Nucleoplasmic Ca(2+)loading is regulated by mobilization of perinuclear Ca(2+)

Regulation of nucleoplasmic calcium (Ca(2+)) concentration may occur by the mobilization of perinuclear luminal Ca(2+)pools involving specific Ca(2+)pumps and channels of both inner and outer perinuclear membranes. To determine the role of perinuclear luminal Ca(2+), we examined freshly cultured 10 day-old embryonic chick ventricular cardiomyocytes. We obtained evidence suggesting the existence of the molecular machinery required for the bi-directional Ca(2+)fluxes using confocal imaging techniques. Embryonic cardiomyocytes were probed with antibodies specific for ryanodine-sensitive Ca(2+)channels (RyR2), sarco/endoplasmic reticulum Ca(2+)ATPase (SERCA2)-pumps, and fluorescent BODIPY derivatives of ryanodine and thapsigargin. Using immunocytochemistry techniques, confocal imaging showed the presence of RyR2 Ca(2+)channels and SERCA2-pumps highly localized to regions surrounding the nucleus, referable to the nuclear envelope. Results obtained from Fluo-3, AM loaded ionomycin-perforated embryonic cardiomyocytes demonstrated that gradual increases of extranuclear Ca(2+)from 100 to 1600 nM Ca(2+)was localized to the nucleus. SERCA2-pump inhibitors thapsigargin and cyclopiazonic acid showed a concentration-dependent inhibition of nuclear Ca(2+)loading. Furthermore, ryanodine demonstrated a biphasic concentration-dependence upon active nuclear Ca(2+)loading. The concomitant addition of thapsigargin or cyclopiazonic acid with ryanodine at inhibitory concentrations caused an significant increase in nuclear Ca(2+)loading at low concentrations of extranuclear added Ca(2+). Our results show that the perinuclear lumen in embryonic chick ventricular cardiomyocytes is capable of autonomously regulating nucleoplasmic Ca(2+)fluxes.     

EGF enhances Ca(2+) mobilization and capacitative Ca(2+) entry in mouse mammary epithelial cells

Effects of epidermal growth factor (EGF) on the intracellular Ca(2+) ([Ca(2+)](i)) responses to nucleotides, Ca(2+) release from thapsigargin-sensitive stores and capacitative Ca(2+) entry were investigated in cultured mouse mammary epithelial cells. EGF treatment induced proliferation of mammary epithelial cells. We checked for mitotic activity by immunocytochemistry with an anti-PCNA (proliferating cell nuclear antigen) antibody, which stains nuclei of the cells in S-phase of cell cycle. EGF treatment apparently increased the number of PCNA-stained cells compared to those treated with differentiating hormones (insulin, prolactin and cortisol) or without any hormone. Application of EGF did not induce any acute [Ca(2+)](i) response. EGF treatment for 1-2 days in culture, however, enhanced [Ca(2+)](i) responses including [Ca(2+)](i) increase by ATP, UTP and other nucelotides, Ca(2+) release from thapsigargin-sensitive stores, as well as capacitative Ca(2+) entry. Genistein, a tyrosine kinase inhibitor, prevented EGF-induced cell proliferation and the [Ca(2+) ](i) responses in a dose-dependent manner. These results indicate that EGF treatment enhances Ca(2+) mobilization and capacitative Ca(2+) entry, well correlated with cellular proliferation in mammary epithelial cells.     

