Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.6.1.3 (ATPase)
 65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In the cat ventricle angiotensin II exerts a positive inotropic effect produced by an increase in intracellular calcium associated with a prolongation of relaxation. The signaling cascades involved in these effects as well as the subcellular mechanisms of the negative lusitropic effect are still not clearly defined. The present study was directed to investigate these issues in cat papillary muscles and isolated myocytes. The functional suppression of the sarcoplasmic reticulum (SR) with either 0.5 microm ryanodine or 0.5 microm ryanodine plus 1 microm thapsigargin or the preincubation of the myocytes with the specific inhibitor of the inositol 1,4,5-triphosphate (IP3) receptors [diphenylborinic acid, ethanolamine ester (2-APB), 5-50 microm] did not prevent the positive inotropic effect and the increment in Ca2+ transient produced by 1 microm angiotensin II. In contrast, protein kinase C (PKC) inhibitors, chelerythrine (20 microm) and calphostin C (1 microm) completely inhibited both, the angiotensin II-induced increase in L-type calcium current and positive inotropic effect. The prolongation of half relaxation time produced by 0.5 microm angiotensin II [207+/-15.4 msec (control) to 235+/-19.98 msec (angiotensin II), P<0.05] was completely blunted by PKC inhibition. This antirelaxant effect, which was independent of intracellular pH changes, was associated with a prolongation of the action potential duration and was preserved after either the inhibition of the SR and the SR Ca2+ ATPase (ryanodine plus thapsigargin) or of the reverse mode of the Na+/Ca2+ exchanger (KB-R7943, 5 microm). We conclude that in feline myocardium the positive inotropic and negative lusitropic effects of angiotensin II are both entirely mediated by PKC without any significant participation of the IP3 limb of the phosphatidylinositol/phospholipase C cascade. The results suggest that the antirelaxant effect of angiotensin II might be determined by the decrease in Ca2+ efflux through the Na+/Ca2+ exchanger produced by the angiotensin II-induced prolongation of the action potential duration.
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PMID:Positive inotropic and negative lusitropic effect of angiotensin II: intracellular mechanisms and second messengers. 1170 41

Alpha(1)-aderenoceptor-mediated constriction of rabbit inferior vena cava (IVC) is signaled by asynchronous wavelike Ca(2+) oscillations in the in situ smooth muscle. We have shown previously that a putative nonselective cationic channel (NSCC) is required for these oscillations. In this report, we show that the application of 2-aminoethoxyphenyl borate (2-APB) to antagonize inositol 1,4,5-trisphosphate (InsP(3))-sensitive Ca(2+) release channels (IP(3)R channels) can prevent the initiation and abolish ongoing alpha(1)-aderenoceptor-mediated tonic constriction of the venous smooth muscle by inhibiting the generation of these intracellular Ca(2+) concentration ([Ca(2+)](i)) oscillations. The observed effects of 2-APB can only be attributed to its selective inhibition on the IP(3)R channels, not to its slight inhibition of the L-type voltage-gated Ca(2+) channel and the sarco(endo)plasmic reticulum Ca(2+) ATPase. Furthermore, 2-APB had no effect on the ryanodine-sensitive Ca(2+) release channel and the store-operated channel (SOC) in the IVC. These results indicate that the putative NSCC involved in refilling the sarcoplasmic reticulum (SR) and maintaining the tonic contraction is most likely an SOC-type channel because it appears to be activated by IP(3)R-channel-mediated SR Ca(2+) release or store depletion. This is in accordance with its sensitivity to Ni(2+) and La(3+) (SOC blockers). More interestingly, RT-PCR analysis indicates that transient receptor potential (Trp1) mRNA is strongly expressed in the rabbit IVC. The Trp1 gene is known to encode a component of the store-operated NSCC. These new data suggest that the activation of both the IP(3)R channels and the SOC are required for PE-mediated [Ca(2+)](i) oscillations and constriction of the rabbit IVC.
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PMID:Sequential opening of IP(3)-sensitive Ca(2+) channels and SOC during alpha-adrenergic activation of rabbit vena cava. 1195 42

