Percentage Of Responding Cells Research Articles

Brainstem pain-modulating neurons are sensitized to visual light in persistent inflammation

Many individuals with chronic pain report abnormal sensitivity to visual light, referred to as “photosensitivity” or “photophobia,” yet how processing of light and nociceptive information come together remains a puzzle. Pain-modulating neurons in the rostral ventromedial medulla (RVM) have been shown to respond to bright visual light in male rats: activity of pain-enhancing ON-cells is increased, while that of pain-inhibiting OFF-cells is decreased. Since the RVM is the output node of a well-known pain modulation pathway, light-related input to these neurons could contribute to photosensitivity. The purpose of the present study was to fully characterize RVM ON– and OFF-cell responses to visual light by defining stimulus–response curves in male and female rats across a range of intensities (30 to 16,000 lx). We also determined if light-evoked responses are altered in animals subjected to persistent inflammation. We found that ON– and OFF-cells responded to relatively dim light (<1000 lx in naïve animals), with no difference between the sexes in threshold for light-evoked changes in firing or the percentage of responsive cells. Second, light-evoked suppression of OFF-cell firing was enhanced in persistent inflammation, with no change in light-evoked activation of ON-cells. These data indicate that pain-modulating neurons can be engaged by dim light, even under normal conditions. Further, they suggest that decreased descending inhibition during light exposure could contribute to reduced nociceptive thresholds in chronic pain states, resulting in light-induced somatic discomfort and aversion to light. Lastly, our findings argue for differences in how light and somatic stimuli engage RVM, and suggest that light-related input acts as a “top-down” regulatory input to RVM.

Carboxamido steroids inhibit the opening properties of transient receptor potential ion channels by lipid raft modulation

Transient Receptor Potential (TRP) cation channels, like the TRP Vanilloid 1 (TRPV1) and TRP Ankyrin 1 (TRPA1), are expressed on primary sensory neurons. These thermosensor channels play a role in pain processing. We have provided evidence previously that lipid raft disruption influenced the TRP channel activation, and a carboxamido-steroid compound (C1) inhibited TRPV1 activation. Therefore, our aim was to investigate whether this compound exerts its effect through lipid raft disruption and the steroid backbone (C3) or whether altered position of the carboxamido group (C2) influences the inhibitory action by measuring Ca2+ transients on isolated neurons and calcium-uptake on receptor-expressing CHO cells. Membrane cholesterol content was measured by filipin staining and membrane polarization by fluorescence spectroscopy. Both the percentage of responsive cells and the magnitude of the intracellular Ca2+ enhancement evoked by the TRPV1 agonist capsaicin were significantly inhibited after C1 and C2 incubation, but not after C3 administration. C1 was able to reduce other TRP channel activation as well. The compounds induced cholesterol depletion in CHO cells, but only C1 induced changes in membrane polarization. The inhibitory action of the compounds on TRP channel activation develops by lipid raft disruption, and the presence and the position of the carboxamido group is essential.

Characterization of Calcium Responses and Electrical Activity in Differentiating Mouse Neural Progenitor Cells In Vitro

In vitro methods for developmental neurotoxicity (DNT) testing have the potential to reduce animal use and increase insight into cellular and molecular mechanisms underlying chemical-induced alterations in the development of functional neuronal networks. Mouse neural progenitor cells (mNPCs) differentiate into nervous system-specific cell types and have proven valuable to detect DNT using biochemical and morphological techniques. We therefore investigated a number of functional neuronal parameters in primary mNPCs to explore their applicability for neurophysiological in vitro DNT testing. Immunocytochemistry confirmed that mNPCs express neuronal, glial, and progenitor markers at various differentiation durations (1, 7, 14, and 21 days). Because intracellular calcium ([Ca(2+)]i) plays an essential role in neuronal development and function, we measured stimulus-evoked changes in [Ca(2+)]i at these differentiation durations using the Ca(2+)-responsive dye Fura-2. Increases in [Ca(2+)]i (averages ranging from 65 to 226 nM) were evoked by depolarization, ATP, l-glutamic acid, acetylcholine, and dopamine (up to 87%, 57%, 93%, 28%, and 37% responding cells, respectively) and to a lesser extent by serotonin and gamma-aminobutyric acid (both up to 10% responding cells). Notably, the changes in percentage of responsive cells and their response amplitudes over time indicate changes in the expression and functionality of the respective neurotransmitter receptors and related calcium signaling pathways during in vitro differentiation. The development of functional intercellular signaling pathways was confirmed using multielectrode arrays, demonstrating that mNPCs develop electrical activity within 1-2 weeks of differentiation (55% active wells at 14 days of differentiation; mean spike rate of 1.16 spikes/s/electrode). The combined data demonstrate that mNPCs develop functional neuronal characteristics in vitro, making it a promising model to study chemical-induced effects on the development of neuronal function.

