli, through a [Ca2+]i-insensitive mechanism.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1995

Dual regulation of L-type Ca2+ channels by serotonin 2 receptor stimulation in vascular smooth muscle cells.

The purpose of the present study was to investigate regulation of voltage-dependent Ca2+ channels by serotonin in rat aortic smooth muscle cells in primary culture. L- and T-type Ca2+ currents (ICa) were recorded using the whole cell voltage-clamp method. Without pretreatment, in 25 of 30 cells examined, 10 microM serotonin decreased L-type ICa to various extents (-14 to -72%). However, in the remaining five cells, serotonin increased L-type ICa 21 +/- 4%. Thus, in 30 cells, serotonin decreased L-type ICa an average of 22 +/- 5%. In the presence of intracellular heparin (100 micrograms/ml), a blocker of inositol 1,4,5-trisphosphate binding to its receptor, serotonin increased L-type ICa in all cells 29 +/- 3% (n = 6). When stored Ca2+ was depleted by pretreatment either with 20 microM ryanodine and 20 mM caffeine or with 100 nM A-23187, serotonin also increased L-type ICa in all cells 30 +/- 5 (n = 4) or 37 +/- 5% (n = 12), respectively. In the presence of heparin, the serotonin-induced increase of L-type ICa was prevented by 100 nM staurosporine (2 +/- 3%; n = 6, P < 0.01). The serotonin-induced decrease of L-type ICa was significantly augmented by 100 nM staurosporine (-43 +/- 10%; n = 5). Phorbol 12,13-dibutylate (PDBu; 1 microM) increased L-type ICa 29 +/- 3% (n = 6), and serotonin did not further increase L-type ICa after its potentiation by PDBu.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

The effects of endothelin on vascular tonus.

Using front-surface fluorometry and fura-2, the effect of endothelin (ET) on the cytosolic Ca concentration, (Ca)i, in smooth muscle cells and endothelial cells was determined. Both the contraction of smooth muscle cells and the release of endothelium-derived relaxing factor (EDRF) from endothelial cells are regulated by changes in (Ca)i. During contractions induced by U-46619, a thromboxane A2 analog, low doses of ET-1 induced an EDRF-dependent reduction of (Ca)i and force in porcine coronary arterial smooth muscles. Using porcine aortic valvular strips, we recorded the signals of (Ca)i in endothelial cells in situ. ET-1 induced an influx of Ca, which was markedly inhibited by pertussis toxin (IAP), thus indicating that this influx was regulated by an IAP-sensitive G-protein. BQ-123, a selective ETA receptor antagonist, partially inhibited the elevation of (Ca)i induced by ET-1, but did not affect the elevation of (Ca)i induced by ET-3. The sequence of cDNA encoding the porcine ETA receptor has been previously determined, and RT-PCR confirmed that ETA receptor mRNA was present in the endothelial cells on the aortic side of the valvular strips. Therefore, in addition to ETB receptors, functioning ETA receptors and ETA receptor mRNA can also be found in endothelial cells in situ. Thus, ET-1 may play an important role in controlling the coronary artery tonus not only by acting directly on smooth muscle cells to increase the force in a paracrine manner, but also by acting on endothelial cells to release EDRF in an autocrine manner, resulting in relaxation of smooth muscle cells.

Animals↗
PubMed1994

Myosin phosphorylation and Ca2+ sensitization in porcine coronary arterial smooth muscle stimulated with endothelin-1.

We examined the mechanism of endothelin-1-induced contraction in the porcine coronary artery by assessing the changes in myosin light chain phosphorylation and cytosolic Ca2+ concentration ([Ca2+]i) determined by fura-2 and front-surface fluorometry. Endothelin-1 induced a rapid phosphorylation of myosin both in the presence and in the absence of extracellular Ca2+. During the later phase of contraction (5-30 min), the tension and [Ca2+]i were maintained, while the phosphorylated myosin decreased in the presence of extracellular Ca2+. At a similar extent of [Ca2+]i elevation, endothelin-1 induced a greater tension development than did 118 mM K(+)-depolarization and this was not attributed to a greater degree of phosphorylation of myosin. In the Ca(2+)-depleted strips, endothelin-1 induced a slow and transient contraction without an increase in either [Ca2+]i or myosin phosphorylation. Therefore, in addition to the activation of mechanisms of [Ca2+]i-myosin phosphorylation, endothelin-1 may activate additional calcium regulatory mechanisms which may thus increase the Ca2+ sensitivity of contractile elements while also enhance and prolong the contractions in the porcine coronary artery.