2-Aminoethoxydiphenyl Borate (2-APB) has been extensively used recently as a membrane permeable modulator of inositol-1,4,5-trisphosphate-sensitive Ca2+ channels and store-operated Ca2+ entry. Here, we report that 2-APB is also an inhibitor of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) Ca2+ pumps, and additionally increases ion leakage across the phospholipid bilayer. Therefore, we advise caution in the interpretation of results when used in Ca2+ signalling experiments. The inhibition of 2-APB on the SERCA Ca2+ pumps is isoform-dependent, with SERCA 2B being more sensitive than SERCA 1A (IC50 values for inhibition being 325 and 725 micro m, respectively, measured at pH 7.2). The Ca2+-ATPase is also more potently inhibited at lower pH (IC50 = 70 micro m for SERCA1A at pH 6). 2-APB decreases the affinity for Ca2+ binding to the ATPase by more than 20-fold, and also inhibits phosphoryl transfer from ATP (by 35%), without inhibiting nucleotide binding. Activity studies performed using mutant Ca2+-ATPases show that Tyr837 is critical for the inhibition of activity by 2-APB. Molecular modeling studies of 2-APB binding to the Ca2+ ATPase identified two potential binding sites close to this residue, near or between transmembrane helices M3, M4, M5 and M7. The binding of 2-APB to these sites could influence the movement of the loop between M6 and M7 (L6-7), and reduce access of Ca2+ to their binding sites.
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PMID:Inhibition of SERCA Ca2+ pumps by 2-aminoethoxydiphenyl borate (2-APB). 2-APB reduces both Ca2+ binding and phosphoryl transfer from ATP, by interfering with the pathway leading to the Ca2+-binding sites. 1215 64

In small segments of circular smooth muscle bundle isolated from the guinea-pig gastric antrum, depolarization of the tissue with intracellular current stimuli evoked regenerative slow potentials after a refractory period of 5-10 s. The refractory period changed inversely with the amplitude and duration of the stimulating depolarization. Thapsigargin (an inhibitor of calcium-ATPase at internal stores), 2-aminoethoxydiphenyl borate (2-APB, an inhibitor of inositol 1,4,5-trisphosphate (IP3)-receptor-mediated Ca2+ release), and carbonyl cyanide m-chlorophenyl-hydrazone (a mitochondrial protonophore) reduced the amplitude of slow potentials, with no significant alteration of the refractory period. Bisindolylmaleimide I or chelerythrine (inhibitors of protein kinase C, PKC) increased the refractory period and inhibited the amplitude of slow potentials. These results indicate that the refractory period and amplitude of slow potentials are related to the activation of PKC and the amount of Ca2+ released from the internal stores through activation of IP3 receptors, respectively. Acetylcholine (ACh) reduced the refractory period and increased the amplitude of slow potentials: the former was antagonized by chelerythrine and the latter by 2-APB. The results suggest that ACh has dual actions; stimulation of the metabolism of inositol phosphate and activation of PKC. Phorbol-12-myristate-13-acetate, a selective stimulant of PKC, at low concentrations (< 10 nM) mimicked the actions of ACh and at high concentrations reduced the frequency of slow potentials and increased the refractory period. The possible involvement of the concentration-dependent differences in the actions of phorbol ester on the translocation of PKC was considered.
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PMID:Excitation of smooth muscles isolated from the guinea-pig gastric antrum in response to depolarization. 1218 Dec 88

2-Aminoethoxydiphenylborate (2-APB) inhibits the extent of inositol 1,4,5-trisphosphate (InsP(3))-induced Ca(2+) release from cerebellar microsomes with a potency that is dependent upon the InsP(3) concentration used. At high InsP(3) concentrations (10 microM), the concentration of 2-APB required to cause half-maximal InsP(3)-induced Ca(2+) release (IC(50)) was greater than 1 mM, while at 0.25 microM InsP(3) this reduced to 220 microM. The fact that the inhibition of the extent of InsP(3)-induced Ca(2+) release (IICR) by 2-APB was not restored to control levels by high concentrations of InsP(3), in addition to the fact 2-APB did not substantially inhibit [3H]InsP(3) binding to its receptor, indicates that the inhibition is not competitive in nature. Since the cooperativity of IICR as a function of InsP(3) was reduced in the presence of 2-APB (Hill coefficient changing from 1.9 in the absence of 2-APB to 1.4 in the presence of 1 mM 2-APB), this suggests that it is acting as an allosteric inhibitor. 2-APB also reduces the rate constants for IICR. In cerebellar microsomes this release process is biphasic in nature, with a fast and slow phase. 2-APB appears particularly to affect the fast-phase component. Although 2-APB does not inhibit the ryanodine receptor, it does inhibit the Ca(2+) ATPase activity as well store-operated Ca(2+) entry channels, which may limit its use as a specific membrane permeant InsP(3) receptor inhibitor.
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PMID:Inhibition of the type 1 inositol 1,4,5-trisphosphate receptor by 2-aminoethoxydiphenylborate. 1222 Jun 21