MicroRNA133 and microRNA499 exert synergistic effect on cardiac differentiation

Purpose: It has been demonstrated that miR1 and miR133 are involved in cardiac development and stem cell differentiation, while miR499 enhances the differentiation of embryonic and adult stem cells into cardiomyocytes (CMC). However, it is currently unknown if these miRNA may act synergistically to improve differentiation efficiency. Methods: Mouse P19 cells transduced with a viral vector expressing GFP under the control of a cTnI promoter were primed with 0.5% of DMSO and used as cardiogenic differentiation model (P19 CTRL). MiR499 was transiently over-expressed in P19 cells individually or together with miR1 or miR133. Moreover, we differentiated P19 cells with pre-miR precursors without DMSO. Cardiac differentiation was quantified by: 1) count of spontaneous beating areas; 2) qPCR of the cardiac markers Nkx2.5, GATA4, MEF2c, MLC2v, Cx43 and cTnT; 3) Western Blot and ICC of cTnT and Cx43; 4) measure of inducibility of RyR-mediated Ca++ transients in P19 exposed to caffeine; 5) recording of mechanical activity after Ca++ depletion and in the presence of nifedipine or ryanodine. Results: The overexpression of miR499 alone increased the number of beating CMC by 1.7 fold compared with P19 CTRL (p<0.05). The association of miR499+133, but not miR499+1, exerted synergistic effects further increasing the number of beating CMC (2.02 fold vs. P19 CTRL, p<0.01). qPCR showed that miR499+133 enhance the expression of GATA4, Nkx2.5, Cx43 and cTnT compared with P19 CTRL (p<0.01). WB and ICC for Cx43 and cTnT confirmed these findings. Caffeine responsiveness was increased 2.6 fold by miR499+133 compared with P19 CTRL (32.5 vs 12.5%, p<0.05). Cyclic contractions were reversibly abolished by extracellular Ca++ depletion and by both nifedipine and ryanodine. Strikingly, the percentage of responsive cells to caffeine was significantly higher in P19 cells treated with pre-miR499+133 but no DMSO compared with P19 CTRL (p<0.05). PCR and WB demonstrated that the over-expression of early and late cardiac genes in P19 cells treated with miRNA precursors and not exposed to DMSO and ICC showed the expression of Cx43 and cTnT proteins, SERCA2a and Cav1.2. Conclusions: Our results show that miR133 and miR499 play synergistic effect in cardiac differentiation to the point that no chemical trigger is required. Our findings may contribute to improve the efficiency of stem cell therapy for cardiac repair.