Animals↗
PubMed1994

Motilin induces the endothelium-dependent relaxation of smooth muscle and the elevation of cytosolic calcium in endothelial cells in situ.

Using front-surface fluorometry with fura-2 and porcine aortic valvular strips, we investigated the effect of motilin, a gastrointestinal peptide, on the cytosolic Ca2+ concentration ([Ca2+]i) of endothelial cells in situ. Motilin induced a biphasic elevation of [Ca2+]i of the endothelial cells on the porcine aortic valvular strips. To investigate the physiological role of these Ca2+ transients in the endothelial cells, we determined the effect of motilin on [Ca2+]i as well as the tension of the smooth muscles in the porcine coronary strips with an intact endothelium. Motilin decreased [Ca2+]i and the tension of the coronary smooth muscle precontracted by U46619, a thromboxane A2 analogue, in an endothelium-dependent manner. In the presence of indomethacin (a cyclooxygenase inhibitor), motilin induced an endothelium-dependent relaxation of the coronary strips which was partially inhibited by N omega-nitro-L-arginine (L-NNA; a NO synthase inhibitor). These results thus indicate that motilin induces Ca2+ transients of the endothelial cells while it also induces vasorelaxation, which may be mediated by both L-NNA sensitive and resistant factors that are derived from the endothelium.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1994

Protein kinase C and protein kinase A regulate the expression of angiotensin II receptor mRNA in smooth muscle cells.

Using reverse transcription polymerase chain reaction and fura-2 microfluorometry of intracellular Ca2+ concentrations, we investigated the effects of angiotensin II and cyclic AMP on the expression of angiotensin II type 1 receptor mRNA and the relationship between angiotensin II receptor mRNA level and physiological responsiveness in rat aortic smooth muscle cells in primary culture. Angiotensin II (1 microM) induced a time- and dose-dependent transient decrease in the level of angiotensin II receptor mRNA. The maximal decrease (50 +/- 13% of control) occurred at 6 h, followed by a gradual return to the control level within 24 h. H-7 (30 microM), a relatively specific protein kinase C inhibitor, inhibited decrease in the expression of angiotensin II receptor mRNA induced by 6 h angiotensin II treatment, while 6 h stimulation by 0.3 microM phorbol 12-myristate 13-acetate also induced a decrease (35 +/- 8% of control) in the expression of angiotensin II receptor mRNA. An increase in cellular cyclic AMP induced by 10 microM forskolin plus 10 microM 3-isobutyl-1-methyl-xanthine, decreased the angiotensin II receptor mRNA level to 50 +/- 13% of the control at 6 h and increased it to 219 +/- 39% of the control at 48 h of treatment. Angiotensin II-induced decrease and cyclic AMP-induced increase in the expression of angiotensin II receptor mRNA were accompanied by a reduction and enhancement in [Ca2+]i transients induced by angiotensin II, respectively. In contrast with angiotensin II receptor mRNA level (102 +/- 8% of control), the [Ca2+]i transients were markedly decreased in angiotensin II plus H-7 treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1994

Cytosolic Ca2+ transients are not required for platelet-derived growth factor to induce cell cycle progression of vascular smooth muscle cells in primary culture. Actions of tyrosine kinase.