Intracellular Ca(2+) regulates responsiveness of cardiac L-type Ca(2+) current to protein kinase A: role of calmodulin

The goal of this study was to determine whether the protein kinase A (PKA) responsiveness of the cardiac L-type Ca(2+) current (ICa) is affected during transient increases in intracellular Ca(2+) concentration. Ventricular myocytes were isolated from 3- to 4-day-old neonatal rats and cultured on aligned collagen thin gels. When measured in 1 or 2 mM Ca(2+) external solution, the aligned myocytes displayed a large ICa that was weakly regulated (20% increase) during stimulation of PKA by 2 microM forskolin. In contrast, application of forskolin caused a 100% increase in ICa when the external Ca(2+) concentration was reduced to 0.5 mM or replaced with Ba(2+). This Ca(2+)-dependent inhibition was also observed when the cells were treated with 1 microM isoproterenol, 100 microM 3-isobutyl-1-methylxanthine, or 500 microM 8-bromo-cAMP. The responsiveness of ICa to PKA was restored during intracellular dialysis with a calmodulin (CaM) inhibitory peptide but not during treatment with inhibitors of protein kinase C, Ca(2+)/CaM-dependent protein kinase, or calcineurin. Adenoviral-mediated expression of a CaM molecule with mutations in all four Ca(2+)-binding sites also increased the PKA sensitivity of ICa. Finally, adult mouse ventricular myocytes displayed a greater response to forskolin and cAMP in external Ba(2+). Thus Ca(2+) entering the myocyte through the voltage-gated Ca(2+) channel regulates the PKA responsiveness of ICa.     

Intracellular Ca(2+) and Zn(2+) signals during monochloramine-induced oxidative stress in isolated rat colon crypts

During acute exacerbations of inflammatory bowel diseases, oxidants are generated through the interactions of bacteria in the lumen, activated granulocytes, and cells of the colon mucosa. In this study we explored the ability of one such class of oxidants, represented by monochloramine (NH(2)Cl), to serve as agonists of Ca(2+) and Zn(2+) accumulation within the colonocyte. Individual colon crypts prepared from Sprague-Dawley rats were mounted in perfusion chambers after loading with fluorescent reporters fura 2-AM and fluozin 3-AM. These reporters were characterized, in situ, for responsiveness to Ca(2+) and Zn(2+) in the cytoplasm. Responses to different concentrations of NH(2)Cl (50, 100, and 200 microM) were monitored. Subsequent studies were designed to identify the sources and mechanisms of NH(2)Cl-induced increases in Ca(2+) and Zn(2+) in the cytoplasm. Exposure to NH(2)Cl led to dose-dependent increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) in the range of 200-400 nM above baseline levels. Further studies indicated that NH(2)Cl-induced accumulation of Ca(2+) in the cytoplasm is the result of release from intracellular stores and basolateral entry of extracellular Ca(2+) through store-operated channels. In addition, exposure to NH(2)Cl resulted in dose-dependent and sustained increases in intracellular Zn(2+) concentration ([Zn(2+)](i)) in the nanomolar range. These alterations were neutralized by dithiothreitol, which shields intracellular thiol groups from oxidation. We conclude that Ca(2+)- and Zn(2+)-handling proteins are susceptible to oxidation by chloramines, leading to sustained, but not necessarily toxic, increases in [Ca(2+)](i) and [Zn(2+)](i). Under certain conditions, NH(2)Cl may act not as a toxin but as an agent that activates intracellular signaling pathways.     

Possible quorum sensing in marine snow bacteria: production of acylated homoserine lactones by Roseobacter strains isolated from marine snow

We report here, for the first time, that bacteria associated with marine snow produce communication signals involved in quorum sensing in gram-negative bacteria. Four of 43 marine microorganisms isolated from marine snow were found to produce acylated homoserine lactones (AHLs) in well diffusion and thin-layer chromatographic assays based on the Agrobacterium tumefaciens reporter system. Three of the AHL-producing strains were identified by 16S ribosomal DNA gene sequence analysis as Roseobacter spp., and this is the first report of AHL production by these alpha-PROTEOBACTERIA: It is likely that AHLs in Roseobacter species and other marine snow bacteria govern phenotypic traits (biofilm formation, exoenzyme production, and antibiotic production) which are required mainly when the population reaches high densities, e.g., in the marine snow community.     

