Atrial natriuretic peptide blunts the cellular effects of cyclosporine in smooth muscle

The effect of cyclosporine A to enhance vasoconstrictor-induced calcium (Ca2+) mobilization in vascular smooth muscle cells may contribute to important side effects in cyclosporine therapy such as hypertension and nephrotoxicity. On the other hand, atrial natriuretic peptide (ANP) is known to diminish vasoconstrictor-stimulated Ca2+ mobilization. The present study, therefore, examined the interaction of cyclosporine and ANP on Ca2+ kinetics in cultured rat vascular smooth muscle cells. Intracellular free calcium concentrations ([Ca2+]i) were measured using fura-2. 45Ca2+ was used to estimate Ca2+ efflux and cellular Ca2+ influx. Preincubation of the cells with cyclosporine (10 micrograms/ml) for 12 minutes lowered basal [Ca2+]i from 48 +/- 4 to 28 +/- 3 nM (p < 0.01). However, in the presence of cyclosporine, the angiotensin II (10(-8) M)-stimulated rise of [Ca2+]i was increased from 296 +/- 22 to 460 +/- 47 nM (p < 0.001). ANP (5 x 10(-9) M) blocked the Ca2+ mobilization by angiotensin II (71 +/- 7 versus 69 +/- 7 nM, NS) and also completely inhibited the effect of angiotensin II in the presence of cyclosporine (77 +/- 5 versus 78 +/- 5 nM, NS). Basal efflux as well as angiotensin II-stimulated 45Ca2+ efflux were not altered by preincubation with cyclosporine, indicating that the effect of cyclosporine on [Ca2+]i was not due to an inhibition of 45Ca2+ efflux.(ABSTRACT TRUNCATED AT 250 WORDS)     

A randomized study of combined spinal-epidural analgesia versus intravenous meperidine during labor: impact on cesarean delivery rate

We recently reported on the successful generation of immortalized (CEPI-17-CL4) cells from primary human corneal epithelial (P-CEPI) cells which exhibited phenotypic, immunohistochemical and metabolic characteristics akin to the P-CEPI cells. The aims of the present studies were to investigate the ligand binding and functional coupling of the histamine receptors to various biochemical and physiological systems in the P-CEPI and CEPI-17-CL4 cells and to relate these findings to the normal and/or pathophysiological role of histamine on the human ocular surface. Specific [3H]-pyrilamine binding to CEPI-17-CL4 cell homogenates comprised >93% of the total binding and represented interaction with an apparent single population of high affinity (Kd=3.76+/-0.78 nM; n=4) and saturable (Bmax = 1582+/-161 fmol g(-1) tissue) number of histamine-1 (H1) receptor binding sites on CEPI-17-CL4 cell homogenates. The H1-receptor selective antagonists, pyrilamine (Ki=3.6+/-0.84 nM, n=4) and triprolidine (Ki = 7.7+/-2.6 nM, n=3), potently displaced [3H]-pyrilamine binding, while the H2- and H3-receptor selective antagonists, ranitidine and clobenpropit, were weak inhibitors (K(i)s>13 microM). Histamine induced phosphoinositide (PI) hydrolysis 2.7-4.4 fold above basal levels and with a potency of 14.9+/-4.9 microM (n=9) and 4.7+/-0.2 microM (n=9) in P-CEPI and CEPI-17-CL4 cells, respectively. Histamine-induced PI turnover was antagonized by H1-receptor selective antagonist, triprolidine, with a potency (Ki) of 3.2+/-0.66 nM (n=10) and 3.03+/-0.8 nM (n=4) in P-CEPI and CEPI-17-CL4 cells, respectively, but weakly effected by 10 microM cimetidine and clobenpropit, H2- and H3-receptor antagonists. The PI turnover response was attenuated by pre-treatment of the cells with the selective phospholipase C inhibitor, U73122 (1-(6-((17beta-3-methoxyestra- 1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) (IC50=4.8+/-2.4 microM, n = 3). Histamine stimulated intracellular Ca2+ ([Ca2+]i) mobilization in CEPI-17-CL4 cells with a potency of 6.3+/-1.5 microM (n=4). The histamine-induced [Ca2+]i mobilization was reduced by about 28% following pre-incubation of the cells with 4 mM EGTA. While triprolidine completely inhibited histamine-induced [Ca2+]i mobilization, it did not influence the bradykinin-induced [Ca2+]i mobilization response. Histamine (EC50s = 1.28-2.77 microM, n=3-4) concentration-dependently stimulated the release of interleukin-6 (IL-6), IL-8 and granulocyte macrophage colony-stimulating factor, but it did not significantly alter release of tumour necrosis factor-alpha, PGE2 or collagenase-1 (matrix metalloproteinase-1; MMP-1) from CEPI cells. However, IL-1 (10 ng ml(-1)), foetal bovine serum (10%) and phorbol-12-myristate-13-acetate (3 microg ml(-1)) were effective positive control secretagogues of all the cytokines, PGE2 and MMP-1, respectively, from these cells. It is concluded that the CEPI cells express H1-histamine receptors which are positively coupled to PI turnover and [Ca2+]i mobilization which may be directly or indirectly responsible for the release of various cytokines from these cells at physiologically and/or pathologically relevant concentrations.     

