Intracellular Free Calcium Levels Research Articles

Inhibition of LDL oxidation and oxidized LDL-induced cytotoxicity by dihydropyridine calcium antagonists.

The antioxidant activity of dihydropyridine calcium channel antagonists was evaluated based on LDL oxidation kinetics, oxidative cell injury associated with reactive species generation, and increases in free intracellular calcium (Ca2+) levels. Interactions with ascorbic acid were studied under conditions representative of LDL oxidation in plasma and tissue. Analysis of antioxidant activity utilized measurements of one-electron oxidation potentials and scavenging of peroxy radical-mediated oxidation. LDL antioxidant potency was determined spectrophotometrically using copper-mediated oxidation kinetics in the absence and presence of 100 microM ascorbic acid. Prevention of oxidant-induced endothelial cell injury was determined from the formation of reactive oxygen species generation and increases in intracellular free calcium concentrations following addition of oxidized LDL or linoleic acid hydroperoxide. Felodipine and amlodipine effectively inhibit peroxyl radical-mediated oxidation in lipoproteins and cells that is markedly enhanced in the presence of ascorbic acid. In the presence of ascorbic acid, inhibition of LDL oxidation is over four times greater than in LDL treated without antioxidants, and oxidized LDL and linoleic acid hydroperoxide-induced reactive oxygen species formation is effectively suppressed in cells. Inhibition of intracellular calcium increases was achieved using nM concentrations of felodipine or amlodipine. The additive effect for ascorbic acid and the calcium channel antagonist is postulated to involve a combination of peroxide-degrading and peroxyl radical scavenging reactions, demonstrating the importance of lipid peroxides during LDL oxidation and oxidized LDL-induced cytotoxicity. Cytoprotection is associated with inhibition of oxidant-induced increases in intracellular free calcium. Both the cytoprotective and LDL antioxidant activity for these compounds is manifested at concentrations approaching the therapeutic levels found in plasma.

The role of Src family kinases in starfish egg fertilisation

A common early feature in the activation of all eggs during fertilisation is an increase in the level of intra-cellular free calcium (Ca2+) that, in most species, propagates as a wave across the egg (reviewed in Strieker, 1999). In echinoderms, this Ca2+ release is the result of a signal transduction cascade that requires phospholipase Cγ (PLCγ)-mediated production of inositol trisphosphate (IP3) (Carroll et al., 1997, 1999). PLCγ is most commonly regulated by tyrosine phosphorylation (Rhee & Bae, 1997), indicating that a tyrosine kinase is a likely upstream regulator of PLCγ enzymatic activity at fertilisation. In support of this hypothesis, an increase in tyrosine kinase activity and an increase in tyrosine-phosphorylated proteins at fertilisation has been observed in echinoderm eggs (Satoh & Garbers, 1985; Ciapa & Epel, 1991; Kinsey, 1997). Moreover, the tyrosine kinase inhibitors genistein (Shen et al., 1999) and PP1 (Abassi et al., 2000) have been used to show that in sea urchin eggs a tyrosine kinase activity is required for normal Ca2+ release in response to fertilisation.In eggs of the starfish Asterina miniata, a Src-type tyrosine kinase has been identified as a potential regulator of PLCγ activity at fertilisation (Giusti et al., 1999a). This kinase exhibits a rapid fertilisation-dependent association specifically with the Src Homology 2 (SH2) domains of PLCγ. Moreover, the timing of this association correlates with an increase in the tyrosine kinase activity bound to the PLCγ SH2 domains, and neither the Src kinase nor the associated kinase activity was observed to associate with the PLCγ SH2 domains after treating eggs with the calcium ionophore A23187 (Giusti et al., 1999a). These data identify an egg Src family kinase as a potential upstream regulator of PLCγ during starfish egg fertilisation.