We investigated interrelations among changes in cytosolic Ca2+ concentrations ([Ca2+]i), tyrosine phosphorylation, and the cell cycles of rat aorta smooth muscle cells in primary culture, as stimulated with platelet-derived growth factor (PDGF). Changes in [Ca2+]i were monitored using the microfluorometry of Fura-2. The phase of the cell cycle and the extent of tyrosine phosphorylation were examined by immunocytochemical analysis of monoclonal antibodies against cell cycle-specific nuclear antigens and against phosphotyrosine, respectively. Prior to the application of PDGF, the cell cycle was synchronized in the G0 phase by serum deprivation for 24 h. In the presence of extracellular Ca2+, PDGF induced an initial transient (first component) and a subsequent lower steady-state (second component) elevation of [Ca2+]i. NiCl2 and the removal of extracellular Ca2+ inhibited the second, but not the first, component. The first component was inhibited by pretreatment with ryanodine. These results are compatible with the notion that the first and second components may be mediated mainly through the release of intracellular Ca2+ and the influx of extracellular Ca2+, respectively. After pretreatment with ryanodine and in the presence of NiCl2, PDGF also stimulated the entry of G0 cells into G1 phase, but there were no [Ca2+]i transients. Genistein, a tyrosine kinase blocker, inhibited tyrosine phosphorylation induced by PDGF and blocked the first, but not the second, component of [Ca2+]i elevation induced by PDGF. However, genistein did not inhibit the release of intracellular Ca2+ induced by angiotensin II or by caffeine. Genistein prevented G0 cells from entering the G1 phase, as induced by PDGF, but this was not the case when serum was reapplied to the growth medium. Similar results were obtained with another tyrosine kinase blocker, tyrphostin. These data suggest that in vascular smooth muscle cells: 1) an increase in [Ca2+]i is not required for competent (G0 to G1) cell proliferation induced by PDGF; and 2) tyrosine kinase plays an important role in the release of intracellular Ca2+ and in cell proliferation, as induced by PDGF.

Angiotensin II↗
PubMed1994

Actions of a new Ca2+ channel antagonist, CD832, on two types of Ca2+ channels in smooth muscle.

The purpose of this study was to determine the characteristics of blockade of L- and T-type Ca2+ channels by a new Ca2+ channel antagonist, 3-(2-nicotinoylamino)ethyl 5-(3-nitrooxypropyl) 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate hydrochloride (CD832), in comparison with those of nifedipine, in rat aortic smooth muscle cells in primary culture. In a whole-cell configuration under voltage-clamp, CD832 and nifedipine dose dependently blocked both L- and T-type Ca2+ currents (L- and T-type ICa) without shifting the axis of the current-voltage relations. The 50% inhibition concentration (IC50) of CD832 was 310 nM for L-type and 1.1 microM for T-type ICa. The IC50 of nifedipine was 36 nM for L-type and 2.7 microM for T-type ICa. CD832 blocked both types of ICa from the first step pulses after drug application, shifted their steady-state inactivation curves to the left, and did not significantly accelerate the current decay. The effect of CD832 reached its maximum within 6 min and was hardly washed out, while that of nifedipine reached a maximum within 30 s and could be washed out within 3 min. These results suggest that (1) CD832 blocks L-type ICa less potently than does nifedipine, (2) CD832 blocks T-type ICa more potently than does nifedipine, (3) CD832 blocks not only the resting state but, more preferentially, the inactivated state of L- and T-type Ca2+ channels, and (4) CD832 has a slow and long-acting activity in blocking both types of ICa as compared with nifedipine.

Animals↗
PubMed1994

Relationship between cytosolic calcium concentration and force in the papaverine-induced relaxation of medial strips of pig coronary artery.