Ex vivo real-time observation of Ca2 + signaling in living bone in response to shear stress applied on the bone surface

Bone cells respond to mechanical stimuli by producing a variety of biological signals, and one of the earliest events is intracellular calcium ([Ca2+]i) mobilization. Our recently developed ex vivo live [Ca2+]i imaging system revealed that bone cells in intact bone explants showed autonomous [Ca2+]i oscillations, and osteocytes specifically modulated these oscillations through gap junctions. However, the behavior and connectivity of the [Ca2+]i signaling networks in mechanotransduction have not been investigated in intact bone. We herein introduce a novel fluid-flow platform for probing cellular signaling networks in live intact bone, which allows the application of capillary-driven flow just on the bone explant surface while performing real-time fluorogenic monitoring of the [Ca2+]i changes. In response to the flow, the percentage of responsive cells was increased in both osteoblasts and osteocytes, together with upregulation of c-fos expression in the explants. However, enhancement of the peak relative fluorescence intensity was not evident. Treatment with 18 α-GA, a reversible inhibitor of gap junction, significantly blocked the [Ca2+]i responsiveness in osteocytes without exerting any major effect in osteoblasts. On the contrary, such treatment significantly decreased the flow-activated oscillatory response frequency in both osteoblasts and osteocytes. The stretch-activated membrane channel, when blocked by Gd3+, is less affected in the flow-induced [Ca2+]i response. These findings indicated that flow-induced mechanical stimuli accompanied the activation of the autonomous [Ca2+]i oscillations in both osteoblasts and osteocytes via gap junction-mediated cell–cell communication and hemichannel. Although how the bone sense the mechanical stimuli in vivo still needs to be elucidated, the present study suggests that cell-cell signaling via augmented gap junction and hemichannel-mediated [Ca2+]i mobilization could be involved as an early signaling event in mechanotransduction.

Casein phosphopeptides modulate proliferation and apoptosis in HT-29 cell line through their interaction with voltage-operated L-type calcium channels

At the intestinal level, proliferation and apoptosis are modulated by the extracellular calcium concentration; thus, dietary calcium may exert a chemoprotective role on normal differentiated intestinal cells, while it may behave as a carcinogenesis promoter in transformed cells. Calcium in milk is associated with casein and casein phosphopeptides (CPPs), hence is preserved from precipitation. CPPs were demonstrated to induce uptake of extracellular calcium ions by in vitro intestinal tumor HT-29 cells but only upon differentiation. Here, the hypothesis that CPPs could differently affect proliferation and apoptosis in undifferentiated and differentiated HT-29 cells through their binding with calcium ions was investigated. Results showed that CPPs protect differentiated intestinal cells from calcium overload toxicity and prevent their apoptosis favoring proliferation while inducing apoptosis in undifferentiated tumor cells. The CPP effect on undifferentiated HT-29 cells, similar to that exerted by ethyleneglycol-O, O′-bis(2-aminoethyl)-N, N, N′, N′-tetraacetic acid (EGTA), is presumably due to the ability in binding the extracellular calcium. The effect on differentiated HT-29 cells is coupled to the interaction of CPPs with the voltage-operated L-type calcium channels, known to activate calcium entry into the cells under depolarization and to exert a mitogenic effect: the use of an agonist potentiates the cell response to CPPs, while the antagonists abolish the response to CPPs (36% of examined cells) or reduce both the percentage of responsive cells and the increase of intracellular calcium concentration. Taken together, these results confirm the potentialities of CPPs as nutraceuticals/functional food and also as modulators of cellular processes connected to the expression of a cancer phenotype.

Extracellular acidosis activates ASIC-like channels in freshly isolated cerebral artery smooth muscle cells

