Sensitive Intracellular Stores Research Articles

Garcinielliptone FC, a polyisoprenylated benzophenone from Platonia insignis Mart., promotes vasorelaxant effect on rat mesenteric artery

Polyisoprenylated benzophenones represent a group of chemical compounds commonly identified in Clusiaceae species and are responsible for a large amount of biological activities. In this work, the vasorelaxant effect induced by garcinielliptone FC (GFC) isolated from Platonia insignis Mart. (Clusiaceae), a monotype species from Platonia genus, was investigated. GFC promoted an endothelium-independent vasorelaxation on phenylephrine (PHE, 10− 5 mol L− 1)-induced vasoconstriction, but not on KCl (80 mmol L− 1)-induced vasoconstriction, on rat superior mesenteric artery rings. In addition, a concentration-dependent decrease of PHE- or serotonin-induced cumulative concentration–response curves was observed for GFC, and a slight decrease of pD2 value on CaCl2-induced vasoconstriction. In a Ca2+-free medium, GFC interfered in calcium mobilisation from PHE (10− 5 mol L− 1)-sensitive intracellular stores. GFC-induced vasorelaxant effect is probably mediated by a dual effect on mobilisation of calcium intracellular stores and attenuation of transmembrane calcium influx.

Fibroblast Circadian Rhythms of PER2 Expression Depend on Membrane Potential and Intracellular Calcium

The suprachiasmatic nucleus (SCN) of the hypothalamus synchronizes circadian rhythms of cells and tissues throughout the body. In SCN neurons, rhythms of clock gene expression are suppressed by manipulations that hyperpolarize the plasma membrane or lower intracellular Ca2+. However, whether clocks in other cells also depend on membrane potential and calcium is unknown. In this study, the authors investigate the effects of membrane potential and intracellular calcium on circadian rhythms in mouse primary fibroblasts. Rhythms of clock gene expression were monitored using a PER2::LUC knockin reporter. Rhythms were lost or delayed at lower (hyperpolarizing) K+ concentrations. Bioluminescence imaging revealed that this loss of rhythmicity in cultures was due to loss of rhythmicity of single cells rather than loss of synchrony among cells. In lower Ca2+ concentrations, rhythms were advanced or had shorter periods. Buffering intracellular Ca2+ by the calcium chelator 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis acetoxymethyl ester (BAPTA-AM) or manipulation of inositol triphosphate (IP3)-sensitive intracellular calcium stores by thapsigargin delayed rhythms. These results suggest that the circadian clock in fibroblasts, as in SCN neurons, is regulated by membrane potential and Ca2+. Changes in intracellular Ca2+ may mediate the effects of membrane potential observed in this study. (Author correspondence: welshdk@ucsd.edu)

Calcium influx-mediated signaling is required for complete mouse egg activation

Mammalian fertilization is accompanied by oscillations in egg cytoplasmic calcium (Ca(2+)) concentrations that are critical for completion of egg activation. These oscillations are initiated by Ca(2+) release from inositol 1,4,5-trisphosphate (IP(3))-sensitive intracellular stores. We tested the hypothesis that Ca(2+) influx across the plasma membrane was a requisite component of egg activation signaling, and not simply a Ca(2+) source for store repletion. Using intracytoplasmic sperm injection (ICSI) and standard in vitro fertilization (IVF), we found that Ca(2+) influx was not required to initiate resumption of meiosis II. However, even if multiple oscillations in intracellular Ca(2+) occurred, in the absence of Ca(2+) influx, the fertilized eggs failed to emit the second polar body, resulting in formation of three pronuclei. Additional experiments using the Ca(2+) chelator, BAPTA/AM, demonstrated that Ca(2+) influx is sufficient to support polar body emission and pronucleus formation after only a single sperm-induced Ca(2+) transient, whereas BAPTA/AM-treated ICSI or fertilized eggs cultured in Ca(2+)-free medium remained arrested in metaphase II. Inhibition of store-operated Ca(2+) entry had no effect on ICSI-induced egg activation, so Ca(2+) influx through alternative channels must participate in egg activation signaling. Ca(2+) influx appears to be upstream of CaMKIIγ activity because eggs can be parthenogenetically activated with a constitutively active form of CaMKIIγ in the absence of extracellular Ca(2+). These results suggest that Ca(2+) influx at fertilization not only maintains Ca(2+) oscillations by replenishing Ca(2+) stores, but also activates critical signaling pathways upstream of CaMKIIγ that are required for second polar body emission.