Inhibition of bradykinin-induced calcium increase by phosphatase inhibitors in neuroblastoma x glioma hybrid NG108-15 cells

Prior treatment of NG108-15 cells with phosphatase inhibitors including okadaic acid and calyculin A inhibited the elevation of cytosolic Ca2+ concentration ([Ca2+]i) induced by bradykinin by approximately 63%. This inhibition was dependent on the concentration of okadaic acid with an IC50 of 0.15 nM. Okadaic acid treatment only lowered the maximal response of [Ca2+]i increase and had no effect on the EC50 value for bradykinin regardless of the presence of extracellular Ca2+. Neither the capacity of 45Ca2+ accumulation within intracellular nonmitochondrial Ca2+ stores nor the magnitude of [Ca2+]i increase induced by thapsigargin was reduced by the treatment of okadaic acid. In contrast, the same phosphatase inhibitor treatment inhibited the bradykinin-evoked inositol 1,4,5-trisphosphate (IP3) generation, the Mn2+ influx, and the capacity of mitochondrial Ca2+ accumulation. Furthermore, the sensitivity of IP3 in the Ca2+ release was suppressed by okadaic acid pretreatment. Our results suggest that the reduction of bradykinin-induced [Ca2+]i rise by the promotion of protein phosphorylation was attributed to the reduced activity of phospholipase C, the decreased sensitivity to IP3, and the slowed rate of Ca2+ influx. Thus, phosphorylation plays a role in bradykinin-sensitive Ca2+ signaling cascade in NG108-15 cells.     

Different mechanisms for [Ca2+]i oscillations induced by carbachol and high concentrations of [Ca2+]o in the rat ventricular myocyte

1. The purpose of the present study was to explore the different mechanisms of [Ca2+]i oscillations induced by high concentrations of either carbachol (CCh) or extracellular Ca2+ ([Ca2+]o). First, we compared the oscillations induced by CCh at concentrations of 100-300 micromol/L and [Ca2+]o (5 mmol/L) in the single rat ventricular myocyte. Second, we studied CCh- and [Ca2+]o-induced [Ca2+]i oscillations following either interference with the production of inositol trisphosphate (IP3), reductions in cytosolic Ca2+ ([Ca2+]i), inhibition of Ca2+ influx and Na+-Ca2+ exchange or depletion of Ca2+ from its intracellular store. 2. The [Ca2+]i oscillations induced by CCh were frequent and were superimposed on [Ca2+]i transients in electrically stimulated cells, whereas those induced by high [Ca2+]o were occasional and occurred in quiescent cells and between [Ca2+]i transients in electrically stimulated cells. In both cases, [Ca2+]i oscillations were preceded by an increase in resting levels of [Ca2+]i. 3. Carbachol-induced [Ca2+]i oscillations were accompanied by an increase in amplitude and prolongation of the time of decline to 80% of the peak of the [Ca2+]i transient, while high [Ca2+]o-induced [Ca2+]i oscillations were the opposite. 4. A reduction of [Ca2+]o to 0.1 mmol/L and treatment with Ni2+ or ryanodine or 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid AM (BAPTA-AM) abolished the [Ca2+]i oscillations induced by both CCh and high [Ca2+]o. 5. The calcium channel blockers verapamil and nifedipine and inhibitors of phospholipase C (neomycin and U-73122) abolished the [Ca2+]i oscillations induced by CCh; Li+ accelerated the onset of the [Ca2+]i oscillations induced by CCh. 6. These observations suggest that the mechanisms responsible for the [Ca2+]i oscillations induced by CCh and high [Ca2+]o are different from each other. Other than an increase in extracellular Ca2+ influx as a mechanism common for both CCh- and high [Ca2+]o-induced [Ca2+]i oscillations, the CCh-induced [Ca2+]i oscillations involve influx of Ca2+ via L-type Ca2+ channels, Na+-Ca2+ exchange, mobilization of intracellular Ca2+ and IP3 production.     

Different mechanisms are involved in intracellular calcium increase by insulin-like growth factors 1 and 2 in articular chondrocytes: voltage-gated calcium channels, and/or phospholipase C coupled to a pertussis-sensitive G-protein

This study describes the mechanisms involved in the IGF-1 and IGF-2-induced increases in intracellular calcium concentration [Ca2+]i in cultured chondrocytes and the involvement of type 1 IGF receptors. It shows that IGF-1, IGF-2, and insulin increased the cytosolic free calcium concentration [Ca2+]i in a dose-dependent manner, with a plateau from 25 to 100 ng/ml for both IGF-1 and IGF-2 and from 1 to 2 micrograms/ml for insulin. The effect of IGF-1 was twice as great as the one of IGF-2, and the effect of insulin was 40% lower than IGF-1 effect. Two different mechanisms are involved in the intracellular [Ca2+]i increase. 1) IGF-1 and insulin but not IGF-2 involved a Ca2+ influx through voltage-gated calcium channels: pretreatment of the cells by EGTA and verapamil diminished the IGF-1 or insulin-induced [Ca2+]i but did not block the effect of IGF-2. 2) IGF-1, IGF-2, and insulin also induced a Ca2+ mobilization from the endoplasmic reticulum: phospholipase C (PLC) inhibitors, neomycin, or U-73122 partially blocked the intracellular [Ca2+]i increase induced by IGF-1 and insulin and totally inhibited the effect of IGF-2. This Ca2+ mobilization was pertussis toxin (PTX) dependent, suggesting an activation of a PLC coupled to a PTX-sensitive G-protein. Lastly, preincubation of the cells with IGF1 receptor antibodies diminished the IGF-1-induced Ca2+ spike and totally abolished the Ca2+ influx, but did not modify the effect of IGF-2. These results suggest that IGF-1 action on Ca2+ influx involves the IGF1 receptor, while part of IGF-1 and all of IGF-2 Ca2+ mobilization do not implicate this receptor.     