Phosphate induced developmental arrest of hamster two-cell embryos is associated with disrupted ionic homeostasis

Culture of hamster embryos with 0.35 mM inorganic phosphate results in developmental arrest at the 2-cell stage. These arrested 2-cell embryos were found to have significantly elevated levels of both intracellular pH and intracellular free calcium. Culture of 2-cell embryos with both glucose and phosphate did not further alter intracellular ionic homeostasis. Developmental arrest of 2-cell embryos was dependent on the concentration of phosphate used. Culture with 1.25 microM phosphate did not alter development, while concentrations of 2.5 microM and 5.0 microM resulted in a percentage of embryos arresting development at the 2-cell stage. Analysis of intracellular levels of pH and calcium after culture with different phosphate concentrations revealed a significant negative correlation between intracellular calcium levels and development beyond the 2-cell stage. There was no correlation between the increase in intracellular pH and embryo development in the presence of phosphate. The increase in intracellular calcium levels after culture with phosphate appears to be derived from intracellular pools, as preventing the influx of extracellular calcium did not alter development beyond the 2-cell stage. Therefore, it is apparent that a disruption in ionic homeostasis is associated with developmental arrest of hamster embryos cultured with phosphate.

Target Cells of 3-Melanocyte-Stimulating Hormone Detected through Intracellular Ca2+ Responses in Immature Rat Pituitary Constitute a Fraction of All Main Pituitary Cell Types, but Mostly Express Multiple Hormone Phenotypes at the Messenger Ribonucleic Acid Level. Refractoriness to Melanocortin-3 Receptor Blockade in the Lacto-Somatotroph Lineage

γ3-MSH has recently been shown to be a biologically active peptide in the rat anterior pituitary. It induces a sustained rise in intracellular free calcium levels ([Ca2+]i) in a relatively small population of immature pituitary cells. The present study was intended to identify the target cells of this peptide and to discern the signal-transducing melanocortin (MC) receptor. In dispersed pituitary cells from 14-day-old rats, increasing doses ofγ 3-MSH (0.1, 1, and 10 nm) evoked a sustained oscillating[ Ca2+]i rise in an increasing number of cells (up to 14.5%). Within the responsive cells, 53% showed GH immunoreactivity (-ir), 12% showed PRL-ir, 2% showed TSHβ-ir, 5% showed LHβ-ir, and 10% showed ACTH-ir, whereas 18% did not express any hormone-ir to a detectable level. As assessed by single cell RT-PCR for the presence of pituitary hormone messenger RNA (mRNA), 26% of theγ 3-MSH-responsive cells contained only GH mRNA, 5% contained only PRL mRNA, and 4% contained only TSHβ mRNA. Twenty-two percent contained mRNA of GH, PRL, and TSHβ in various dual or triple combinations. About 24% of the γ3-MSH-responsive cells expressed POMC mRNA, mostly together with other mRNAs, i.e. with GH mRNA and/or PRL mRNA or with mRNA of GH, PRL, and TSHβ. Eighteen percent of the responsive cells expressed LHβ, all of them together with mRNA of GH, PRL, and TSHβ in various combinations. The absence of hormone mRNA was found in less than 1% of the responsive cells. In cells chosen at random (representative of the total pituitary cell population), the proportion of cells expressing two or multiple hormone mRNAs was twice as low as that in the γ3-MSH-responsive population, whereas the proportion of cells expressing a single hormone mRNA was twice as high (about two thirds of all cells). Moreover, unlike in theγ 3-MSH-responsive cell population, randomly chosen cells were found that coexpressed POMC mRNA with LHβ mRNA. The effect of γ3-MSH on [Ca2+]i was blocked by the MC-3 receptor antagonist SHU9119 (used up to a 1000-fold excess) in 46% or less of the responsive cells. SHU9119 failed to block the[ Ca2+]i response to γ3-MSH in PRL-, GH-, and TSHβ-ir cells, but it did block the response in most ACTH-ir cells and in cells expressing no hormone to a detectable level. Single cell RT-PCR revealed that expression of MC-3 receptor mRNA was detected in only 16% of γ3-MSH-responsive cells. The present data suggest that the target cells of γ3-MSH in terms of[ Ca2+]i responses in the immature rat pituitary constitute subpopulations of all main pituitary cell types, including nonhormonal (or low expression hormonal) cells. However, in contrast to the total pituitary cell population, most of these cells display multilineage gene activation at the mRNA level, i.e. express mRNA of GH, PRL, TSHβ, POMC, and LHβ in dual, triple, or quadruple combinations. Although γ3-MSH may act through the MC-3 receptor in a portion of these cells, most of these cells (mainly in the lacto-somatotroph lineage) may transduce the signal through another receptor or through an MC-3 receptor with unconventional binding characteristics.