Characteristics of spontaneous calcium oscillations in renal tubular epithelial cells

The kidney is a major organ involved in calcium (Ca(2+)) metabolism. Ca(2+) is transported through renal tubular epithelial cells. The intracellular free calcium concentration ([Ca(2+)](i)) is tightly controlled at a low concentration, but transient increases and oscillations in [Ca(2+)](i) are induced by various conditions. In this study, we investigated the mechanisms underlying the spontaneous [Ca(2+)](i) oscillations observed in MDCK cells. [Ca(2+)](i) was monitored in fura-2-loaded Madin-Darby canine kidney (MDCK) cells using a calcium imaging system. We investigated the mechanism by which [Ca(2+)](i) changed by applying drugs or by changing the extracellular Ca(2+) concentration. Spontaneous [Ca(2+)](i) oscillations occurred in MDCK cells. The oscillations occurred irregularly and were not transmitted to neighboring cells. Spontaneous [Ca(2+)](i) oscillations in MDCK cells were initiated by Ca(2+) release from ryanodine/IP(3)-sensitive intracellular calcium stores, and their frequency was largely unaffected by the extracellular Ca(2+) concentration. Moreover, the frequency of the oscillations was increased by extracellular nucleotide, but was decreased when the nucleotides were removed. Our study suggested that [Ca(2+)](i) release from ryanodine/IP(3)-sensitive intracellular calcium stores mediates spontaneous [Ca(2+)](i) oscillations in MDCK cells. Calcium oscillations may be associated with the function of the renal tubular epithelial cells.

Investigation of Mechanisms Involved in (−)‐Borneol‐Induced Vasorelaxant Response on Rat Thoracic Aorta

The monoterpene (-)-borneol is present in essential oils of several medicinal plants. The aim of this study was to evaluate (-)-borneol effects on rat thoracic aorta artery rings. The cumulative addition of (-)-borneol (10(-9) -3 × 10(-4) M) on a phenylephrine-induced pre-contraction (10(-6) M) promoted a vasorelaxant effect in a concentration-dependent manner and independent of vascular endothelium. A similar effect was obtained on KCl-induced pre-contractions (80 mM). (-)-Borneol (10(-5) -3 × 10(-4 ) M) inhibited contractions induced by cumulative addition of CaCl2 (10(-6) -3 × 10(-2) M) in depolarizing medium without Ca(2+) in a concentration-dependent manner. On S-(-) Bay K 8644-induced pre-contractions (10(-7) M), (-)-borneol did not induce significant changes compared with KCl-induced pre-contractions. In a Ca(2+) -free medium, (-)-borneol (10(-5) , 10(-4) or 10(-3) M) interfered in calcium mobilization from phenylephrine (10(-6) M)- or caffeine (20 mM)-sensitive intracellular stores. The involvement of K(+) channels was evaluated by tetraethylammonium (3 mM), 4-aminopyridine (1 mM) and glibenclamide (10(-5) M) pre-treatment, and (-)-borneol-induced vasorelaxation was markedly attenuated. Thus, this vasorelaxant effect can probably be attributed to calcium influx blockade through voltage-operated calcium channels (CaV L), calcium mobilization from intracellular stores and potassium channels activation.