High magnesium concentration inhibits ligand-stimulated calcium influx and hormone secretion in rat pituitary lactotropes with involvement of intracellular free magnesium

The effects of extracellular magnesium concentration ([Mg2+]ex) on thyrotropin-releasing hormone (TRH)-stimulated intracellular free calcium mobilization and prolactin secretion were investigated concomitantly with measurement of the intracellular free magnesium concentration ([Mg2+]i). TRH-stimulated intracellular free calcium mobilization was significantly inhibited when the medium was replaced by high Mg2+ medium ([Mg2+]ex = 10 mM) in normal Ca2+ medium. The inhibitory effects of high Mg2+ became apparent concomitantly with an increase in [Mg2+]i from 0.7 to 1.3 mM. High Mg2+ significantly inhibited TRH-induced PRL secretion in a dose-dependent manner in normal Ca2+ medium. TRH-stimulated inositol triphosphate (IP3) production was rather augmented by the replacement with high Mg2+ medium. In summary, high Mg2+ inhibits Ca2+ influx stimulated by TRH in the rat pituitary lactotropes, possibly with the involvement of [Mg2+]i increase. These results have general importance in relation to high Mg(2+)-induced suppression of the biological functions of cells.     

Role of endoscopic injection therapy in the treatment of bleeding peptic ulcer

Transforming growth factor beta (TGF beta) was examined regarding its regulation of the mitogen EGF. A431 human epidermoid carcinoma cells were treated with TGF beta and epidermal growth factor (EGF) (10 ng/ml each) to determine if TGF beta modulates EGF-induced Ca2+ signaling and c-Fos oncoprotein levels. Changes in [Ca2+]i were determined by digital imaging analysis or photon counting. In HBSS + Ca2+ (1.37 mM), EGF treatment resulted in a transient increase in [Ca2+]i from 75 to 150 nM, which lasted approximately 3.5 min and re-equilibrated to 90 nM. In nominally Ca(2+)-free (2-5 muM) HBSS, EGF caused a [Ca2+]i elevation that peaked at 140 nM and returned to baseline. TGF beta in HBSS + Ca2+ did not elicit a [Ca2+]i increase, although affinity labeling revealed types I, II, and III TGF beta receptors. TGF beta added simultaneously with EGF in HBSS + Ca2+ caused a gradual rise in [Ca2+]i from 50 to 100 nM over 16 min. Pretreatment with TGF beta (3 h; 10 ng/ml) abolished the EGF-induced [Ca2+]i elevation. EGF or TGF beta treatments increased c-Fos immunoreactivity by around 1 h. In summary, EGF elevated [Ca2+]i in the presence or absence of [Ca2+]e, resulting in high [Ca2+]n, associated with tyrosine and threonine phosphorylation, and increased c-Fos oncoprotein immunoreactivity. TGF beta did not increase [Ca2+]i but did increase c-Fos; TGF beta + EGF added simultaneously altered the EGF-induced [Ca2+]i elevation, and TGF beta pretreatment eliminated EGF-induced [Ca2+]i elevation. This suggests that TGF beta can regulate EGF in A431 cells and that increased c-Fos may not be mediated by Ca2+.     

Stimulation of Fc(alpha) receptors induces tyrosine phosphorylation of phospholipase C-gamma(1), phosphatidylinositol phosphate hydrolysis, and Ca2+ mobilization in rat and human mesangial cells

Although knowledge of IgA Fc receptor (Fc(alpha)R) structure and gene organization has progressed in the past few years, signal transduction pathways elicited by its activation have hardly been studied. Previously, we have demonstrated that mesangial cells (MC) possess Fc(alpha)R stimulation triggers several biologic responses. In this work, we studied the early biochemical signals triggered by Fc(alpha)R stimulation in MC. MC incubation with aggregated IgA (AIgA) elicited a dose-dependent increase in cytosolic Ca2+ ([Ca2+]i). The response was rapid and transient, and slowly fell to the original baseline. Ca2+ mobilization was dependent on the Fc region of the IgA, because Fc, but neither Fab fragment nor carbohydrates, inhibited the [Ca2+] rise. The initial induction of [Ca2+]i rise was due to Ca2+ mobilization from inositol trisphosphate (IP3)-sensitive intracellular stores, while sustained levels were maintained through extracellular Ca2+ influx. Stimulation of Fc(alpha)R also resulted in production of IP3, temporally correlated with Ca2+ mobilization. Protein tyrosine kinase inhibitors abolished [Ca2+]i rise, indicating that tyrosine phosphorylation of some substrates is required for Ca2+ mobilization. Stimulation through Fc(alpha)R gave rise to a marked increase in tyrosine phosphorylation of several proteins, including the 147-kDa band, similar in size to phospholipase C-gamma(1) (PLC-gamma(1)). Tyrosine phosphorylation of PLC-gamma(1) reached a maximum 30 s after stimulation, as determined by immunoprecipitation and Western blot. Collectively, these results indicate that the Fc(alpha)R signaling pathway in MC involves PLC-(gamma(1) activation, IP3 formation, and Ca2+ mobilization, and is linked to activation of tyrosine kinases.     