Target cells of gamma3-melanocyte-stimulating hormone detected through intracellular Ca2+ responses in immature rat pituitary constitute a fraction of all main pituitary cell types, but mostly express multiple hormone phenotypes at the messenger ribonucleic acid level. Refractoriness to melanocortin-3 receptor blockade in the lacto-somatotroph lineage.

Gamma3-MSH has recently been shown to be a biologically active peptide in the rat anterior pituitary. It induces a sustained rise in intracellular free calcium levels ([Ca2+]i) in a relatively small population of immature pituitary cells. The present study was intended to identify the target cells of this peptide and to discern the signal-transducing melanocortin (MC) receptor. In dispersed pituitary cells from 14-day-old rats, increasing doses of gamma3-MSH (0.1, 1, and 10 nM) evoked a sustained oscillating [Ca2+]i rise in an increasing number of cells (up to 14.5%). Within the responsive cells, 53% showed GH immunoreactivity (-ir), 12% showed PRL-ir, 2% showed TSHbeta-ir, 5% showed LHbeta-ir, and 10% showed ACTH-ir, whereas 18% did not express any hormone-ir to a detectable level. As assessed by single cell RT-PCR for the presence of pituitary hormone messenger RNA (mRNA), 26% of the gamma3-MSH-responsive cells contained only GH mRNA, 5% contained only PRL mRNA, and 4% contained only TSHbeta mRNA. Twenty-two percent contained mRNA of GH, PRL, and TSHbeta in various dual or triple combinations. About 24% of the gamma3-MSH-responsive cells expressed POMC mRNA, mostly together with other mRNAs, i.e. with GH mRNA and/or PRL mRNA or with mRNA of GH, PRL, and TSHbeta. Eighteen percent of the responsive cells expressed LHbeta, all of them together with mRNA of GH, PRL, and TSHbeta in various combinations. The absence of hormone mRNA was found in less than 1% of the responsive cells. In cells chosen at random (representative of the total pituitary cell population), the proportion of cells expressing two or multiple hormone mRNAs was twice as low as that in the gamma3-MSH-responsive population, whereas the proportion of cells expressing a single hormone mRNA was twice as high (about two thirds of all cells). Moreover, unlike in the gamma3-MSH-responsive cell population, randomly chosen cells were found that coexpressed POMC mRNA with LHbeta mRNA. The effect of gamma3-MSH on [Ca2+]i was blocked by the MC-3 receptor antagonist SHU9119 (used up to a 1000-fold excess) in 46% or less of the responsive cells. SHU9119 failed to block the [Ca2+]i response to gamma3-MSH in PRL-, GH-, and TSHbeta-ir cells, but it did block the response in most ACTH-ir cells and in cells expressing no hormone to a detectable level. Single cell RT-PCR revealed that expression of MC-3 receptor mRNA was detected in only 16% of gamma3-MSH-responsive cells. The present data suggest that the target cells of gamma3-MSH in terms of [Ca2+]i responses in the immature rat pituitary constitute subpopulations of all main pituitary cell types, including nonhormonal (or low expression hormonal) cells. However, in contrast to the total pituitary cell population, most of these cells display multilineage gene activation at the mRNA level, i.e. express mRNA of GH, PRL, TSHbeta, POMC, and LHbeta in dual, triple, or quadruple combinations. Although gamma3-MSH may act through the MC-3 receptor in a portion of these cells, most of these cells (mainly in the lacto-somatotroph lineage) may transduce the signal through another receptor or through an MC-3 receptor with unconventional binding characteristics.

Distinct signal thresholds for the unique antigen receptor-linked gene expression programs in mature and immature B cells.

Although it is well established that immature B lymphocytes are exquisitely sensitive to tolerance induction compared with their mature counterparts, the molecular basis for this difference is unknown. We demonstrate that signaling by B cell antigen receptors leads to distinct and mutually exclusive biologic responses in mature and immature B cells: upregulation of CD86, CD69, and MHC class II in mature cells and receptor editing in immature cells. These responses can be induced simply by elevation of intracellular free calcium levels, as occurs after receptor aggregation. Importantly, induction of immature B cell responses requires much smaller increases in intracellular free calcium than does induction of mature B cell responses. These differences in biologic response and sensitivity to intracellular free calcium likely contributes to selective elimination at the immature stage of even those B cells that express low affinity for self-antigens.