2-Aminoethoxydiphenyl-borate (2-APB) increases excitability in pyramidal neurons

Calcium ions (Ca 2+) released from inositol trisphosphate (IP 3)-sensitive intracellular stores may participate in both the transient and extended regulation of neuronal excitability in neocortical and hippocampal pyramidal neurons. IP 3 receptor (IP 3R) antagonists represent an important tool for dissociating these consequences of IP 3 generation and IP 3R-dependent internal Ca 2+ release from the effects of other, concurrently stimulated second messenger signaling cascades and Ca 2+ sources. In this study, we have described the actions of the IP 3R and store-operated Ca 2+ channel antagonist, 2-aminoethoxydiphenyl-borate (2-APB), on internal Ca 2+ release and plasma membrane excitability in neocortical and hippocampal pyramidal neurons. Specifically, we found that a dose of 2-APB (100 μM) sufficient for attenuating or blocking IP 3-mediated internal Ca 2+ release also raised pyramidal neuron excitability. The 2-APB-dependent increase in excitability reversed upon washout and was characterized by an increase in input resistance, a decrease in the delay to action potential onset, an increase in the width of action potentials, a decrease in the magnitude of afterhyperpolarizations (AHPs), and an increase in the magnitude of post-spike afterdepolarizations (ADPs). From these observations, we conclude that 2-APB potently and reversibly increases neuronal excitability, likely via the inhibition of voltage- and Ca 2+-dependent potassium (K +) conductances.

Nitric oxide regulates growth cone filopodial dynamics via ryanodine receptor‐mediated calcium release

Nitric oxide (NO) is a gaseous intercellular messenger involved in numerous processes during development, including wiring of the nervous system. Neuronal growth cones are responsible for establishing the correct connectivity in the nervous system, but how NO might affect neuronal pathfinding is not fully understood. We have demonstrated in a previous study that local application of a NO donor, NOC-7, via micropipette onto individual growth cones from Helisoma trivolvis B5 neurons results in an increase in filopodial length, a decrease in filopodial number and an increase in the intracellular calcium concentration ([Ca(2+)](i)). Moreover, these NO-induced effects were demonstrated to be mediated via an intracellular cascade involving soluble guanylyl cyclase, protein kinase G (PKG) and cyclic adenosine diphosphate ribose (cADPR). We now demonstrate that the increase in the [Ca(2+)](i) that results from local NO application is mediated via release from ryanodine receptor (RyR)-sensitive intracellular stores. We also show that PKG and RyRs are localized within growth cones and microinjection of cADPR mimics the effects of NO, providing further support that the NO-induced effects are mediated via cADPR. Lastly, we provide evidence that calcium influx across the plasma membrane is a necessary component of the NO-induced calcium increase; however, this calcium influx is secondary to the RyR-induced calcium release from intracellular stores. This study details a signalling pathway by which NO can cause changes in growth cone morphology and thus provides a mechanism by which NO could affect neuronal wiring by acting locally on individual growth cones during the pathfinding process.

Nicotinic receptor‐mediated enhancement of long‐term potentiation involves activation of metabotropic glutamate receptors and ryanodine‐sensitive calcium stores in the dentate gyrus

Little is known about the mechanisms underlying the enhancement of long-term potentiation (LTP) by nicotine. In the present study, the mechanisms of nicotinic enhancement of LTP were investigated in the rat dentate gyrus in vitro. Acute application of nicotine enhanced LTP induction, an action requiring activation of alpha7 nicotinic acetylcholine receptors (nAChRs), as it was blocked by the nAChR antagonist methyl-lycaconitine, mimicked by the acetylcholine receptor agonist choline and absent in mutant mice null for alpha7 nAChR. Nicotinic enhancement of LTP was both dependent on N-methyl-D-aspartate receptor activation, as no LTP was induced in the presence of nicotine and an N-methyl-D-aspartate receptor antagonist, and expressed post-synaptically, as no change in paired-pulse ratio accompanied nicotinic enhancement of LTP. The nicotinic-enhanced component of LTP, unlike control LTP, was dependent on activation of metabotropic glutamate receptors (mGluRs), being inhibited by the group I/II antagonist LY341495 and the mGluR5 antagonist MPEP, and also dependent on influx of Ca via L-type Ca channels and release from ryanodine (RyR)-sensitive intracellular stores, being prevented by nifedipine and RyR, respectively. It is suggested that nicotinic activation of the Ca-permeable alpha7 nAChRs fills RyR Ca stores and release of Ca from such stores by high-frequency stimulation via Ca-induced Ca release and activation of mGluRs induces an additional component of LTP which summates with control LTP. Chronic application of nicotine in vivo also enhanced LTP induction in slices and was dependent on activation of mGluRs and Ca release from RyR-sensitive intracellular stores, although acutely applied nicotine was not required for such enhanced LTP.