Presence of m3 subtype muscarinic acetylcholine receptors and receptor-mediated increases in the cytoplasmic concentration of Ca2+ in Jurkat, a human leukemic helper T lymphocyte line

Recent studies have demonstrated the presence and the regulatory function of several neurotransmitters in the immune system. In the present study, we examined the presence of acetylcholine receptors, using pharmacological and molecular biological assays, and their transmembrane control and functions, using a biochemical assay, in a cloned human leukemic helper T lymphoma cell line, Jurkat. Several muscarinic agonists, such as acetylcholine, carbachol, muscarine, and oxotremorine-M (Oxo-M), at 100 microM caused a transient elevation of the free cytosolic Ca2+ concentration ([Ca2+]i), in contrast to the tonic elevation of [Ca2+]i induced by 10 micrograms/ml phytohemagglutinin (PHA). It appeared that the elevation induced by Oxo-M, the most potent [Ca2+]i elevator, was more effectively inhibited by p-fluorohexahydrosiladifenidol hydrochloride (p-F-HHSiD) and 4-diphenylacetoxy-N-methylpiperidine methiodine than by pirenzepine and 11-2[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro- 6H-pyrido[2,3-b] [1,4]benzodiazepine-6-one (AF-DX 116), suggesting that a pharmacological M3 subtype of muscarinic receptors is involved in the elevation of [Ca2+]i. Northern blot analysis showed that the m3 type of receptors are expressed in Jurkat cells. Scatchard analysis of [3H]quinuclidinyl benzilate binding to intact cells indicated a Kd of 14.1 nM and a Bmax of 45,370 binding sites/cell. [3H]Quinuclidinyl benzilate binding to cell membranes was also inhibited by p-F-HHSiD rather than by pirenzepine and AF-DX 116. Oxo-M induced formation of inositol trisphosphate, and 5'-O-(2-thio)diphosphate inhibited the formation. Cholera toxin treatment inhibited the PHA-induced [Ca2+]i rise but did not affect the Oxo-M-induced rise. Neither pertussis nor butulinus (type C) toxin affected the rise induced by Oxo-M or PHA. Thus, bacterial toxin-insensitive GTP-binding proteins seem to be involved in the Oxo-M-induced increase in [Ca2+]i. Treatment with 12-O-tetradecanoylphorbol 13-acetate abolished the Oxo-M-induced [Ca2+]i rise but did not affect that induced by PHA. m3 Muscarinic receptors thus appear to cause Ca2+ mobilization from intracellular stores via bacteria toxin-insensitive GTP-binding proteins, phospholipase C activation, and inositol trisphosphate formation in Jurkat cells. Protein kinase C seems to negatively modulate the m3 receptor system.     

Ca2+ signaling induced by sphingosylphosphorylcholine and sphingosine 1-phosphate via distinct mechanisms in rat glomerular mesangial cells

BACKGROUND: To elucidate the molecular mechanism underlying sphingosine 1-phosphate (S1P) and sphingosylphosphorylcholine (SPC) mediated signaling, we compared their effects with those of adenosine triphosphate (ATP) and angiotensin II (Ang II) on the cytosolic free Ca2+ concentration ([Ca2+]i), inositol 1,4, 5-trisphosphate (IP3) generation and arachidonic acid release in rat glomerular mesangial cells. METHODS: The fluorescent Ca2+ indicator, Fura-2, was used to measure the [Ca2+]i changes in cultured rat glomerular mesangial cells either in suspension or attached to the coverslips. RESULTS: SPC 5 microM, S1P 5 microM, ATP 100 microM and Ang II 90 nM all induced increases in the [Ca2+]i, and the effect showed marked homologous desensitization, while heterologous desensitization was less. After the initial exposure of the cells to SPC, the increase in [Ca2+]i induced by subsequent addition of ATP or Ang II was only reduced by about 14.3% and 4.8%, respectively. After the initial exposure to S1P, a greater reduction was seen (42. 1% and 47.7%, respectively). Both arachidonic acid release and IP3 generation were activated by all four agonists with an identical rank order of effectiveness of SPC > S1P > ATP = Ang II; both were pertussis toxin-sensitive and cholera toxin-resistant. The arachidonic acid release induced by all four agonists showed identical susceptibility to removal of extracellular Ca2+, whereas IP3 generation displayed differential extracellular Ca2+ dependence. Only SPC-induced IP3 generation was highly sensitive to extracellular Ca2+ level, and this Ca2+ dependence was abolished after pretreatment of cells with arachidonyl trifluoromethyl ketone (AACOCF3), a phospholipase A2 inhibitor. Furthermore, the Mn2+ influx was markedly greater in SPC-stimulated cells than in either control or other agonist-stimulated cells, and was decreased by prior exposure of cells to AACOCF3. After phospholipase A2 was inhibited or in the absence of extracellular Ca2+, SPC displayed identical effectiveness as S1P on desensitizing the action of ATP or Ang II on the increase in [Ca2+]i. Conclusions. Our results indicate that all four agents primarily activate phospholipase C through their receptor occupancies, but that SPC alone also induces further significant Mn2+ influx and IP3 generation attributable to its primary stimulatory effect on arachidonic acid release. Thus, the heterologous desensitization to ATP or Ang II induced by SPC was less profound than that induced by S1P, since SPC induced a Ca2+ influx.     