Intracellular Ca(2+) and Ca(2+)/calmodulin-dependent kinase II mediate acute potentiation of neurotransmitter release by neurotrophin-3

Neurotrophins have been shown to acutely modulate synaptic transmission in a variety of systems, but the underlying signaling mechanisms remain unclear. Here we provide evidence for an unusual mechanism that mediates synaptic potentiation at the neuromuscular junction (NMJ) induced by neurotrophin-3 (NT3), using Xenopus nerve-muscle co-culture. Unlike brain-derived neurotrophic factor (BDNF), which requires Ca(2+) influx for its acute effect, NT3 rapidly enhances spontaneous transmitter release at the developing NMJ even when Ca(2+) influx is completely blocked, suggesting that the NT3 effect is independent of extracellular Ca(2+). Depletion of intracellular Ca(2+) stores, or blockade of inositol 1, 4, 5-trisphosphate (IP3) or ryanodine receptors, prevents the NT3-induced synaptic potentiation. Blockade of IP3 receptors can not prevent BDNF-induced potentiation, suggesting that BDNF and NT3 use different mechanisms to potentiate transmitter release. Inhibition of Ca(2+)/calmodulin-dependent kinase II (CaMKII) completely blocks the acute effect of NT3. Furthermore, the NT3-induced potentiation requires a continuous activation of CaMKII, because application of the CaMKII inhibitor KN62 reverses the previously established NT3 effect. Thus, NT3 potentiates neurotransmitter secretion by stimulating Ca(2+) release from intracellular stores through IP3 and/or ryanodine receptors, leading to an activation of CaMKII.     

Intracellular Ca(2+) channel immunoreactivity in neuroendocrine axon terminals

The concentration of neuroendocrine terminals in the neurohypophysis facilitates the identification and localization of Ca(2+) channel subtypes near neuroendocrine release sites. Immunoblots of rat neurohypophysial tissue identified the alpha(1)1.3, alpha(1)2.1, alpha(1)2.2, and alpha(1)2.3 Ca(2+) channel subunits. Immunofluorescence staining of axon terminal plasma membranes was weak, suggesting that Ca(2+) channels are dispersed. This contrasts with the highly punctate alpha(1)2.2 immunoreactivity in bovine chromaffin cells; the neurohypophysial terminals may therefore lack the specialized release zones found in those cells. Immunofluorescence and immunogold labeling identify dense core granule-like structures in the terminal cytoplasm containing multiple Ca(2+) channel types. Ca(2+) channels in internal membranes may play an important role in channel targeting and distribution in neuroendocrine cells.     

Intracellular Ca(2+) release as irreversible Markov process

In striated muscles, intracellular Ca(2+) release is tightly controlled by the membrane voltage sensor. Ca(2+) ions are necessary mediators of this control in cardiac but not in skeletal muscle, where their role is ill-understood. An intrinsic gating oscillation of Ca(2+) release-not involving the voltage sensor-is demonstrated in frog skeletal muscle fibers under voltage clamp. A Markov model of the Ca(2+) release units is shown to reproduce the oscillations, and it is demonstrated that for Markov processes to have oscillatory transients, its transition rates must violate thermodynamic reversibility. Such irreversibility results in permanent cycling of the units through a ring of states, which requires a source of free energy. Inhibition of the oscillation by 20 to 40 mM EGTA or partial depletion of Ca(2+) in the sarcoplasmic reticulum (SR) identifies the SR [Ca(2+)] gradient as the energy source, and indicates a location of the critical Ca(2+)-sensing site at distances greater than 35 nm from the open channel. These results, which are consistent with a recent demonstration of irreversibility in gating of cardiac Ca(2+) sparks, (Wang, S.-Q., L.-S. Song, L. Xu, G. Meissner, E. G. Lakatta, E. Ríos, M. D. Stern, and H. Cheng. 2002. Biophys. J. 83:242-251) exemplify a cell-wide oscillation caused by coupling between ion permeation and channel gating.     