Origin of spontaneous rhythmicity in smooth muscle

Rhythmic electrical activity is a feature of most smooth muscles but the mechanical consequences can vary from regular rapid phasic contractions to sustained contracture. For many years it was thought that spontaneous electrical activity originated in smooth muscle cells but recently it has become apparent that there are specialized pacemaker cells in many organs that are morphologically and functionally distinct from smooth muscle and that the former cells are the source of spontaneous electrical activity. Such a pacemaker function is well documented for the ICC of the gastrointestinal tract but evidence is accumulating that ICC-like cells play a similar role in other types of smooth muscle. We have recently shown that there are specialized pacemaking cells in the rabbit urethra which are spontaneously active when freshly isolated, readily distinguishable from smooth muscle cells under bright field illumination and relatively easy to study using patch-clamp and confocal imaging techniques. Recent results suggest that calcium oscillations in isolated rabbit urethral interstitial cells are initiated by calcium release from ryanodine sensitive intracellular stores, that oscillation frequency is very sensitive to the external calcium concentration and that conversion of the primary oscillation to a propagated calcium wave depends upon IP3-induced calcium release.

GAP43 stimulates inositol trisphosphate-mediated calcium release in response to hypotonicity.

The identification of osmo/mechanosensory proteins in mammalian sensory neurons is still elusive. We have used an expression cloning approach to screen a human dorsal root ganglion cDNA library to look for proteins that respond to hypotonicity by raising the intracellular Ca(2+) concentration ([Ca(2+)](i)). We report the unexpected identification of GAP43 (also known as neuromodulin or B50), a membrane-anchored neuronal protein implicated in axonal growth and synaptic plasticity, as an osmosensory protein that augments [Ca(2+)](i) in response to hypotonicity. Palmitoylation of GAP43 plays an important role in the protein osmosensitivity. Depletion of intracellular stores or inhibition of phospholipase C (PLC) activity abrogates hypotonicity-evoked, GAP43-mediated [Ca(2+)](i) elevations. Notably, hypotonicity promoted the selective association of GAP43 with the PLC-delta(1) isoform, and a concomitant increase in inositol-1,4,5-trisphosphate (IP(3)) formation. Collectively, these findings indicate that hypo-osmotic activation of GAP43 induces Ca(2+) release from IP(3)-sensitive intracellular stores. The osmosensitivity of GAP43 furnishes a mechanistic framework that links axon elongation with phospho inositide metabolism, spontaneous triggering of cytosolic Ca(2+) transients and the regulation of actin dynamics and motility at the growth cone in response to temporal and local mechanical forces.

A comparison between the distinct inward currents activated in rat cultured dorsal root ganglion neurones by intracellular flash photolysis of two forms of caged cyclic guanosine monophosphate

Whole cell inward currents activated by intracellular photorelease of cyclic guanosine monophosphate (cGMP) were investigated in cultured dorsal root ganglion (DRG) neurones. The actions of two distinct types of caged cGMP (NPE-caged cGMP and a highly water-soluble caged cGMP) were compared. Rapidly activating inward currents were evoked by cGMP in a subpopulation (12.5%) of neurones and these currents may be due to activity of cyclic nucleotide-gated channels. In contrast in 52% of DRG neurones intracellular photorelease of cGMP activated a delayed Ca 2+-dependent inward current through the generation of cyclic ADPribose and mobilisation of Ca 2+ from ryanodine sensitive intracellular stores. Similar delayed inward currents were activated by both caged compounds but only NPE-caged cGMP evoked rapidly activating currents. Cyclic GMP appears to increase excitability in some DRG neurones by diverse mechanisms.