Extracellular calcium is required for the polychlorinated biphenyl-induced increase of intracellular free calcium levels in cerebellar granule cell culture

Recent studies from the laboratory indicate that polychlorinated biphenyl (PCB) congeners can alter signal transduction and calcium homeostasis in neuronal preparations. These effects were more pronounced for the ortho-substituted, non-coplanar congeners, although the mechanisms underlying these effects are not clear. In the present study the time-course and concentration-dependent effects of coplanar and non-coplanar PCBs on intracellular free calcium concentration ([Ca 2+] i) in cerebellar granule cell cultures were compared using the fluorescent probe fura-2. The ortho-substituted congeners 2,2′-dichlorobiphenyl (DCB) and 2,2′,4,6,6′-pentachlorobiphenyl (PeCB) caused a gradual increase of [Ca 2+] i while the non- ortho-substituted congeners 4,4′-DCB and 3,3′,4,4′,5-PeCB had no effect. The increase of [Ca 2+] i produced by 2,2′-DCB was time- and concentration-dependent. Further studies examined possible mechanisms for this rise in [Ca 2+] i. In contrast to the muscarinic agonist carbachol, the effects of 2,2′-DCB on [Ca 2+] i were not blocked by thapsigargin and required the presence of extracellular calcium. The effects of ortho-substituted PCBs may depend on their ability to inhibit calcium sequestration as 2,2′-DCB significantly inhibited 45Ca 2+-uptake by microsomes and mitochondria while 3,3′,4,4′,5-PeCB had no effect. In addition, 2,2′-DCB significantly increased the binding of [ 3H]inositol 1,4,5-trisphosphate to receptors on cerebellar microsomes, suggesting another possible mechanism by which ortho-substituted PCBs can mobilize [Ca 2+] i. These results show that PCBs increase [Ca 2+] i in vitro via a mechanism that requires extracelluar calcium, and support previous structure-activity studies indicating that ortho-substituted PCBs are more potent than non- ortho-substituted PCBs.

Pneumadin-evoked intracellular free Ca 2+ responses in rat aortic smooth muscle cells: effect of dexamethasone

The direct vascular effect of pneumadin (PN) was determined by studying the changes in intracellular free calcium ([Ca 2+] i) levels in cultured rat aortic smooth muscle cells maintained between the second and fifth passages. PN evoked a rapid, concentration-dependent, biphasic increase in [Ca 2+] i. The [Ca 2+] i level rose from a basal value of 108 nM to a maximum increase in peak value of 170 nM. Although the level of maximal [Ca 2+] i response evoked by PN was less than with other vasoactive agonists, it was more potent ( ec 50 0.5 nM) than even endothelin-1 ( ec 50 3.1 nM). At concentrations > 100 nM, [Ca 2+] i elevations induced by PN above basal levels were no longer observed. Pretreatment with dexamethasone (100 nM for 24 hr) resulted in a significant increase ( P < 0.01) in the peak [Ca 2+] i response (310 nM) to PN. However, the biphasic pattern in the peak [Ca 2+] i responses encountered with increasing concentrations of PN remained unaffected. The exaggerated [Ca 2+] i response to PN was abolished by preincubation of cells with either the glucocorticoid antagonist mifepristone (RU 486) or the protein synthesis inhibitor cycloheximide. Inclusion of either an AT 1 antagonist (losartan), a V 1 selective vasopressin antagonist (d(Ch 2) 5 Tyr (Me) AVP), or an α-adrenoceptor antagonist (phentolamine) failed to affect the increases in [Ca 2+] i induced by PN. PN-evoked increases in inositol 1,4,5-trisphosphate levels paralleled the [Ca 2+] i changes. These data suggest that PN increases Ca 2+ mobilization in rat aortic smooth muscle cells via activation of phospholipase C coupled receptors. This effect is up-regulated by dexamethasone.

A transient and a persistent calcium release are induced by chlorocresol in cultivated mouse myotubes.