Recent studies suggest that certain acid-sensing ion channels (ASIC) are expressed in vascular smooth muscle cells (VSMCs) and are required for VSMC functions. However, electrophysiological evidence of ASIC channels in VSMCs is lacking. The purpose of this study was to test the hypothesis that isolated cerebral artery VSMCs express ASIC-like channels. To address this hypothesis, we used RT-PCR, Western blotting, immunolabeling, and conventional whole cell patch-clamp technique. We found extracellular H(+)-induced inward currents in 46% of cells tested (n = 58 of 126 VSMCs, pH 6.5-5.0). The percentage of responsive cells and the current amplitude increased as the external H(+) concentration increased (pH(6.0), n = 28/65 VSMCs responsive, mean current density = 8.1 +/- 1.2 pA/pF). Extracellular acidosis (pH(6.0)) shifted the whole cell reversal potential toward the Nernst potential of Na(+) (n = 6) and substitution of extracellular Na(+) by N-methyl-d-glucamine abolished the inward current (n = 6), indicating that Na(+) is a major charge carrier. The broad-spectrum ASIC blocker amiloride (20 microM) inhibited proton-induced currents to 16.5 +/- 8.7% of control (n = 6, pH(6.0)). Psalmotoxin 1 (PcTx1), an ASIC1a inhibitor and ASIC1b activator, had mixed effects: PcTx1 either 1) abolished H(+)-induced currents (11% of VSMCs, 5/45), 2) enhanced or promoted activation of H(+)-induced currents (76%, 34/45), or 3) failed to promote H(+) activation in nonresponsive VSMCs (13%, 6/45). These findings suggest that freshly dissociated cerebral artery VSMCs express ASIC-like channels, which are predominantly formed by ASIC1b.

Induced Adaptive Resistance to Nitrooxidative Stress in the CNS: Therapeutic Implications

NO is a free radical that is normally released and utilized by the CNS for a variety of normal physiological functions such as neurotransmission and differentiation. However, if NO released in inappropriate locations, in excess or at a high flux rate, it is toxic. In the CNS, after injury or during disease, NO is released by astrocytes, activated microglia, or macrophages that have migrated across the blood-brain barrier, and is concomitant with massive cell death. Nitrotyrosine formation, a marker for NO-mediated damage, is seen in CNS injury and in many neurodegenerative diseases such as Alzheimers, ALS, multiple sclerosis and spinal injury. In our lab we have found that motor neurons pretreated with sub-toxic doses of NO gain resistance to normally toxic doses of NO. This phenomenon, induced adaptive resistance (IAR), is demonstrated by a significant decrease in the percentage of apoptotic cells in response to a toxic dose of NO. IAR is dependent on the heme-metabolizing enzyme, heme oxygenase-1 (HO1), as indicated by 1) an increase in HO1 expression, 2) loss of resistance in CNS cells incubated with HO1 inhibitors, and 3) lack of native NO resistance in cells isolated from spinal cords of HO1-null mice. The overall aim of this review is to elucidate and dissect the IAR phenomenon and the HO1 signal transduction axis on which it depends, and by so doing, begin to understand native resistance mechanisms in the CNS that can be manipulated to protect neurons against NO-mediated damage seen in CNS injury and neurodegenerative disease.

ATP Release through Connexin Hemichannels in Corneal Endothelial Cells

Intercellular Ca(2+) wave propagation is a distinct form of cell-cell communication. In corneal endothelial cells, intercellular Ca(2+) wave propagation evoked by a point mechanical stimulus (PMS) is partially mediated by adenosine triphosphate (ATP) release and subsequent activation of P2Y receptors. This study was conducted to investigate the possibility that extrajunctional connexons (hemichannels) play a role in ATP release during PMS-induced Ca(2+) wave propagation in bovine corneal endothelial cells (BCECs). A Ca(2+) wave was evoked by a PMS applied to a single cell in a monolayer of cultured BCECs. Changes in [Ca(2+)](i) in the mechanically stimulated cell (MS cell) and in the neighboring (NB) cells were visualized by fluorescence imaging using the Ca(2+)-sensitive dye Fluo-4. From these images, the maximum normalized fluorescence (NF), the percentage of responsive cells (%RC), and the total area of cells reached by the Ca(2+) wave (active area [AA], in square micrometers) were calculated. Intercellular dye transfer, generally attributed to gap junctional coupling, was assessed by fluorescence recovery after photobleaching (FRAP) using 6-carboxyfluorescein diacetate. Opening of hemichannels was investigated by measuring cellular uptake of the fluorescent dye Lucifer yellow, which is known to permeate hemichannels. ATP release was measured by luciferin-luciferase bioluminescence. Flufenamic acid (FFA; 50 microM) and the connexin mimetic peptide Gap26 (300 microM), known blockers of hemichannels, significantly reduced AA in confluent monolayers as well as in contact-free cells. Neither FFA nor Gap26 affected the FRAP, indicating that reduction in AA of the PMS-induced wave by these agents is not due to a block of gap junction channels. FFA as well as Gap26 inhibited the increase in AA of the wave that was observed when cells were pretreated with the ectonucleotidase inhibitor ARL-67156 (100 microM). These findings suggest that the hemichannel blockers reduce the Ca(2+) wave propagation by inhibiting ATP release. Consistent with this finding, PMS or exposure to Ca(2+)-free solution (a maneuver known to induce the opening of hemichannels) led to ATP release; moreover, the release was inhibited by the hemichannel blockers. The extracellular ATP levels in response to both PMS and extracellular Ca(2+) removal were strongly enhanced by ARL-67156, and this effect was inhibited by FFA as well as by Gap26. Moreover, pretreatment of subconfluent BCEC monolayers with FFA or Gap26 inhibited the uptake of Lucifer yellow induced by removal of extracellular Ca(2+). Hemichannels contribute to ATP release on mechanical stimulation in BCECs. The released ATP contributes to propagation of the Ca(2+) wave.