Inositol 1,4,5-trisphosphate [correction of tris-phosphate] activation of inositol trisphosphate [correction of tris-phosphate] receptor Ca2+ channel by ligand tuning of Ca2+ inhibition

Inositol 1,4,5-trisphosphate (IP3) [corrected] binding to its receptors (IP3R) in the endoplasmic reticulum (ER) activates Ca2+ release from the ER lumen to the cytoplasm, generating complex cytoplasmic Ca2+ concentration signals including temporal oscillations and propagating waves. IP3-mediated Ca2+ release is also controlled by cytoplasmic Ca2+ concentration with both positive and negative feedback. Single-channel properties of the IP3R in its native ER membrane were investigated by patch clamp electrophysiology of isolated Xenopus oocyte nuclei to determine the dependencies of IP3R on cytoplasmic Ca2+ and IP3 concentrations under rigorously defined conditions. Instead of the expected narrow bell-shaped cytoplasmic free Ca2+ concentration ([Ca2+]i) response centered at approximately 300 nM-1 microM, the open probability remained elevated (approximately 0.8) in the presence of saturating levels (10 microM) of IP3, even as [Ca2+]i was raised to high concentrations, displaying two distinct types of functional Ca2+ binding sites: activating sites with half-maximal activating [Ca2+]i (Kact) of 210 nM and Hill coefficient (Hact) approximately 2; and inhibitory sites with half-maximal inhibitory [Ca2+]i (Kinh) of 54 microM and Hill coefficient (Hinh) approximately 4. Lowering IP3 concentration was without effect on Ca2+ activation parameters or Hinh, but decreased Kinh with a functional half-maximal activating IP3 concentration (KIP3) of 50 nM and Hill coefficient (HIP3) of 4 for IP3. These results demonstrate that Ca2+ is a true receptor agonist, whereas the sole function of IP3 is to relieve Ca2+ inhibition of IP3R. Allosteric tuning of Ca2+ inhibition by IP3 enables the individual IP3R Ca2+ channel to respond in a graded fashion, which has implications for localized and global cytoplasmic Ca2+ concentration signaling and quantal Ca2+ release.     

An immunosuppressive agent, FTY720, increases intracellular concentration of calcium ion and induces apoptosis in HL-60

We previously reported that FTY720 is an efficient inducer of apoptosis in lymphocytes and cultured cell lines. In the present study, HL-60 human promyerocytoma cells also induced apoptosis through in vitro treatment with the drug, demonstrating extensive DNA fragmentation 6 hr after incubation. The major target of FTY720 was the common signalling pathway of apoptosis, since a rapid (< 1 min) increase in the intracellular Ca2+ concentration ([Ca2+]i) was found in the cells treated with the drug. Calcium chelation in the culture medium with EGTA did not affect the [Ca2+]i mobilization. A phospholipase C inhibitor, U73122, inhibited the increase in [Ca2+]i as well as the fragmentation of the nuclear DNA, whereas U73343, a non-effective analogue of U73122, had little effect. These results suggest that FTY720-induced apoptosis is mediated through an activation of phospholipase C and the subsequent release of Ca2+ from intracellular calcium pools. In addition, the treatment of HL-60 with pertussis toxin (PTX) did not inhibit Ca2+ mobilization or apoptosis, suggesting that the activation of phospholipase C is independent of PTX-sensitive G-proteins.     

Ca2+ signalling by endothelin receptors in rat and human cultured airway smooth muscle cells