Studies on the role of calcium in the 5-HT-stimulated release of glutamate from C6 glioma cells

We recently reported that 5-hydroxytryptamine 2A (5-HT 2A) receptor activation on cultured glial cells induces glutamate release [J. Neurosci. Res. 67 (2002) 399]. Here we use C6 glioma cells to examine the role of calcium in this response. 5-Hydroxytryptamine (5-HT) increases glutamate release from C6 glioma cells, an effect blocked by low calcium conditions. The calcium ionophores ionomycin and calcimycin also released glutamate from C6 glioma cells in a Ca 2+-dependent manner. The effect of 5-HT was reduced by the phospholipase C inhibitor U73122 (1-[6[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1 H-pyrrole-2,5-dione), but not its inactive enantomer U73343(1-[6[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidinedione). The protein kinase C inhibitors staurosporine and calphostin C had no effect on the response to 5-HT, whereas the response was blocked by thapsigargin and caffeine. Neither the L-type calcium channel blockers, nifedipine and verapamil, nor the N-type calcium channel blocker ω-conotoxin GVIA inhibited the effect of 5-HT, whereas NiCl 2 and KCl blocked the response to 5-HT. We conclude that the 5-HT-induced efflux of glutamate from C6 glioma cells is Ca 2+-dependent and involves, at least in part, the mobilisation of Ca 2+ from inositol (1,4,5) tris phosphate (IP 3) sensitive intracellular stores.

Threshold conditions for synaptically evoking Ca(2+) waves in hippocampal pyramidal neurons.

Regenerative Ca(2+) release from inositol 1,4,5-trisphosphate (IP(3))-sensitive intracellular stores in the form of Ca(2+) waves leads to large-amplitude [Ca(2+)](i) increases in the apical dendrites of hippocampal CA1 pyramidal neurons. Release is generated following synaptic activation of group I metabotropic glutamate (mGlu) receptors. We systematically examined the conditions for evoking these waves in transverse slices from 2- to 3-wk-old rats. Using a sharpened asymmetrical bipolar tungsten stimulating electrode placed in the stratum radiatum, we varied the lateral position of the electrode, the number of stimulating pulses, the train frequency, and stimulus current. Several trends were clear. Increasing the frequency of stimulation from 20 to 100 Hz, keeping the total number of pulses constant, lowered the required stimulus current. Stimulation at frequencies below 20 Hz made it difficult to evoke release. Increasing the number of stimulation pulses, keeping the frequency constant, lowered the threshold current. A minimum of five pulses at 100 Hz was required to evoke release reliably, but several examples of success with three pulses were recorded. Theta-burst stimulation was as effective as tetanic stimulation. Placing the point of the stimulation electrode closer to the pyramidal neuron made it easier to evoke release, although stimulation at a lateral distance of 500 microm with unsharpened electrodes was sometimes successful. The simplest explanation for these results is that a bolus of IP(3) must be produced quickly in a restricted region of the dendrites to generate Ca(2+) waves. The conditions necessary for evoking regenerative Ca(2+) release have many parallels (and some differences) with the conditions required to evoke long-term potentiation in these cells following tetanic stimulation.

Mitochondrial polarization in rat hippocampal astrocytes is resistant to cytosolic Ca(2+) loads.