Intracellular Ca(2+) dynamics and sarcomere length in single ventricular myocytes

The measurements of the sarcomere length in dissociated cardiac ventricular myocytes are discussed using mainly our own experimental data. The striation periodicity of these unloaded cells was found to be that which is to be expected of a myocyte free of the ultrastructural constraints imposed upon it by the normal syncytial matrix of the ventricular wall. The sarcomere length and [Ca(2+)] relationship was consistent as expected from the intact tissue, when it was measured soon after partial rupturing the cell membrane. Miniature fluctuations of individual sarcomere length were demonstrated during rest, which was augmented by the Ca(2+) overload. The [Ca(2+)] could be estimated from the measurements of sarcomere length during the positive staircase of contraction. The usefulness of the optical measurement of sarcomere pattern was indicated.     

Hypoxia-induced alterations in Ca(2+) mobilization in brain microvascular endothelial cells

To investigate the possible cellular mechanisms of the ischemia-induced impairments of cerebral microcirculation, we investigated the effects of hypoxia/reoxygenation on the intracellular Ca(2+) concentration ([Ca(2+)](i)) in bovine brain microvascular endothelial cells (BBEC). In the cells kept in normal air, ATP elicited Ca(2+) oscillations in a concentration-dependent manner. When the cells were exposed to hypoxia for 6 h and subsequent reoxygenation for 45 min, the basal level of [Ca(2+)](i) was increased from 32.4 to 63.3 nM, and ATP did not induce Ca(2+) oscillations. Hypoxia/reoxygenation also inhibited capacitative Ca(2+) entry (CCE), which was evoked by thapsigargin (Delta[Ca(2+)](i-CCE): control, 62.3 +/- 3.1 nM; hypoxia/reoxygenation, 17.0 +/- 1.8 nM). The impairments of Ca(2+) oscillations and CCE, but not basal [Ca(2+)](i), were restored by superoxide dismutase and the inhibitors of mitochondrial electron transport, rotenone and thenoyltrifluoroacetone (TTFA). By using a superoxide anion (O(2)(-))-sensitive luciferin derivative MCLA, we confirmed that the production of O(2)(-) was induced by hypoxia/reoxygenation and was prevented by rotenone and TTFA. These results indicate that hypoxia/reoxygenation generates O(2)(-) at mitochondria and impairs some Ca(2+) mobilizing properties in BBEC.     

Intracellular Ca(2+) dynamics and the stability of ventricular tachycardia

Ventricular fibrillation (VF), the major cause of sudden cardiac death, is typically preceded by ventricular tachycardia (VT), but the mechanisms underlying the transition from VT to VF are poorly understood. Intracellular Ca(2+) overload occurs during rapid heart rates typical of VT and is also known to promote arrhythmias. We therefore studied the role of intracellular Ca(2+) dynamics in the transition from VT to VF, using a combined experimental and mathematical modeling approach. Our results show that 1) rapid pacing of rabbit ventricular myocytes at 35 degrees C led to increased intracellular Ca(2+) levels and complex patterns of action potential (AP) configuration and the intracellular Ca(2+) transients; 2) the complex patterns of the Ca(2+) transient arose directly from the dynamics of intracellular Ca(2+) cycling, and were not merely passive responses to beat-to-beat alterations in AP; 3) the complex Ca(2+) dynamics were simulated in a modified version of the Luo-Rudy (LR) ventricular action potential with improved intracellular Ca(2+) dynamics, and showed good agreement with the experimental findings in isolated myocytes; and 4) when incorporated into simulated two-dimensional cardiac tissue, this action potential model produced a form of spiral wave breakup from VT to a VF-like state in which intracellular Ca(2+) dynamics played a key role through its influence on Ca(2+)-sensitive membrane currents such as I(Ca), I(NaCa), and I(ns(Ca)). To the extent that spiral wave breakup is useful as a model for the transition from VT to VF, these findings suggest that intracellular Ca(2+) dynamics may play an important role in the destabilization of VT and its degeneration into VF.     