The effect of 4-chloro-m-cresol (4-CmC), a stabilizing agent used in commercial preparations of the muscle relaxant succinylcholine, on intracellular free calcium levels in cultivated mouse myotubes was studied. Calcium signals were monitored with an inverted microscope equipped for fluorescence photometry using fura-2 as the calcium indicator. Upon bath application of 500 microM 4-CmC for 90 s, two separate calcium signals, a transient and a sustained one, could be regularly discriminated. First, with a delay of 2 s, the intracellular calcium concentration increased from 41+/-13 to 541+/-319 nM, peaked after 2-5 s and declined within 10 s to nearly resting values (n=36). Then, after a delay of up to 20 s, intracellular calcium rose quickly again to almost the same value and stayed elevated as long as the drug was applied. Upon drug removal, intracellular calcium rapidly decreased to a new level that was always slightly higher than the original base line. At 250 microM 4-CmC, the response was small, whereas at 500 microM it was at its maximum. Thus, the concentration-response curve was very steep. Replacement of extracellular calcium by EGTA and application of calcium channel blockers revealed that, for both the transient and the sustained response, calcium was released from intracellular stores. Pre-treatment with thapsigargin (0.1 microM) or ryanodine (10 microM) abolished both signal components. Repeated short-term applications of 4-CmC suggest that the two components may arise from different systems.

Lymphocyte intracellular free calcium concentration is increased in preeclampsia

Objectives: We tested 2 hypotheses: (1) Preeclampsia is characterized by an increase in intracellular free calcium concentration in lymphocytes. (2) Levels of intracellular free calcium are influenced by the calcium concentration in the extracellular milieu or by parathyroid hormone. Study Design: Intracellular free calcium concentrations were measured in 4 groups of women: nonpregnant women (n = 25), normotensive pregnant women (n = 30), pregnant women with chronic hypertension (n = 15), and women with preeclampsia (n = 15). Intracellular free calcium concentration was measured in the basal state, at varying extracellular calcium ion concentrations, and in the presence of exogenous parathyroid hormone. Results: Women with preeclampsia had the highest basal lymphocyte intracellular free calcium concentration (121 ± 7 nmol/L, mean ± SEM) compared with normotensive pregnant women during the third trimester (94 ± 3 nmol/L, P < .001) and pregnant women in the third trimester with chronic hypertension (100 ± 3 nmol/L, P < .01). During the third trimester normotensive women and women with chronic hypertension had significantly higher basal intracellular free calcium concentrations than were found in women during the first trimester. Exposure of lymphocytes to an extracellular milieu of low calcium concentration resulted in an increase in intracellular free calcium concentration. Incubation with parathyroid hormone had no effect on intracellular free calcium concentration. Conclusions: Lymphocyte intracellular free calcium concentration is increased in preeclampsia and not in chronic hypertensive pregnancy and is greater during the third trimester than during the first trimester. Extracellular calcium depletion increases lymphocyte intracellular free calcium concentration. These data support the idea that a calcium deficit leading to an increased intracellular free calcium concentration during late pregnancy contributes to the pathogenesis of preeclampsia. (Am J Obstet Gynecol 1999;180:1209-14.)

The effects of vasopressin on hippocampal slices from trained rats.

The effects of vasopressin on hippocampal slices from control and pretrained rats were compared. Prolonged presentation of both combined and noncombined conditioned stimuli (CS) and unconditioned stimuli (US) increases the sensitivity of hippocampal neurons to vasopressin. The effect of this was that responses to application of DG-AVP after training and active control treatment was biphasic, consisting of initial excitation and subsequent inhibition; similar effects were seen in control slices when the peptide concentration was increased. Increases in the excitatory phase occurred with presentation of the CS and US and were independent of the training factor. The inhibitory phase increased to a greater extent on presentation of combinations of the CS and US. It is suggested that increases in depression in response to application of vasopressin in trained animals, as compared with active controls, are due to a higher level of free intracellular calcium occurring as a result of the training procedure.

Functional and molecular characterization of a muscarinic receptor type and evidence for expression of choline-acetyltransferase and vesicular acetylcholine transporter in human granulosa-luteal cells.