ATP-Dependent Paracrine Intercellular Communication in Cultured Bovine Corneal Endothelial Cells

Intercellular communication (IC) in nonexcitable cells is mediated through gap junctions and/or through the release of paracrine mediators. This study was conducted to investigate adenosine-5' triphosphate (ATP)-dependent paracrine IC in the propagation of Ca2+ waves in confluent monolayers of cultured bovine corneal endothelial cells (BCECs). A Ca2+ wave was induced by point mechanical stimulation (PMS) of a single cell by indentation with a glass micropipette (approximately 1 microm tip) for <1 second. Dynamic changes in [Ca2+]i in the mechanically stimulated (MS) cell and in the neighboring (NB) cells were visualized with a confocal microscope, using a fluorescent dye. Normalized fluorescence (NF), calculated as the ratio of the average fluorescence of a cell to the average under resting conditions, was used as a measure of [Ca2+]i. Expression of P2Y receptors and ecto-adenosine triphosphatases (ATPases) was investigated by RT-PCR. ATP release in response to PMS was measured by luciferin-luciferase (LL) bioluminescence. BCECs subjected to PMS showed a transient [Ca2+]i increase. Under control conditions, the maximum NF in the MS cell occurred within 600 ms, and the fluorescence returned to baseline within 170 seconds. NB cells also presented a [Ca2+]i increase with a transient characterized by decreasing maximum NF and increasing latency as a function of the distance from the MS cell. These transients propagated as an intercellular Ca2+ wave to a distance of five or six NB cells away from the MS cell, covering areas (called active areas, AAs) up to 77,000 +/- 3,200 microm2 (N=21). The percentage of responsive cells (defined as cells showing maximum NF >1.1) decreased with increasing distance from the MS cell. The Ca2+ wave crossed cell-free lanes. Pretreatment of cells with the nonselective purinergic receptor antagonist suramin (200 microM), exogenous apyrases, which break down nucleotides (10 U/mL), or the PLC inhibitor U-73122 (10 microM) reduced the wave propagation, whereas the ecto-ATPase inhibitor ARL-67156 (100 microM) significantly enhanced it. ATP-dependent LL bioluminescence increased after PMS. RT-PCR showed mRNAs for P2Y1 and P2Y2 receptors and ecto-ATPases in BCECs. PMS of BCECs induces release of ATP and a concomitant intercellular Ca2+ wave, even in the absence of direct cell-cell contacts. The AA of the wave is modulated by agents that affect P2Y receptor activity. Thus, PMS-induced intercellular Ca2+ wave propagation in BCECs involves ATP-dependent paracrine IC.