1. The aim of the current study was to characterize the ET receptor subtypes in cultured airway smooth muscle cells derived from rat trachea and human bronchus using radioligand binding techniques and to investigate the coupling of ET receptors to intracellular calcium signalling mechanisms using endothelin receptor-selective agonists (sarafotoxin S6c) and antagonists (BQ-123, BQ-788) and digital image fluorescence microscopy. 2. Confluent rat airway smooth muscle cells in culture possessed a mixed ET receptor population (30% ETA : 70% ETB), with a density of approximately 3400+/-280 ETA and 8000+/-610 ETB receptors/cell (n = 3 experiments). The density of ETB, but not ETA receptors increased substantially in serum-containing medium. However, a 2-day period of serum deprivation, which inhibited cellular growth, substantially reduced ETB receptor density such that the ET receptor subtype proportions were approximately equal (55% ETA; 45% ETB) and similar to those previously observed in intact rat tracheal smooth muscle. 3. Challenge of rat airway smooth muscle cells in culture with endothelin- 1 elicited a concentration-dependent biphasic increase in [Ca2+]i (EC50: 16 nM), that comprised an initial transient peak [Ca2+]i increase (typically 350 nM) followed by a modest sustained component. The endothelin-1-induced biphasic [Ca2+]i increase was primarily due to ETA receptor activation, although a modest and inconsistent ETB response was observed. The ETA-mediated [Ca2+]i increase was due primarily to the mobilization of IP3-sensitive and to a lesser extent ryanodine-sensitive intracellular calcium stores. In contrast, ETB receptor activation was exclusively coupled to extracellular calcium influx. 4. Somewhat surprisingly, human airway smooth muscle cells in culture contained a homogeneous population of ETA receptors at a density of 6100+/-800 receptors cell(-1) (n = 3 experiments). Serum deprivation was without effect on either ET receptor subtype proportion or ETA receptor density. Challenge of human airway smooth muscle cells with endothelin-1 provoked a concentration-dependent increase in [Ca2+]i (EC50: 15 nM), with a peak [Ca2+]i increase to greater than 700 nM. Furthermore, the ETA-mediated calcium response in these human airway smooth muscle cells in culture was entirely dependent upon the mobilization of calcium from intracellular stores. 5. In summary, rat cultured tracheal airway smooth muscle cells contained both ETA and ETB receptors. ETA receptors, the numbers of which remained constant during cell growth, were linked to the release of Ca2+ from intracellular stores and a strong rise in [Ca2+]i in the majority of airway smooth muscle cells. In stark contrast, the numbers of ETB receptors increased significantly during cell growth, an effect that was diminished substantially by incubation in serum-free medium. Moreover, despite the greater number of ETB receptors, their activation in a small number of airway smooth muscle cells produced only a weak rise in [Ca2+]i, which appeared to be attributable to the influx of extracellular Ca2+. In contrast, the populations of ET receptors and their linkage to [Ca2+]i were markedly different in the human cultured airway smooth muscle cells used in the current study compared to that previously observed in intact human isolated bronchial smooth muscle.     

Effect of cilostazol, a phosphodiesterase type III inhibitor, on histamine-induced increase in [Ca2+]i and force in middle cerebral artery of the rabbit

1. The effect of cilostazol, an inhibitor of phosphodiesterase type III (PDE III), on the contraction induced by histamine was studied by making simultaneous measurements of isometric force and the intracellular concentration of Ca2+ ([Ca2+]i) in endothelium-denuded muscle strips from the peripheral part of the middle cerebral artery of the rabbit. 2. High K+ (80 mM) produced a phasic, followed by a tonic increase in both [Ca2+]i and force. Cilostazol (10 microM) did not modify the resting [Ca2+]i, but it did significantly decrease the tonic contraction induced by high K+ without a corresponding change in the [Ca2+]i response. 3. Histamine (3 microM) produced a phasic, followed by a tonic increase in both [Ca2+]i and force. Cilostazol (3 and 10 microM) significantly reduced both the phasic and tonic increases in [Ca2+]i and force induced by histamine, in a concentration-dependent manner. 4. Rp-adenosine-3':5'-cyclic monophosphorothioate (Rp-cAMPS, 0.1 mM), a PDE-resistant inhibitor of protein kinase A (and as such a cyclic AMP antagonist), did not modify the increases in [Ca2+]i and force induced by histamine alone, but it did significantly decrease the cilostazol-induced inhibition of the histamine-induced responses. 5. In Ca2+-free solution containing 2 mM EGTA, both histamine (3 microM) and caffeine (10 mM) transiently increased [Ca2+]i and force. Cilostazol (1-10 microM) (i) significantly reduced the increases in [Ca2+]i and force induced by histamine, and (ii) significantly reduced the increase in force but not the increase in [Ca2+]i induced by caffeine. 6. In ryanodine-treated strips, which had functionally lost the histamine-sensitive Ca2+ storage sites, histamine (3 microM) slowly increased [Ca2+]i and force. Cilostazol (3 and 10 microM) lowered the resting [Ca2+]i, but did not modify the histamine-induced increase in [Ca2+]i, suggesting that functional Ca2+ storage sites are required for the cilostazol-induced inhibition of histamine-induced Ca2+ mobilization. 7. The [Ca2+]i-force relationship was obtained in ryanodine-treated strips by applying ascending concentrations of Ca2+ (0.16-2.6 mM) in Ca2+-free solution containing 100 mM K+. Histamine (3 microM) shifted the [Ca2+]i-force relationship to the left and increased the maximum Ca2+-induced force. Under the same conditions, whether in the presence or absence of 3 microM histamine, cilostazol (3-10 microM) shifted the [Ca2+]i-force relationship to the right without producing a change in the maximum Ca2+-induced force. 8. It is concluded that, in smooth muscle of the peripheral part of the rabbit middle cerebral artery, cilostazol attenuates the histamine-induced contraction both by inhibiting histamine-induced Ca2+ mobilization and by reducing the myofilament Ca2+ sensitivity. It is suggested that the increase in the cellular concentration of cyclic AMP that will follow the inhibition of PDE III may play an important role in the cilostazol-induced inhibition of the histamine-contraction.     