Within muscular equivalents of cat lower esophageal sphincter (LES), the circular muscle develops greater spontaneous tone, whereas the sling muscle is more responsive to cholinergic stimulation. Smooth muscle contraction involves a combination of calcium release from stores and of calcium entry via several pathways. We hypothesized that there are differences in the sources of Ca(2+) used for contraction in sling and circular muscles and that these differences could contribute to functional asymmetry observed within LES. Contraction of muscle strips from circular and sling regions of LES was assessed in the presence of TTX. In Ca(2+)-free Krebs, tone was inhibited to a greater degree in circular than sling muscle. L-type Ca(2+) channel blockade with nifedipine or verapamil inhibited tone in LES circular but not sling muscle. Sarcoplasmic reticulum (SR) Ca(2+)-ATPase inhibitor cyclopiazonic acid (CPA) caused greater increase in tone in sling than in circular muscle. The phospholipase C inhibitor U-73122 and the SR inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] receptor blocker 2-aminoethoxydiphenyl borate (2-APB) inhibited tone in circular and sling muscles, demonstrating that continuous release of Ca(2+) from Ins(1,4,5)P(3)-sensitive stores is important in tone generation in both muscles. In Ca(2+)-free Krebs, ACh-induced contractions (AChC) were inhibited to a greater degree in sling than circular muscles. However, nifedipine and verapamil greatly inhibited AChC in the circular but not sling muscle. Depletion of SR Ca(2+) stores with CPA or inhibition of Ins(1,4,5)P(3)-mediated store release with either U-73122 or 2-APB inhibited AChC in both muscles. We demonstrate that LES circular and sling muscles 1) use intracellular and extracellular Ca(2+) sources to different degrees in the generation of spontaneous tone and AChC and 2) use different Ca(2+) entry pathways. These differences hold the potential for selective modulation of LES tone in health and disease.
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PMID:Calcium source diversity in feline lower esophageal sphincter circular and sling muscle. 1456 70

The present study describes the first characterization of Ca(2+)-activated Cl(-) currents (I(ClCa)) in single smooth muscle cells from a murine vascular preparation (portal veins). I(ClCa) was recorded using the perforated patch version of the whole cell voltage-clamp technique and was evoked using membrane depolarization. Generation of I(ClCa) relied on Ca(2+) entry through dihydropyridine-sensitive Ca(2+) channels because I(ClCa) was abolished by 1 microM nicardipine and enhanced by raising external Ca(2+) concentration or by application of BAY K 8644. I(ClCa) was characterized by the sensitivity to Cl(-) channel blockers and the effect of altering the external anion on reversal potential. Activation of I(ClCa) after membrane depolarization was dependent on Ca(2+) release from intracellular stores. Thus the amplitude of I(ClCa) was diminished by the SR-ATPase inhibitor cyclopiazonic acid, the inositol 1,4,5-trisphosphate receptor antagonist 2-aminoethoxydiphenyl borate (2-APB), and the ryanodine receptor blocker tetracaine. The degree of inhibition produced by the application of 2-APB and tetracaine together was significantly greater than the effect of each agent applied alone. In current-clamp mode, injection of depolarizing current elicited a biphasic action potential, with the later depolarization being sensitive to niflumic acid (NFA; 10 microM). In isometric tension recordings, NFA inhibited spontaneous contractions. These data support a role for this conductance in portal vein excitability.
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PMID:Activation of chloride currents in murine portal vein smooth muscle cells by membrane depolarization involves intracellular calcium release. 1535 51

The existence of functionally distinct intracellular Ca(2+) stores has been proposed in some types of smooth muscle. In this study, we sought to examine Ca(2+) stores in the gallbladder by measuring intracellular Ca(2+) concentration ([Ca(2+)](i)) in fura 2-loaded isolated myocytes, membrane potential in intact smooth muscle, and isometric contractions in whole mount preparations. Exposure of isolated myocytes to 10 nM CCK caused a transient elevation in [Ca(2+)](i) that persisted in Ca(2+)-free medium and was inhibited by 2-aminoethoxydiphenylborane (2-APB). Application of caffeine induced a rapid spike-like elevation in [Ca(2+)](i) that was insensitive to 2-APB but was abolished by pretreatment with 10 muM ryanodine. These data support the idea that both inositol trisphosphate (IP(3)) receptors (IP(3)R) and ryanodine receptors (RyR) are present in this tissue. When caffeine was applied in Ca(2+)-free solution, the [Ca(2+)](i) transients decreased as the interval between Ca(2+) removal and caffeine application was increased, indicating a possible leakage of Ca(2+) in these stores. The refilling of caffeine-sensitive stores involved sarcoendoplasmic reticulum Ca(2+)-ATPase activation, similar to IP(3)-sensitive stores. The moderate Ca(2+) elevation caused by CCK was associated with a gallbladder contraction, but caffeine or ryanodine failed to induce gallbladder contraction. Nevertheless, caffeine caused a concentration-dependent relaxation in gallbladder strips either under resting tone conditions or precontracted with 1 muM CCK. Taken together, these results suggest that, in gallbladder smooth muscle, multiple pharmacologically distinct Ca(2+) pools do not exist, but IP(3)R and RyR must be spatially separated because Ca(2+) release via these pathways leads to opposite responses.
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PMID:Characterization of intracellular Ca(2+) stores in gallbladder smooth muscle. 1549 78