Characterization of nerve growth factors (NGFs) from snake venoms by use of a novel, quantitative bioassay utilizing pheochromocytoma (PC12) cells overexpressing human trkA receptors

Snake venoms are a very abundant source of nerve growth factors (NGF). NGFs of Elapidae showing 65% sequence homology with mouse or human NGF, while the Viperidae NGF shows N-glycosylation (Asn-21) typical of these mammalian NGFs. Snake NGF-induced neurite outgrowth (neurotropic activity) was measured in the past by using PC12 cell or dorsal root ganglion bioassays.The present study was aimed at comparing, by dose-response experiments, the neurotropic activity of cobra and vipera versus mammalian NGFs, by using a novel bioassay involving PC12 cells genetically engineered to overexpress NGF-trkA receptors of human origin.These cells respond to NGF by differentiation (morphologically expressed as neurite outgrowth) by a process mediated by NGF-trkA receptors. This process was evaluated by two different criteria: (1) elongation of neurites (E), and (2) Percentage of responsive cells (PRC) determined by digital acquisition of data and computer analysis. We found that snake venom NGFs were less potent than mouse NGF, and that cobra NGF was more potent than vipera NGF. These data indicate the following order of NGF activity towards recombinant human trkA receptors: recombinant human NGF>mouse NGF>cobra NGF>vipera NGF. The neurotropic efficacy of these NGFs was found to be similar, reaching 80–90% of maximal activity obtained with all NGF forms. Interestingly, cobra (but not vipera) NGF demonstrated prolonged neurotropic activity compared with mouse NGF.The results of the present study indicate that cobra and vipera venom NGFs represent natural agonists of human trkA-receptor of a lower potency, but of similar efficacy, compared with mammalian NGFs. These compounds are important pharmacological tools to characterize the trkA receptor structure-function relationship, and to develop novel neurotropic drugs.

A calcium-permeable channel activated by muscarinic acetylcholine receptors and InsP3 in developing chick ciliary ganglion neurons

The electrical responses elicited by the muscarinic cholinergic pathway have been studied in cultured embryonic chick ciliary ganglion (CG) neurons. Neurons obtained from E7–E8 ganglia were maintained in serum-free medium for 1 to 3 days. Stimulation with 50 μM muscarine induced depolarizing responses in about 30% of the cells tested. In voltage clamp experiments at a holding potential of −50 mV, an inward current could be recorded in the same percentage of cells in response to muscarinic stimulation. In single channel experiments, with standard physiological solution in the pipette, muscarine transiently activated an inward conducting channel. Cell-attached recordings with 100 mM CaCl 2 in the pipette provided evidence that muscarinic agonists can activate a cationic calcium-permeable channel. Two main conductance levels could be detected, of 2.3±0.6 and 5.6±0.6 pS, respectively. In excised patches, addition of 5–20 μM inositol 1,4,5-trisphosphate (InsP 3) to the bath reactivated a channel that could be blocked by heparin and whose characteristics were very similar to those of the channel seen in response to muscarinic stimulation. A channel with similar properties has been previously shown to be activated by basic fibroblast growth factor (bFGF) and InsP 3 in the same preparation.

Norepinephrine- and K+-Induced Mn2+-Dependent Contractions and the Dynamics of Intracellular Mn2+ Changes in Dispersed Smooth Muscle Cells From Ca2+-Depleted Mn2+-Loaded Vas Deferens of the Guinea Pig

The cellular mechanisms of norepinephrine (NE)-induced Mn2+-dependent contractions were investigated in dispersed smooth muscle cells from guinea pig vas deferens by characterizing the effects of NE and K+ on cell length observed by videotape microscopy and on intracellular Mn2+ and Ca2+ concentration ([Mn2+]i and [Ca2+]i) observed by confocal microscopy. Both stimulants induced slow sustained contraction in Ca2+-depleted, Mn2+-accumulated cells (Mn2+-loaded cells), whereas they induced biphasic contractions in normal cells that were neither Ca2+-depleted nor Mn2+-loaded. In both conditions, the number of cells responding to NE as well as the magnitude of NE-induced contractions increased in a dose-dependent manner. Contractions induced by K+ in Mn2+-loaded strip preparations were markedly smaller than those induced by NE. Although individual K+-induced contractions in responsive Mn2+-loaded cells were as large as those induced by NE, a much smaller percentage of Mn2+-loaded cells was responsive to K+ than to NE. These results are consistent with the idea that the contractions of strip preparations may reflect the magnitude of the contractions of individual cells as well as the percentage of responsive cells in the preparations. Inconsistent with the contractions, the [Mn2+]i rise induced by K+ was larger than that induced by NE, and the percentage of cells responsive to K+ was larger than that responsive to NE. These results suggest that NE may increase the Mn2+ sensitivity of contractile elements.