Luminal Ca(2+) and the activity of sarcoplasmic reticulum Ca(2+) pumps modulate histamine-induced all-or-none Ca(2+) release in smooth muscle cells

We have studied histamine (HA)-evoked intracellular Ca(2+) release in single, freshly isolated myocytes from the guinea pig urinary bladder. Short applications of histamine (5 s) produced a thapsigargin (TG)-sensitive transient increase in intracellular calcium concentration ([Ca(2+)](i)). It was established that histamine and caffeine (Caff) released Ca(2+) from the same intracellular stores in these cells. Reducing the Ca(2+) content of internal stores by incubating cells with U-73343 or cyclopiazonic acid (CPA) inhibited the histamine-evoked Ca(2+) release in 69% and 60% of cells, respectively. Under these conditions, all cells released Ca(2+) in response to either caffeine or acetylcholine (ACh). However, decreasing internal Ca(2+) stores by removing external Ca(2+) inhibited histamine-induced Ca(2+) mobilization in only 22% of cells. A similar small fraction of cells was inhibited when sarcoplasmic reticulum (SR) Ca(2+) pumps were quickly blocked to avoid a significant reduction of luminal Ca(2+). In conclusion, lowering the luminal Ca(2+) content in combination with an impairment of the SR Ca(2+) pump activity significantly diminishes the ability of histamine to evoke an all-or-none intracellular Ca(2+) release.     

Intracellular Ca(2+) oscillations induced by over-expressed Ca(V)3.1 T-type Ca(2+) channels in NG108-15 cells

T-type Ca²⁺ channel family includes three subunits Ca_V3.1, Ca_V3.2 and Ca_V3.3 and have been shown to control burst firing and intracellular Ca²⁺ concentration ([Ca²⁺]_i) in neurons. Here, we investigated whether Ca_V3.1 channels could generate a pacemaker current and contribute to cell excitability. Ca_V3.1 clones were over-expressed in the neuronal cell line NG108-15. Ca_V3.1 channel expression induced repetitive action potentials, generating spontaneous membrane potential oscillations (MPOs) and concomitant [Ca²⁺]_i oscillations. These oscillations were inhibited by T-type channels antagonists and were present only if the membrane potential was around −61 mV. [Ca²⁺]_i oscillations were critically dependent on Ca²⁺ influx through Ca_V3.1 channels and did not involve Ca²⁺ release from the endoplasmic reticulum. The waveform and frequency of the MPOs are constrained by electrophysiological properties of the Ca_V3.1 channels. The trigger of the oscillations was the Ca_V3.1 window current. This current induced continuous [Ca²⁺]_i increase at −60 mV that depolarized the cells and triggered MPOs. Shifting the Ca_V3.1 window current potential range by increasing the external Ca²⁺ concentration resulted in a corresponding shift of the MPOs threshold. The hyperpolarization-activated cation current (I_h) was not required to induce MPOs, but when expressed together with Ca_V3.1 channels, it broadened the membrane potential range over which MPOs were observed. Overall, the data demonstrate that the Ca_V3.1 window current is critical in triggering intrinsic electrical and [Ca²⁺]_i oscillations.     

Human umbilical vein endothelial cells (HUVECs) show Ca(2+) mobilization as well as Ca(2+) influx upon hypoxia