Previously, we provided evidence for the presence of a class of muscarinic receptors on human luteinized granulosa cells (human GC) that is linked to transient increases in intracellular free calcium levels, but not to steroid production. The precise nature of the receptor is not known, and neither its function nor the source of its natural ligand acetylcholine (ACh) is clear. To address these issues we used RT-PCR approaches and isolated complementary DNAs corresponding to the M1 receptor subtype from reverse transcribed human GC messenger ribonucleic acids. M1 receptors were further shown by immunocytochemistry, using a M1 receptor antiserum. Single cell calcium measurements showed that the M1 receptor was functionally active and linked to acute increases in intracellular free calcium, as the M1 receptor specific antagonist pirenzepine blocked the Ca2+-mobilizing effect of oxotremorine M (a muscarinic agonist). An unexpected consequence of M1 receptor activation was evidenced by the ability of muscarinic agonists to stimulate the proliferation of human GC within 24 h. In vivo, ACh, the natural ligand of these receptors is thought to be contained in cholinergic nerve fibers innervating the ovary. Surprisingly, the prerequisite for the synthesis of ACh, the enzyme choline-acetyltransferase (ChAT), is also expressed by human GC, as shown by Western blotting and immunocytochemistry. In addition, these cells express another marker for ACh synthesis, namely the gene for the vesicular acetylcholine transporter, as evidenced by RT-PCR cloning, Western blotting, and immunocytochemistry. In conclusion, our data identify the M1 receptor in human GC and point to a novel, trophic role of the neurotransmitter ACh. Furthermore, the presence of the prerequisites of ACh synthesis in human GC indicate that an autocrine/paracrine regulatory loop also exists in the in vivo counterparts of these cells in the ovary, i.e. in the cells of the preovulatory follicle and/or of the young corpus luteum.

Mitochondrial participation in modulation of calcium transients in DRG neurons.

The effects of the mitochondrial Na+/Ca2+ exchange blocker tetraphenylphosphonium (TPP+) and the permeability transition blocker cyclosporinA (CysA) on the ability of mitochondria to participate in the regulation of intracellular calcium were investigated on freshly isolated mice sensory DRG neurons. The free intracellular calcium level ([Ca2+]in) was measured using indo-1 based microfluorimetry. The characteristics of depolarization-induced [Ca2+]in transients were changed in the presence of 25 microM TPP+. The amplitude of [Ca2+]in transients became decreased and the restoration of resting [Ca2+]in level speeded up in the presence of TPP+. Application of 5 microM cyclosporinA induced substantial residual elevation of [Ca2+]in after termination of depolarization. We conclude that the mitochondrial Na+/Ca2+ exchanger mechanism plays an important role in the regulation of calcium signals during neuronal activity, prolonging them by releasing Ca2+ stored during transient peak. Activation of permeability transition pores does not participate in these processes.

Detection of melanocortin-3 receptor mRNA in immature rat pituitary: functional relation to gamma3-MSH-induced changes in intracellular Ca2+ concentration?

We have previously shown that gamma3-MSH stimulates DNA replication in lactotrophs, somatotrophs and thyrotrophs of early postnatal rat pituitary in culture. Since the melanocortin-3 (MC-3) receptor is the only known receptor displaying high affinity for gamma3-MSH, the present study explored whether mRNA of the latter receptor is present in the pituitary and whether the receptor is functional. RT-PCR of RNA extracts from 14-day-old rat pituitary revealed the presence of MC-3 receptor mRNA in both the anterior and the neurointermediate lobe. The identity of the amplified products was confirmed by sequence analysis. Dispersed cells of 14-day-old female rats (24 h in culture) were exposed to gamma3-MSH, and changes in intracellular free calcium levels ([Ca2+]i) were assessed by means of fluo-3 video imaging. Gamma3-MSH evoked a rapid and maintained oscillating [Ca2+]i increase in 5%, 10% and 15% of the cells at a dose of 0.1, 1 and 10 nM, respectively. The MC-3/MC-4 receptor antagonist Ac-Nle4-c[Asp5,(D-Nal(2)7,Lys10]alpha-MSH-(4-10)-NH2 (SHU 9119) blocked the effect of gamma3-MSH in about 50% of the responsive cells. The present data suggest that the MC-3 receptor is expressed in the rat pituitary but that this receptor mediates only half of the effects of its putative ligand, gamma3-MSH, on [Ca2+]i. Part of the effect of gamma3-MSH may be mediated by a MC-3 receptor in a functional state different from the one studied previously in transfected cell lines or by a hitherto unidentified MC receptor.