The influence of physiological Ca(2+)-inducing stimuli and agents mimicking ischemic conditions on mitochondrial potential was studied in postnatal (P1) hippocampal astrocytes. Cytosolic Ca(2+) loads with characteristic kinetics of rise and duration, detected by Fura-2, were provoked by extracellular Ca(2+) influx, release from InsP(3)-sensitive intracellular stores, or inhibition of the reloading of endoplasmic reticulum Ca(2+) stores. Inhibitors of mitochondrial respiration caused only moderate release of Ca(2+) from intracellular stores, inducing a rise of less than 60 nM. The maximal Ca(2+) rise was found with InsP(3)-mediated responses (500 nM; via ATP) or with ionophore (4-Br-A23187)-mediated Ca(2+) influx from extracellular medium (770 nM). Remarkably, all these agents causing significant rise of cytosolic Ca(2+), only minimally depolarized the mitochondria. Membrane potential of mitochondria was monitored by Rh123 or TMRE. Depolarization was only found with very high cytosolic Ca(2+) levels (above 60 microM; measured by fura FF). These were achieved with external Ca(2+) influx by ionophore in combination with inhibition of glycolysis. Thus, mitochondria in the astrocytes are obviously not sensitive to moderate cytosolic Ca(2+) loads, irrespective of the source of Ca(2+). Furthermore, isolated rat brain mitochondria display a low sensitivity of respiratory activity to Ca(2+), which is consistent with the data obtained with the astrocytes in vitro. The capacity of isolated mitochondria to build up a potential was gradually reduced at low micromolar Ca(2+) and totally compromised only at Ca(2+) concentrations in the 100 microM range.

Localization and pharmacological characterization of voltage dependent calcium channels in cultured neocortical neurons

The physiological significance and subcellular distribution of voltage dependent calcium channels was defined using calcium channel blockers to inhibit potassium induced rises in cytosolic calcium concentration in cultured mouse neocortical neurons. The cytosolic calcium concentration was measured using the fluorescent calcium chelator fura-2. The types of calcium channels present at the synaptic terminal were determined by the inhibitory action of calcium channel blockers on potassium-induced [3H]GABA release in the same cell preparation. L-, N-, P-, Q- and R-/T-type voltage dependent calcium channels were differentially distributed in somata, neurites and nerve terminals. ω-conotoxin MVIIC (ω-CgTx MVIIC) inhibited approximately 40% of the Ca2+-rise in both somata and neurites and 60% of the potassium induced [3H]GABA release, indicating that the Q-type channel is the quantitatively most important voltage dependent calcium channel in all parts of the neuron. After treatment with thapsigargin the increase in cytosolic calcium was halved, indicating that calcium release from thapsigargin sensitive intracellular calcium stores is an important component of the potassium induced rise in cytosolic calcium concentration. The results of this investigation demonstrate that pharmacologically distinct types of voltage dependent calcium channels are differentially localized in cell bodies, neurites and nerve terminals of mouse cortical neurons but that the Q-type calcium channel appears to predominate in all compartments.

Characterization and Functional Significance of Calcium Transients in the 2-Cell Mouse Embryo Induced by an Autocrine Growth Factor