Calcium channels in PDGF-stimulated A172 cells open after intracellular calcium release and are not voltage-dependent

Using laser image cytometry and indo-1 fluorescence, we investigated the intracellular free Ca 2+ concentration ([Ca 2+] i) of confluent A172 human glioblastoma cells stimulated by the BB homodimer of platelet-derived growth factor (PDGF-BB). The shape of the calcium transients and the delay time between stimulation and the beginning of the transient varied considerably. The percentage of responsive cells, the peak [Ca 2+] i and the duration of the response were directly related to PDGF-BB dose, while the delay time was inversely related; the maximal response occurred at a PDGF-BB concentration of 20 ng/ml. Studies with EGTA and inorganic calcium-channel blockers (Ni 2+, La 3+) showed that the increase of [Ca 2+] i resulted from initial release of intracellular stores and subsequent calcium influx across the plasma membrane. Opening of calcium channels in the plasma membrane, monitored directly by studying Mn 2+ quenching of indo-1 fluorescence, was stimulated by PDGF-BB and blocked by La 3+; the opening occurred 55 ± 10 s after the initial increase in [Ca 2+] i. Therefore, in these tumor cells, intracellular release always occurs before channel opening in the plasma membrane. Depolarization of cells with high extracellular [K +] did not generally induce calcium transients but did decrease calcium influx. L-type calcium-channel blockers (verapamil, nifedipine, and diltiazem) had little or no effect on the calcium influx induced by PDGF-BB. These results indicate that PDGF-BB induces calcium influx by a mechanism independent of voltage-sensitive calcium channels in A172 human glioblastoma cells.

Attachment of A172 human glioblastoma cells affects calcium signalling: A comparison of image cytometry, flow cytometry, and spectrofluorometry

The intracellular free calcium concentration ([Ca2+]i) of indo-1 loaded A172 human glioblastoma cells stimulated by platelet-derived growth factor (PDGF) was studied in cell suspensions by flow cytometry and spectrofluorometry and in confluent monolayers by laser image cytometry and spectrofluorometry. With all three techniques, the percentage of responsive cells, peak [Ca2+]i, and the duration of response were directly related, and the delay time was inversely related to PDGF dose. The maximum response occurred at a PDGF concentration of about 20 ng/ml. Basal and peak [Ca2+]i did not differ significantly from method to method even though different calibration procedures were used. Cells in suspension monitored by both spectrofluorometry and flow cytometry displayed significantly shorter calcium responses than attached cells. This did not appear to be a direct effect of trypsinization. Spectral analysis of indo-1 in cytoplasm, 40% glycerol, and aqueous solutions showed significant differences in the isosbestic point and quantum efficiency. Calibration of [Ca2+]i with spectrofluorometry is more accurate using the ratio of fluorescence intensities than the fluorescence intensities measured at either 405 or 485 nm.