Tyrosine kinase inhibitors suppress prostaglandin F2alpha-induced phosphoinositide hydrolysis, Ca2+ elevation and contraction in iris sphincter smooth muscle

We investigated the effects of the protein tyrosine kinase inhibitors, genistein, tyrphostin 47, and herbimycin on prostaglandin F2alpha- and carbachol-induced inositol-1,4,5-trisphosphate (IP3) production, [Ca2+]i mobilization and contraction in cat iris sphincter smooth muscle. Prostaglandin F2alpha and carbachol induced contraction in a concentration-dependent manner with EC50 values of 0.92 x 10(-9) and 1.75 x 10(-8) M, respectively. The protein tyrosine kinase inhibitors blocked the stimulatory effects of prostaglandin F2alpha, but not those evoked by carbachol, on IP3 accumulation, [Ca2+]i mobilization and contraction, suggesting involvement of protein tyrosine kinase activity in the physiological actions of the prostaglandin. Daidzein and tyrphostin A, inactive negative control compounds for genistein and tyrphostin 47, respectively, were without effect. Latanoprost, a prostaglandin F2alpha analog used as an antiglaucoma drug, induced contraction and this effect was blocked by genistein. Genistein (10 microM) markedly reduced (by 67%) prostaglandin F2alpha-stimulated increase in [Ca2+]i but had little effect on that of carbachol in cat iris sphincter smooth muscle cells. Vanadate, a potent inhibitor of protein tyrosine phosphatase, induced a slow gradual muscle contraction in a concentration-dependent manner with an EC50 of 82 microM and increased IP3 generation in a concentration-dependent manner with an EC50 of 90 microM. The effects of vanadate were abolished by genistein (10 microM). Wortmannin, a myosin light chain kinase inhibitor, reduced prostaglandin F2alpha- and carbachol-induced contraction, suggesting that the involvement of protein tyrosine kinase activity may lie upstream of the increases in [Ca2+]i evoked by prostaglandin F2alpha. Further studies aimed at elucidating the role of protein tyrosine kinase activity in the coupling mechanism between prostaglandin F2alpha receptor activation and increases in intracellular Ca2+ mobilization and identifying the tyrosine-phosphorylated substrates will provide important information about the role of protein tyrosine kinase in the mechanism of smooth muscle contraction, as well as about the mechanism of the intraocular pressure lowering effect of the prostaglandin in glaucoma patients.     

The effect of a secreted form of beta-amyloid-precursor protein on intracellular Ca2+ increase in rat cultured hippocampal neurones

1. The effects of secreted forms of beta-amyloid-precursor proteins (APP(S)s) on the intracellular Ca2+ concentration ([Ca2+]i) were investigated in rat cultured hippocampal neurones. APP695S, a secretory form of APP695, attenuated the increase in [Ca2+]i evoked by glutamate. In addition, APP695S itself evoked an increase in [Ca2+]i in 1 or 2 day-cultured hippocampal cells, but not in 7 to 13 day-cultured cells. 2. Eighty-one percent of neurones which were immunocytochemically positive for microtubule-associated protein 2 responded to APP695S with an increase in [Ca2+]i. 3. APP695S induced a transient rise in [Ca2+]i even in the absence of extracellular Ca2+ and produced an elevation in inositol-1,4,5-trisphosphate (IP3) in a concentration-dependent manner from 100 to 500 ng ml(-1). In the presence of extracellular Ca2+, APP695S caused a transient rise in [Ca2+]i followed by a sustained phase at high [Ca2+]i, suggesting Ca2+ entry from the extracellular space. 4. The [Ca2+]i elevation was mimicked by amino terminal peptides of APPs, but not by carboxy terminal peptides. 5. These results taken together suggest that APP695S induces an increase in [Ca2+]i in hippocampal neurones through an IP3-dependent mechanism that changes according to the stage of development.     

Thrombin-dependent inhibition of fibrinolysis

AIM: To study the mechanisms underlying oxytocin (Oxy)-induced insulin release. METHODS: In a clonal pancreatic beta-cell line, RINm5F cells. RESULTS: Oxy increased insulin release and [Ca2+]i in a concentration-dependent manner. Oxy-induced insulin release was not altered by pretreatment with pertussis toxin (PT). U-73122 (2-8 mumol.L-1), a phospholipase C (PLC) inhibitor, concentration-dependently inhibited Oxy-induced increases in [Ca2+]i with IC50 value of 2.8 +/- 0.2 mumol.L-1. In addition, U-73122 diminished the Oxy-induced increase in intracellular concentration of inositol 1, 4, 5-triphosphate (IP3). U-73122 at 8 mumol.L-1 totally abolished the Oxy-induced increases in [Ca2+]i and IP3; however it reduced the Oxy-induced increase in insulin release only by 36% and 63% in the monolayer and suspended cell preparations, respectively. CONCLUSION: Oxy increases insulin release through both PLC and non-PLC mediated signal transduction mechanisms.     