Activation of human vascular endothelial cells by factor Xa: effect of specific inhibitors

Recently, human umbilical vein endothelial cells (HUVEC) have been shown to express functional high-affinity receptors for factor Xa, which may be of importance in the regulation of coagulation and homeostasis of the vascular wall. In this paper, we demonstrate that when added to cultured HUVEC, factor Xa was a potent mitogen, stimulating an increase in cell number at a 0.3 to 100 nM concentration. The same doses of factor Xa also increased intracellular free calcium levels and phosphoinositide turnover. When added to confluent HUVEC, factor Xa induced the expression of tissue factor and the release of tissue-type plasminogen activator and plasminogen activator inhibitor-1 without affecting urokinase expression. Indirect (antithrombin-pentasaccharide) and direct (DX9065) inhibitors of factor Xa affected all these activities of factor Xa in a dose-dependent manner. Taken together, these data show that the activities induced by factor Xa on HUVEC were dependent on its catalytic activity and could be inhibited by both direct and indirect factor Xa inhibitors.

Neurotensin stimulation of mast cell secretion is receptor-mediated, pertussis-toxin sensitive and requires activation of phospholipase C.

Pretreatment of isolated rat serosal mast cells with U-73122, an aminosteroid inhibitor of phospholipase C, inhibited histamine secretion in response to neurotensin (NT). This inhibition reached a maximum after 1 h of pretreatment at 37 degrees C and was dependent upon the concentration of U-73122 (IC50 approximately 0.2 microM). The inactive analog, U-73343, had no effect on the secretory response to NT. Pretreatment of mast cells with U-73122 also blocked histamine secretion in response to substance P (SP), mastoparan (MP), compound 48/80, or amidated NT (NT-NH2). Stimulation of mast cells by NT was accompanied by a rise in the level of intracellular free calcium and a rapid (within seconds) increase in the level of inositol trisphosphate (IP3) which was inhibited by pretreatment of the cells with U-73122. Pretreatment of isolated mast cells with pertussis toxin (PTx) blocked histamine release in response to NT as well as to all peptides tested. PTx had no effect on histamine secretion elicited by anti-IgE stimulation of sensitized mast cells. Pretreatment of mast cells with SR 48692, a NT-receptor antagonist, had no effect on histamine release induced by MP. At a high concentration (100 nM) SR 48692 partially inhibited the response to NT-NH2. These results, together with our earlier findings with SR 48692, indicate that the signal transduction pathway in mast cells activated by NT requires a specific NT-receptor, the activation of phospholipase C, and the involvement of a PTx sensitive G protein. The peptides SP and MP, and compound 48/80, while also requiring the activation of PLC and a PTx sensitive G protein, are not inhibited by the NT-R antagonist, SR 48692, suggesting that they exert their actions either via a different mast cell receptor or via a receptor-independent mechanism.

Rapid measurements of intracellular calcium using a fluorescence plate reader.

Intracellular calcium is a universal second messenger that can serve as a broad-based measure of receptor activity. Recent developments in multi-well plate fluorescence readers facilitate measurement of intracellular free-calcium levels and reduce reliance on slower, more cumbersome or expensive data collection methods. In this report, we describe a rapid and sensitive method to assay intracellular calcium ions in human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells from multi-well plates using a fluorometer equipped with on-line injectors. We examine the compatibility of visible-light excitable dyes Calcium Green-1 and Oregon Green 488 BAPTA-1. Using this assay, we were able to detect and quantify activity from muscarinic and beta-adrenergic receptors endogenous to HEK293 cells and detect calcium signals generated by activation of Gi-coupled recombinant mu-opioid and dopamine D2L receptors, and the Gs-coupled melanocortin subtype 4 (MC4) receptor. Fluorescence signals, stable in HEK293 cells, required the use of Oregon Green 488 BAPTA-1 and an inhibitor of organic anion transport in CHO cells. Under appropriate conditions, both cell types can be used to collect complete concentration-response data for a variety of receptors (including a recombinant muscarinic M1 receptor expressed in CHO cells) from a single plate of dye-loaded cells.