Changes in hormone responsiveness of chick oviduct during primary stimulation with estrogen

The induction of ovalbumin mRNA (mRNA ov) and conalbumin mRNA (mRNA con) during primary stimulation of the chick oviduct with estrogen was monitored by hybridization with complementary DNAs (cDNA). Approximately 4 mRNA ov and 30 mRNA con molecules per cell were detected in the unstimulated oviduct and these levels increased to about 250 and 300 molecules/cell, respectively, after 1 day of treatment with estradiol benzoate. The rates of accumulation of both mRNAs were about an order of magnitude lower than the rates observed during secondary stimulation, i.e., in birds withdrawn from prior estrogen treatment and then restimulated. In situ hybridization with cDNA ov revealed that nearly all of the epithelial cells of the oviduct accumulate mRNA ov within a day of estrogen treatment and that the percentage of responsive cells is about the same as in withdrawn oviducts; thus, the submaximal response during primary stimulation cannot be explained by a difference in the number of responsive cells. The number of nuclear estrogen receptors in the oviduct increases from 850 to 4500 molecules per cell within 3 hr of estrogen administration in vivo. These values are greater than the number of nuclear receptors measured during early secondary stimulation, implying that nuclear receptors are not the rate-limiting component during primary stimulation. Therefore, we propose that receptor-induced transcription or mRNA stabilization is suboptimal during primary stimulation. Oviduct explants incubated in culture with 100 n M 17β-estradiol are able to induce mRNA ov and mRNA con. This induction occurs without a significant change in DNA synthesis and is unaffected by hydroxyurea. These observations, taken together with the in vivo studies, indicate that primary stimulation of the oviduct with estrogen is qualitatively similar to secondary stimulation and that the initial accumulation of mRNA ov and mRNA con occurs prior to cell division. Neither progesterone nor glucocorticoids induce mRNA ov or mRNA con during primary stimulation, whereas they are both as effective as estrogen during early secondary stimulation. Thus, during primary stimulation with estrogen, the oviduct becomes responsive to these other steroid hormones. This responsiveness is maintained even in the absence of continued estrogen stimulation. Estrogen must be present for more than 36 hr to allow subsequent secondary stimulation with progesterone. Progesterone receptor levels increase several-fold between 36 and 48 hr after primary stimulation with estrogen; this time correlates well with the time when progesterone begins to show effects on mRNA accumulation and morphogenesis.

Role of visual experience in postcritical-period reversal of effects of monocular deprivation in cat striate cortex.

AbstractWe reported previously that the effects of monocular deprivation on cat striate cortex cells can be partially reversed after the critical period by enucleation of the experienced eye (Kratz et al., '76). The enucleation resulted in a rapid (less than 12 hours) increase in the percent of cells which responded to the deprived eye; however, the receptive field properties of the responsive cells were abnormal. There was no change in this result if the animals were allowed to survive 3 to 16 months after the enucleation, during which the deprived eye remained closed. The present study investigated whether enucleation of the experienced eye extends the period of sensitivity during which connections from the deprived eye can be modified by visual experience. That is, we asked whether opening the deprived eye and allowing it normal visual experience after enucleation of the experienced eye would lead to a further increase in the percentage of responsive cells or produce a modification of receptive field properties.Using single‐cell recording in striate cortex, comparisons were made between monocularly deprived control kittens (MD), kittens which had the experienced eye enucleated at four months of age while the deprived eye remained closed (MD‐DE) or was opened (MD‐DE‐O) for an additional three to four months, and kittens which received reverse‐suture at the same age and for the same duration (MD‐RS). Kittens in both the MD‐DE and MD‐DE‐O groups showed an increase in the percent of cells responsive to the deprived eye compared to MD control kittens, replicating our previous findings. However, there were no differences between the MD‐DE and MD‐DE‐O groups either in terms of percent responsive cells or in the receptive field properties of those which responded. Reverse‐suture also produced a significant, although variable, increase in the percent of cells responding to the initially deprived eye. However, this increase was significantly less than that produced by enucleation of the experienced eye in MD‐DE and MD‐DE‐O animals. Among the cells responsive to the initially deprived eye in the reverse‐suture animals, the receptive field properties remained abnormal.These results suggest that binocular interactions continue to play an active role in the effects of monocular deprivation after the previously defined critical period. However, reversal procedures such as suture or enucleation of the experienced eye do not extend the period in development during which cells in striate cortex can be modified by visual experience. Rather, they may simply release those cells already receiving anatomical connections from the deprived eye from a tonic inhibition mediated by the experienced eye.