Growth of preimplantation embryos is influenced by autocrine trophic factors that need to act by the 2-cell stage, but their mode of action is not yet described. This report shows that late zygote and 2-cell stage mouse embryos responded to embryo-derived platelet-activating factor (PAF) with transient increases in intracellular calcium concentration ([Ca(2+)](i)). [Ca(2+)](i) transients were single global events and were specifically induced by embryo-derived PAF. They were blocked by inhibition of phospholipase C (U 73122) and an inositol trisphosphate (IP(3)) receptor antagonist (xestospongin C), indicating the release of calcium from IP(3)-sensitive intracellular stores. Transients were also inhibited by the absence of calcium from extracellular medium and partially inhibited by treatment with dihydropyridine (nifedipine, 10 micrometer), but not pimozide (an inhibitor of an embryonic T-type calcium channel). (+/-)BAY K8644 (an L-type channel agonist) induced [Ca(2+)](i) transients, yet these were completely inhibited by nifedipine (10 micrometer). The complete inhibition of BAY K8644, but only partial inhibition of PAF by nifedipine shows that L-type channels were only partly responsible for the calcium influx. Depolarization of 2-cell embryos by 50 mm K(+) did not inhibit PAF-induced calcium transients, showing that the influx channels were not voltage-dependent. Depletion of intracellular calcium stores by thapsigargin revealed the presence of store-operated channels. The interdependent requirement for IP(3)-sensitive internal calcium stores and extracellular calcium in the generation of PAF-induced transients may be explained by a requirement for capacitative calcium entry via store-operated channels. A functionally important role for the PAF-induced transients is supported by the observation that inhibition of [Ca(2+)](i) transients by a PAF-antagonist (WEB 2086) or an intracellular calcium chelator (1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetrakis-acetoxymethyl ester; BAPTA-AM) caused marked inhibition of early embryo development. Growth inhibition by BAPTA-AM was relieved by addition of exogenous PAF.

Transduction of basolateral-to-apical signals across epithelial cells: ligand-stimulated transcytosis of the polymeric immunoglobulin receptor requires two signals.

Transcytosis of the polymeric immunoglobulin receptor (pIgR) is stimulated by binding of its ligand, dimeric IgA (dIgA). During this process, dIgA binding at the basolateral surface of the epithelial cell transmits a signal to the apical region of the cell, which in turn stimulates the transport of dIgA-pIgR complex from a postmicrotubule compartment to the apical surface. We have previously reported that the signal of stimulation was controlled by a protein-tyrosine kinase (PTK) activated upon dIgA binding. We now show that this signal of stimulation moves across the cell independently of pIgR movement or microtubules and acts through the tyrosine kinase activity by releasing Ca++ from inositol trisphosphate-sensitive intracellular stores. Surprisingly we have found that a second independent signal is required to achieve dIgA-stimulated transcytosis of pIgR. This second signal depends on dIgA binding to the pIgR solely at the basolateral surface and the ability of pIgR to dimerize. This enables pIgR molecules that have bound dIgA at the basolateral surface to respond to the signal of stimulation once they reach the postmicrotubule compartment. We propose that the use of two signals may be a general mechanism by which signaling receptors maintain specificity along their signaling and trafficking pathways.

Chapter 25 Nucleotide receptor signalling in spinal cord astrocytes: Findings and functional implications

Publisher Summary The chapter focuses on the characterization of Ca 2+ signals evoked by extracellular purine and pyrimidine nucleotides in astrocytes from the spinal cord. The chapter demonstrates that spinal cord astrocytes express two functionally distinct subtypes of nucleotide receptor. One receptor appears to be the cloned P2Y 1 receptor while the other represents a novel P2Y receptor subtype activated by uridine- 5 '-triphosphate (UTP). Each receptor couples to the release of Ca 2+ from IP 3 -sensitive intracellular stores but when subjected to sustained stimulation the two subtypes of receptor induce distinctive Ca 2+ response patterns. The chapter reveals that the differing Ca 2+ signals evoked by the different receptors may have distinct functional consequences in terms of signal discrimination, gene expression, growth, and proliferation. The morphological changes induced by ATP stimulation of astrocytes require prolonged periods of stimulation. Thus, in terms of possible involvement of the two metabotropic P2 receptors, involvement of the P2Y receptor seems unlikely as the Ca 2+ signal evoked by stimulating this receptor subtype fades rapidly with sustained stimulation. On the other hand, the pyrimidinoceptor-triggered Ca 2+ signal persists during the prolonged elevations of extracellular nucleotide.

Btk/Tec kinases regulate sustained increases in intracellular Ca2+ following B-cell receptor activation.