Effects of palmatine on isometric force and intracellular calcium levels of arterial smooth muscle

The effects of palmatine on isometric force and intracellular free calcium levels ([Ca 2+] i) were determined in isolated rat arterial strips. Palmatine dose-dependently relaxed the contractile responses stimulated by phenylephrine (PE) in aortic strips. In contrast, it only partially relaxed aortic strips contracted by 51 mM KCl. Pretreatment with palmatine shifted the dose-response curves of PE both rightwards and downwards in a dose-dependent manner. When Ca 2+-free solution and re-addition of Ca 2+ were applied to assess PE-induced phasic and tonic contractions, palmatine was found to be effective in inhibiting both contractions. The effects of palmatine on intracellular calcium levels were measured with the bioluminescent calcium indicator aequorin in rat tail artery strips. Palmatine caused a concomitant, dose-dependent decrease in PE-activated isometric force and [Ca 2+] i, resulting in small changes in the [Ca 2+] i-force relationship. These results suggest that vasodilatory effect of palmatine was mediated by reducing [Ca 2+] i as well as affecting [Ca 2+] i sensitivity of the contractile apparatus. Palmatine-induced [Ca 2+] i decreases appeared to involve decreases in both Ca 2+ release from intracellular stores and Ca 2+ influx through calcium channels.

Cardiac contractility: how calcium activates the myofilaments.

In both cardiac and skeletal muscle, the force-generating molecular motors (crossbridges) are turned on by increasing the intracellular free calcium level that regulates the troponin-tropomyosin system. However, calcium activation is a two-way process in the sense that activated crossbridges also affect the troponin-tropomyosin system. Here we review the mechanism of calcium action on myofilament proteins, particularly tropomyosin, that affects both the extent and the rate of force development and hence the contractility of the myocardium. At low myoplasmic Ca2+ concentrations tropomyosin is located at the edge of the thin filament, thereby interfering with the formation of strong actin-myosin linkages (blocked state). An increase in Ca2+ activity causes an azimuthal shift of tropomyosin around the filament (by about 30 degrees), thereby increasing the probability of low-force crossbridge interaction, a process which by cooperative effects induces further tropomyosin movement (by an additional 10 degrees) which results in the open state of the filament characterized by forceful crossbridge interaction. (This mechanism may be analogous to that in ligand-gated ion channels, where ligand binding increases the open probability of the pore.) The extent of activation then depends on the free Ca2+ concentration and on the calcium sensitivity of the thin filament that may be affected by protein phosphorylation, crossbridge attachment, the troponin isoform composition of the filament, and novel calcium-sensitizing drugs that act on the contractile or regulatory proteins and thus increase the force of the heart.

Depolarization Stimulates Initial Calcitonin Gene-Related Peptide Expression by Embryonic Sensory NeuronsIn Vitro

The neuropeptide calcitonin gene-related peptide (CGRP) is expressed by one-third of adult rat lumbar dorsal root ganglion (DRG) neurons, many of which mediate pain sensation or cause vasodilation. The factors that regulate the developmental expression of CGRP are poorly understood. Embryonic DRG neurons initially lack CGRP. When these neurons were stimulated in culture by serum or persistent 50 mM KCl application, the same percentage of CGRP-immunoreactive (CGRP-IR) neurons developed in vitro as was seen in the adult DRG in vivo. The addition of the L-type calcium channel blockers, 5 microM nifedipine or 10 microM verapamil, dramatically decreased the proportion of CGRP-IR neurons that developed, although the N-type calcium channel blocker, 2.5 microM omega-conotoxin, was less effective. By contrast, the sodium channel blocker 1 microM tetrodotoxin had no effect on CGRP expression after depolarization. Fura-2 ratiometric imaging demonstrated that mean intracellular free calcium levels increased from 70 to 135 nM with chronic depolarization, and the addition of nifedipine inhibited that increase. Only a subpopulation of neurons had elevated calcium concentrations during chronic depolarization, and they were correlated with CGRP expression. Key signal transduction pathways were tested pharmacologically for their role in CGRP expression after depolarization; the addition of the CaM kinase inhibitor KN-62 reduced the proportion of CGRP-IR neurons to basal levels. By contrast, protein kinase A and protein kinase C were not implicated in the depolarization-induced CGRP increases. These data suggest that depolarization and the subsequent Ca2+-based signal transduction mechanisms play important roles in the de novo expression of CGRP by specific embryonic DRG neurons.