Although L-type voltage-dependent calcium channels play a major role in mediating vascular smooth muscle cell contraction in the renal vasculature, non-L-type calcium entry mechanisms represent a significant component of vasoactive agonist-induced calcium entry in these cells as well. To investigate the role of these non-voltage-dependent calcium entry pathways in the regulation of renal microvascular reactivity, we have characterized the function of store- and receptor-operated channels (SOCs and ROCs) in renal cortical interlobular arteries (ILAs) of rats. Using fura 2-loaded, microdissected ILAs, we find that the L-type channel antagonist nifedipine blocks less than half the rise in intracellular calcium concentration ([Ca(2+)](i)) elicited by norepinephrine. SOCs were activated in these vessels using the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) inhibitors cyclopiazonic acid and thapsigargin and were dose dependently blocked by the SOC antagonists Gd(3+) and 2-aminoethoxydiphenyl borate (2-APB) and the combined SOC/ROC antagonist SKF-96365. Gd(3+) had no effect on the non-L-type Ca(2+) entry activated by 1 microM NE. A low concentration of SKF-96365 that did not affect thapsigargin-induced store-operated Ca(2+) entry blocked 60-70% of the NE-induced Ca(2+) entry. Two different calmodulin inhibitors (W-7 and trifluoperazine) also blocked the NE-induced Ca(2+) entry. These data suggest that in addition to L-type channels, NE primarily activates ROCs rather than SOCs in ILAs and that this receptor-operated Ca(2+) entry mechanism is regulated by calmodulin. Interestingly, 2-APB completely blocked the NE-induced non-L-type Ca(2+) entry, implying that SOCs and ROCs in preglomerular resistance vessels share a common molecular structure.
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PMID:Calmodulin mediates norepinephrine-induced receptor-operated calcium entry in preglomerular resistance arteries. 1570 15

Despite extensive work in the field of glioblastoma research no significant increase in survival rates for this devastating disease has been achieved. It is known that disturbance of intracellular Ca(2+) ([Ca(2+)](i)) and intracellular pH (pH(i)) regulation could be involved in tumor formation. The sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) is a major regulator of [Ca(2+)](i). We have investigated the effect of inhibition of SERCA by thapsigargin (TG) on [Ca(2+)](i) and pH(i) in human primary glioblastoma multiforme (GBM) cells and GBM cell lines, compared with normal human astrocytes, using the fluorescent indicators fura-2 and BCECF, respectively. Basal [Ca(2+)](i) was higher in SK-MG-1 and U87 MG but not in human primary GBM cells compared with normal astrocytes. However, in tumor cells, TG evoked a much larger and faster [Ca(2+)](i) increase than in normal astrocytes. This increase was prevented in nominally Ca(2+)-free buffer and by 2-APB, an inhibitor of store-operated Ca(2+) channels. In addition, TG-activated Ca(2+) influx, which was sensitive to 2-APB, was higher in all tumor cell lines and primary GBM cells compared with normal astrocytes. The pH(i) was also elevated in tumor cells compared with normal astrocytes. TG caused acidification of both normal and all GBM cells, but in the tumor cells, this acidification was followed by an amiloride- and 5-(N,N-hexamethylene)-amiloride-sensitive recovery, indicating involvement of a Na(+)/H(+) exchanger. In summary, inhibition of SERCA function revealed a significant divergence in intracellular Ca(2+) homeostasis and pH regulation in tumor cells compared with normal human astrocytes.
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PMID:Changes in intracellular Ca2+ and pH in response to thapsigargin in human glioblastoma cells and normal astrocytes. 1580 52


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