Bleb formation is an early event of cellular damage observed in a variety of cell types upon hypoxia. Although we previously found that the [Ca(2+)](i) rise before bleb formation only at the same loci of HUVECs upon hypoxia (localized [Ca(2+)](i) rise), the mode of the [Ca(2+)](i) rise remains ill-defined. In order to clarify the mechanisms causing the localized [Ca(2+)](i) rise in hypoxia challenged HUVECs, we studied the effects of several Ca(2+) channel blockers or a Ca(2+) chelator, EGTA, which reduces extracellular Ca(2+) concentration on the hypoxia-induced localized [Ca(2+)](i) rise and bleb formation by employing a confocal laser scanning microscopy (CLSM). After the initiation of hypoxia, [Ca(2+)](i) rose gradually in a localized fashion up to 15 min, which was associated with bleb formation at the same loci. The maximal [Ca(2+)](i) rise was 435 +/- 84 nM at the loci of bleb formation. Ca(2+) channel blockers including Ni(2+) (non-specific, 1 mM), nifedipine (L type, 10 microM), nicardipine (L + T type, 10 microM), and cilnidipine (L + N type, 10 microM) did not inhibit either the localized [Ca(2+)](i) rise or bleb formation. Although both the localized [Ca(2+)](i) rise and bleb formation were inhibited by lowering extracellular Ca(2+) concentration below 100 nM, a diffuse [Ca(2+)](i) rise through the cytoplasm remained without bleb formation, which was inhibited by a phospholipase C (PLC) inhibitor, U73122. In conclusion, hypoxia causes both the Ca(2+) mobilization and the Ca(2+) influx in HUVECs and the Ca(2+) influx through unknown Ca(2+) channels is responsible for the localized [Ca(2+)](i) rise integral to bleb formation.     

Intracellular Ca(2+)-Mg(2+)-ATPase regulates calcium influx and acrosomal exocytosis in bull and ram spermatozoa.

Calcium influx is required for the mammalian sperm acrosome reaction (AR), an exocytotic event occurring in the sperm head prior to fertilization. We show here that thapsigargin, a highly specific inhibitor of the microsomal Ca(2+)-Mg(2+)-ATPase (Ca(2+) pump), can initiate acrosomal exocytosis in capacitated bovine and ram spermatozoa. Initiation of acrosomal exocytosis by thapsigargin requires an influx of Ca(2+), since incubation of cells in the absence of added Ca(2+) or in the presence of the calcium channel blocker, La(3+), completely inhibited thapsigargin-induced acrosomal exocytosis. ATP-Dependent calcium accumulation into nonmitochondrial stores was detected in permeabilized sperm in the presence of ATP and mitochondrial uncoupler. This activity was inhibited by thapsigargin. Thapsigargin elevated the intracellular Ca(2+) concentration ([Ca(2+)](i)), and this increase was inhibited when extracellular Ca(2+) was chelated by EGTA, indicating that this rise in Ca(2+) is derived from the external medium. This rise of [Ca(2+)](i) took place first in the head and later in the midpiece of the spermatozoon. However, immunostaining using a polyclonal antibody directed against the purified inositol 1,4,5-tris-phosphate receptor (IP(3)-R) identified specific staining in the acrosome region, in the postacrosome, and along the tail, but not in the midpiece region. No staining in the acrosome region was observed in sperm without acrosome, indicating that the acrosome cap was stained in intact sperm. The presence of IP(3)-R in the anterior acrosomal region as well as the induction, by thapsigargin, of intracellular Ca(2+) elevation in the acrosomal region and acrosomal exocytosis, implicates the acrosome as a potential cellular Ca(2+) store. We suggest here that the cytosolic Ca(2+) is actively transported into the acrosome by an ATP-dependent, thapsigargin-sensitive Ca(2+) pump and that the accumulated Ca(2+) is released from the acrosome via an IP(3)-gated calcium channel. The ability of thapsigargin to increase [Ca(2+)](i) could be due to depletion of Ca(2+) in the acrosome, resulting in the opening of a capacitative calcium entry channel in the plasma membrane. The effect of thapsigargin on elevated [Ca(2+)](i) in capacitated cells was 2-fold higher than that in noncapacitated sperm, suggesting that the intracellular Ca pump is active during capacitation and that this pump may have a role in regulating [Ca(2+)](i) during capacitation and the AR.     