1. The mechanisms of vasorelaxation induced by papaverine were investigated using front-surface fluorometry and fura-2-loaded medial strips of the pig coronary artery. 2. In the presence of extracellular Ca2+ (1.25 x 10(-3) M), histamine (10(-4) M) induced abrupt elevations of cytosolic calcium concentration, [Ca2+]i reaching a peak within 12 s (the first phase); after making a slight shoulder, [Ca2+]i declined gradually to reach sustained levels (the second phase). Force rapidly rose to reach maximum levels in 3 min, then gradually declined. Papaverine (10(-7)-10(-5) M) inhibited both the first and the second phases of [Ca2+]i elevation and the development of force induced by histamine, in a concentration-dependent manner. 3. In the absence of extracellular Ca2+, histamine (10(-4) M) induced a transient increase in [Ca2+]i and force, both of which were inhibited in a concentration-dependent manner by papaverine (10(-7)-10(-5) M). When papaverine was washed out, a second application of 10(-4) M histamine also induced transient increases in [Ca2+]i and force. The smaller the first response, the greater was the second response. The total amount of [Ca2+]i released from intracellular stores by the first and second application of histamine in the presence of papaverine was smaller than in its absence, thereby indicating a reduction of Ca2+ in the histamine-sensitive store. However, while papaverine (10(-5) M) did not affect the transient increase in [Ca2+]i induced by 2 x 10(-2) M caffeine, contractions were inhibited. 4. For a given level of [Ca2+]i, the force developed with the cumulative application of histamine(10-7 M-10-1 M) was greater than that observed with the cumulative application of extracellular Ca2+(0-7.5 x 10-3 M) during high K+ depolarization. Papaverine (10-7 M-10-5M) suppressed, in a concentration-dependent manner, the increase in [Ca2+]i and the force induced by cumulative applications of both histamine and extracellular Ca2+ during high K+ depolarization. The [Ca2+]i-force curve obtained by depolarization with K+, but not that obtained during histamine application, was shifted to the right by papaverine. Diltiazem, 10-7 M, a concentration causing a similar degree of relaxation to 10-5 M papaverine, did not shift the [Ca2+]i-force curve obtained with high K+. Nitroglycerin (10-6 M)and isoprenaline (10-6 M) shifted the [Ca2+]i-force curve to the right to a greater extent than did 10-5 M papaverine.5. These findings suggest that papaverine relaxes medial strips of the porcine coronary artery by two mechanisms. The first is mainly due to a decrease in [Ca2+]i, not only through inhibiting Ca2+ influx through either voltage-dependent or receptor-operated Ca2+ channels, but also by inhibiting agonist induced intracellular Ca2+ release. This occurs presumably by interference with the signal transduction pathway for histamine and by a depletion of Ca2+ in histamine-sensitive stores. Secondly, the [Ca2+]i-sensitivity of certain contractile mechanisms may be minimally decreased.

Animals↗
PubMed1994

Sensitivity of G-protein involved in endothelin-1-induced Ca2+ influx to pertussis toxin in porcine endothelial cells in situ.

1. We designed a new method to determine quantitatively the intracellular Ca2+ concentration ([Ca2+]i) in endothelial cells in situ, using front-surface fluorometry and fura-2-loaded porcine aortic valvular strips. Using this method, we investigated the characteristics of the G-protein involved in endothelin-1 (ET-1)-induced changes in [Ca2+]i of endothelial cells in situ. 2. Endothelial cells were identified by specific uptake of acetylated-low density lipoprotein labelled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI-Ac-LDL). Double staining with DiI-Ac-LDL and fura-2 showed that the valvular strip was covered with a monolayer of endothelial cells and that the cellular component which contributed to the fura-2 fluorescence, [Ca2+]i signal, was exclusively endothelial cells. 3. ET-1 (10(-7) M) induced an elevation of [Ca2+]i consisting of two components: the first was a rapid and transient elevation to reach a peak, followed by a second, sustained elevation (the second phase). The first phase was composed of extracellular Ca(2+)-independent and -dependent components, while the second phase was exclusively extracellular Ca(2+)-dependent. The extracellular Ca(2+)-independent component of the first phase was due to the release of Ca2+ from intracellular storage sites. The second phase and part of the first phase of [Ca2+]i elevation were attributed to the influx of extracellular Ca2+. The Ca2+ influx component was completely inhibited by 10(-3) M Ni2+ but was not affected by 10(-5) M diltiazem. 4. Pertussis toxin (IAP) markedly inhibited the extracellular Ca2+-dependent elevation of [Ca2+]j, but had no effect on the extracellular Ca2+-independent elevation of [Ca2+], caused by ET-1 (10-7M).5. Bradykinin (10-7 M) or ATP (10- 5M) elevated [Ca2+]i and these responses also consisted of extracellular Ca2+-independent and extracellular Ca2+-dependent components. IAP had no effect on either component of the [Ca2+]i elevation induced by bradykinin or ATP.6. From these findings we conclude that, in porcine endotheliel cells in situ, ET-1 elevates [Ca2+]i as are result of a Ca2+ influx component from the extracellular space and release of intracelluarly stored Ca2+ .The Ca2+ influx is regulated by an IAP-sensitive G-protein, while the release of Ca2+ from the intracellular store is not.