The role of immune complex in otitis media with effusion

Lead characteristically perturbs processes linked to the calcium messenger system. This study was undertaken to determine the role of PKC in the Pb2+ induced rise of [Ca2+]i. [Ca2+]i was measured using the divalent cation indicator, 1,2-bis(2-amino-5-fluorophenoxy) ethane N, N,N',N'-tetraacetic acid (5F-BAPTA) and 19F-NMR in the osteoblast cell line, ROS 17/2.8. Treatment of cells with Pb2+ at 1 and 5 microM produced a rise in [Ca2+]i from a basal level of 125 nM to 170 nM and 230 nM, respectively, while treatment with phorbol 12-myristate 13-acetate (PMA) (10 microM), an activator of PKC, produced a rise in [Ca2+]i to 210 nM. Pretreatment with calphostin C, a potent and highly selective inhibitor of PKC activation failed to produce a change in basal [Ca2+]i and prevented any rise in [Ca2+]i in response to Pb2+. To determine whether Pb2+ acts directly on PKC, we measured the Pb2(+)-dependent activation of phosphatidylserine/diolein-dependent incorporation of 32P from ATP into histone and endogenous TCA precipitable proteins in the 100,000 X g supernatant from homogenized ROS 17/2.8 cells. The free concentrations of Pb2+ and Ca2+ were set using 5F-BAPTA; and [Ca2+] and [Pb2+] in the PKC reaction mixtures were confirmed by 19F-NMR. We found that Pb2+ activates PKC in the range of 10(-11)-10(-7) M, with an activation constant of 1.1 X 10(-10) M, whereas Ca2+ activates PKC in the range from 10(-8) to 10(-3) M, with an activation constant of 3.6 X 10(-7) M. These data suggest that Pb2+ activates PKC in ROS 17/2.8 cells and that Pb2+ activation of PKC mediates the documented rise in [Ca2+]i and, perhaps, other toxic effects of Pb2+.     

Allelic loss on chromosome 18q as a prognostic marker in stage II colorectal cancer

The aim of the study was to elucidate the vasodilatory mechanism due to Cu2+ by assessing nitric oxide (NO) production as determined by NOx (NO, NO2-, and NO3-) that is released from human pulmonary arterial endothelial cell (HPAEC) monolayers using a NO chemiluminescence analyzer, and also to assess Ca2+ movement using 45Ca and fura 2 in HPAEC. Cu2+ (10(-6)-10(-4) M) significantly increased NO production in a dose-dependent manner when extracellular Ca2+ was present. 45Ca influx into the adherent cells was dose-dependently enhanced by Cu(2+) (10(-6)-10(-4) M), but not by Mn(2+), Zn(2+) or Fe(2+). [Ca2+]i, measured by monitoring the fluorescence changes of fura 2, was significantly elevated in the presence of Cu2+. The increase in [Ca2+]i induced by Cu2+ was inhibited by either diethyldithiocarbamate (DDC) or the depletion of extracellular Ca2+. The dihydropyridine receptor agonist, BayK8644, significantly attenuated the Cu2+-induced increase in [Ca2+]i in a dose dependent manner and nitrendipine or nifedipine, the dihydropyridine receptor antagonists, dose-dependently inhibited a Cu2+-induced increase in [Ca2+]i. These results suggest that Cu2+ activates eNOS through the mechanism of [Ca2+]i elevation due to Ca2+ influx into HPAEC and that the Cu2+-induced [Ca2+]i elevation in HPAEC is likely due to activation of the dihydropyridine-like receptors.     

Cyclosporin A inhibits apical secretory K+ channels in rabbit cortical collecting tubule principal cells

We used the cell-attached patch clamp configuration to examine the effect of basolateral cyclosporin A (CsA) exposure on low conductance K+ channels found in the principal cell apical membrane of rabbit cortical collecting tubule (CCT) primary cultures. Baseline K+ channel activity, measured as mean NPo (number of channels x open probability), was 2.7 +/- 1.1 (N = 29). NPo fell by 69% (0.84 +/- 0.32; N = 32) in cultures pretreated with 500 ng/ml CsA for 30 minutes prior to patching. Chelation of intracellular [Ca2+]i (10 mM BAPTA/AM; N = 8) or removal of extracellular Ca2+ (N = 9), but not prevention of [Ca2+]i store release (10 microM TMB-8; N = 7), abolished CsA-induced inhibition. This suggested that CsA effects were mediated by an initial rise in [Ca2+]i via Ca2+ influx. Either 25 nM AVP (N = 10) or 0.25 microM thapsigargin (N = 8) (causing IP3-dependent and -independent release of [Ca2+]i stores, respectively) augmented, while 25 pM (N = 6) or 250 pM AVP (N = 8) reversed CSA-induced channel inhibition. Apical membrane protein kinase C (PKC) activation with 0.1 microM phorbol ester, PMA (N = 8) or 10 microM synthetic diacylglycerol, OAG (N = 7), mimicked (mean NPo = 0.99 +/- 0.40) the inhibitory effect of CsA. Apical PKC inhibition by prolonged apical exposure to PMA (N = 10) or 100 microM D-sphingosine (N = 6) blocked CsA's effect. Cyclic AMP increasing maneuvers, 10 microM forskolin (N = 5) or 0.5 mM db-cAMP (N = 8), stimulated basal K+ channel activity in the absence of CsA. In Conclusion: (1) basolateral exposure to CsA inhibits the activity of apical membrane 13 pS channels responsible for physiologic K+ secretion in rabbit CCT principal cells. (2) The inhibition is mediated by changes in intracellular Ca2+ and activation of apical PKC. (3) Pharmacologic AVP (nM) augments CsA-induced inhibition by releasing intracellular Ca2+ stores; more physiologic AVP (pM) attenuates channel inhibition, probably through cAMP generation. (4) Inhibition of apical secretory K+ channels by CsA likely contributes to decreased kaliuresis and clinical hyperkalemia observed in patients on CsA therapy.