Ca(2+)-calmodulin regulates receptor-operated Ca(2+) entry activity of TRPC6 in HEK-293 cells

Mammalian homologues of the Drosophila transient receptor potential channel (TRPC) are involved in Ca(2+) entry following agonist stimulation of nonexcitable cells. Seven mammalian TRPCs have been cloned but their mechanisms of activation and/or regulation are still the subject of intense research efforts. It has already been shown that calmodulin (CaM) can regulate the activity of Drosophila TRP and TRPL and, more recently, CaM has been shown to interact with mammalian TRPCs. In this study, TRPC6 stably transfected into HEK-293 cells was used to investigate the possible influence of CaM on TRPC6-dependent Ca(2+) entry. Overexpression of TRPC6 in mammalian cells is known to enhance agonist-induced Ca(2+) entry, but not thapsigargin-induced Ca(2+) entry. Here, we show that CaM inhibitors (calmidazolium and trifluoperazine) abolish receptor-operated Ca(2+) entry (ROCE) without affecting thapsigargin-operated Ca(2+) entry and that the activity of CaM is dependent on complexation with Ca(2+). We also show that Ca(2+)-CaM binds to TRPC6 and that the binding can be abolished by CaM inhibitors. These results indicate that CaM is involved in the modulation of ROCE.     

Intracellular Mg(2+) enhances the function of BK-type Ca(2+)-activated K(+) channels.

BK channels modulate neurotransmitter release due to their activation by voltage and Ca(2+). Intracellular Mg(2+) also modulates BK channels in multiple ways with opposite effects on channel function. Previous single-channel studies have shown that Mg(2+) blocks the pore of BK channels in a voltage-dependent manner. We have confirmed this result by studying macroscopic currents of the mslo1 channel. We find that Mg(2+) activates mslo1 BK channels independently of Ca(2+) and voltage by preferentially binding to their open conformation. The mslo3 channel, which lacks Ca(2+) binding sites in the tail, is not activated by Mg(2+). However, coexpression of the mslo1 core and mslo3 tail produces channels with Mg(2+) sensitivity similar to mslo1 channels, indicating that Mg(2+) sites differ from Ca(2+) sites. We discovered that Mg(2+) also binds to Ca(2+) sites and competitively inhibits Ca(2+)-dependent activation. Quantitative computation of these effects reveals that the overall effect of Mg(2+) under physiological conditions is to enhance BK channel function.     

P2Y(2) receptor-stimulated phosphoinositide hydrolysis and Ca(2+) mobilization in tracheal epithelial cells

Extracellular nucleotides have been implicated in the regulation of secretory function through the activation of P2 receptors in the epithelial tissues, including tracheal epithelial cells (TECs). In this study, experiments were conducted to characterize the P2 receptor subtype on canine TECs responsible for stimulating inositol phosphate (InsP(x)) accumulation and Ca(2+) mobilization using a range of nucleotides. The nucleotides ATP and UTP caused a concentration-dependent increase in [(3)H]InsP(x) accumulation and Ca(2+) mobilization with comparable kinetics and similar potency. The selective agonists for P1, P2X, and P2Y(1) receptors, N(6)-cyclopentyladenosine and AMP, alpha,beta-methylene-ATP and beta, gamma-methylene-ATP, and 2-methylthio-ATP, respectively, had little effect on these responses. Stimulation of TECs with maximally effective concentrations of ATP and UTP showed no additive effect on [(3)H]InsP(x) accumulation. The response of a maximally effective concentration of either ATP or UTP was additive to the response evoked by bradykinin. Furthermore, ATP and UTP induced a cross-desensitization in [(3)H]InsP(x) accumulation and Ca(2+) mobilization. These results suggest that ATP and UTP directly stimulate phospholipase C-mediated [(3)H]InsP(x) accumulation and Ca(2+) mobilization in canine TECs. P2Y(2) receptors may be predominantly mediating [(3)H]InsP(x) accumulation, and, subsequently, inositol 1,4,5-trisphosphate-induced Ca(2+) mobilization may function as the transducing mechanism for ATP-modulated secretory function of tracheal epithelium.