Bruton's tyrosine kinase (Btk) is essential for B-lineage development and represents an emerging family of non-receptor tyrosine kinases implicated in signal transduction events initiated by a range of cell surface receptors. Increased dosage of Btk in normal B cells resulted in a striking enhancement of extracellular calcium influx following B-cell antigen receptor (BCR) cross-linking. Ectopic expression of Btk, or related Btk/Tec family kinases, restored deficient extracellular Ca2+ influx in a series of novel Btk-deficient human B-cell lines. Btk and phospholipase Cgamma (PLCgamma) co-expression resulted in tyrosine phosphorylation of PLCgamma and required the same Btk domains as those for Btk-dependent calcium influx. Receptor-dependent Btk activation led to enhanced peak inositol trisphosphate (IP3) generation and depletion of thapsigargin (Tg)-sensitive intracellular calcium stores. These results suggest that Btk maintains increased intracellular calcium levels by controlling a Tg-sensitive, IP3-gated calcium store(s) that regulates store-operated calcium entry. Overexpression of dominant-negative Syk dramatically reduced the initial phase calcium response, demonstrating that Btk/Tec and Syk family kinases may exert distinct effects on calcium signaling. Finally, co-cross-linking of the BCR and the inhibitory receptor, FcgammaRIIb1, completely abrogated Btk-dependent IP3 production and calcium store depletion. Together, these data demonstrate that Btk functions at a critical crossroads in the events controlling calcium signaling by regulating peak IP3 levels and calcium store depletion.

Calcium mobilization contributes to pressure-mediated afferent arteriolar vasoconstriction.

Preglomerular responses to vasoactive agonists utilize calcium released from intracellular stores and activation of calcium influx pathways to elicit vasoconstriction. The current study was performed to determine the role of calcium release from intracellular stores on the afferent arteriolar response to increases in perfusion pressure. Experiments were performed, in vitro, using the blood perfused, juxtamedullary nephron technique combined with videomicroscopy. The response of afferent arterioles to 30 mm Hg increases in perfusion pressure was determined before and after depletion of intracellular calcium pools with a 10-minute preincubation with 1 micromol/L thapsigargin or 100 micromol/L cyclopiazonic acid. Afferent arteriolar diameter averaged 20.2+/-1.0 microm (n=19) at a control perfusion pressure of 100 mm Hg. Increasing perfusion pressure to 130 and 160 mm Hg reduced afferent caliber by 10.7+/-1.0% (P<.05 versus con) and by 24.7+/-1.6% (P<.05 versus diameter at 130 mm Hg); respectively. Thapsigargin significantly increased afferent diameter by 21+/-2% (n=6) at 100 mm Hg and prevented pressure-induced autoregulatory responses. Afferent diameter averaged 24.3+/-1.7, 24.5+/-1.8 and 24.3+/-1.8 microm at perfusion pressures of 100, 130 and 160 mm Hg; respectively. Cyclopiazonic acid treatment also inhibited autoregulatory behavior but did not alter resting vessel diameter. Afferent arteriolar diameter (n=6) averaged 21.4+/-1.9 microm at 100 mm Hg and 20.9+/-2.1 and 20.5+/-2.2 microm at 130 and 160 mm Hg; respectively. Additional studies were performed to assess the role of phospholipase C activity in pressure-mediated autoregulatory behavior of afferent arterioles. Step increases in perfusion pressure decreased afferent diameter by 10.7+/-3.8 and 21.7+/-4.1%; respectively. Administration of the phospholipase C inhibitor, U-73122, (5 micromoles/L) did not significantly alter baseline diameter but did attenuate the pressure-mediated vasoconstrictor response. Increasing perfusion pressure to 130 and 160 mm Hg reduced afferent diameter by only 6.5+/-1.5 and 10.0+/-2.0%; respectively. These data demonstrate that interruption of calcium mobilization with thapsigargin, cyclopiazonic acid, or phospholipase C inhibition markedly attenuates pressure-mediated afferent arteriolar vasoconstriction and suggests that autoregulatory adjustments in afferent arteriolar diameter involve calcium release from inositoltrisphosphate(IP3)-sensitive intracellular stores.