Animals↗
PubMed1994

The effects of a novel vasodilator, LP-805, on cytosolic Ca2+ concentrations and on tension in rabbit isolated femoral arteries.

1. LP-805, 8-tert-butyl-6,7-dihydropyrrolo-[3,2-e]-5-methylpyrazolo- [1,5a]-pyrimidine-3-carbonitrile, is a newly synthesized potent vasodilator. To investigate the cellular mechanisms of vasorelaxation induced by LP-805, we simultaneously determined the effects of LP-805 on cytosolic Ca2+ concentrations ([Ca2+]i) and on tension of smooth muscle of rabbit femoral arterial strips, with or without the endothelium, using front-surface fluorometry and fura-2. 2. In the absence of the endothelium, LP-805, in a concentration-dependent manner, decreased [Ca2+]i and tension during the contraction induced by K(+)-depolarization, at relatively low concentrations ([K+]o < or = 30 mM). The decreases in [Ca2+]i and tension were fully antagonized by treatment with 2 x 10(-6) M glibenclamide. The [Ca2+]i-tension relationship in the LP-805-induced relaxation was similar to that of K(+)-depolarization-induced contractions. 3. LP-805, in a concentration-dependent manner (IC50 for inhibition of tension; 1.7 x 10(-6) M), decreased both [Ca2+]i and tension during the steady-state of contractions induced by 1 x 10(-7) M noradrenaline (NA) in the strips without the endothelium. Glibenclamide completely inhibited these reductions of [Ca2+]i and tension. At the steady-state of relaxation induced by LP-805 during NA-induced contraction, [Ca2+]i-tension relation was shifted to the left of that obtained with high K(+)-induced contraction. 4. NA induced transient increases in [Ca2+]i and tension in the absence of extracellular Ca2+. LP-805 (up to 3 x 10(-6) M) had no effect on these intracellular Ca2+ mobilisation and tension development induced by NA. 5. In strips with an intact endothelium, LP-805 decreased both [Ca2+]i and tension during contraction induced by 1 x 10(-7) M NA. The concentration-response curve for inhibition of [Ca2+]i and tension obtained in the presence of the endothelium was shifted to the left from that obtained in the absence of endothelium. IC50 for the inhibition of tension obtained in the strips with the endothelium was 4.0 x 10(-7) M. Treatment with 1 x 10(-4) M NG-nitro-L-arginine (L-NOARG) attenuated reductions of both [Ca2+]i and tension induced by LP-805 and the concentration-response curve shifted to the right and overlapped that obtained in the absence of the endothelium. Treatment with glibenclamide almost fully overcame the reduction of [Ca2+]i induced by LP-805, while the reversion of tension was 50% at most. 6. In the presence of the endothelium with L-NOARG, LP-805 reduced the tension to the extent of that expected from the reduction of [Ca2'ji, as based on the [Ca2+]i-tension relationship obtained with LP-805 in the absence of endothelium. On the contrary, in the presence of the endothelium without L-NOARG, LP-805 induced a greater reduction of tension than expected from the reduction of [Ca2+J1.This effect became more apparent after treatment with glibenclamide.7. These results suggest that: (1) LP-805 relaxes smooth muscle mainly by activating ATP-sensitive K+channels of smooth muscle and by releasing endothelium-derived relaxing factor (EDRF). (2) Activation of ATP-sensitive K+ channels decrease [Ca2+]i and thereby relax smooth muscle with no effect on Ca2"-sensitivity of the contractile apparatus of smooth muscle or on the agonist-induced Ca2"-release process. (3) EDRF induced by LP-805 relaxes smooth muscle not only by decreasing [Ca2+]i but also decreasing Ca2+-sensitivity of the contractile apparatus of smooth muscle. In the presence of an intact endothelium, a decrease in Ca2+-sensitivity of the contractile apparatus may play an important role in LP-805-induced relaxation.

Animals↗