Calcium Channel Agonist Bay K8644 Research Articles

A Silicon-Based Field-Effect Biosensor for Drug-Induced Cardiac Extracellular Calcium Ion Change Detection.

Calcium ions participate in the regulation of almost all biological functions of the body, especially in cardiac excitation-contraction coupling, acting as vital signaling through ion channels. Various cardiovascular drugs exert their effects via affecting the ion channels on the cell membrane. The current strategies for calcium ion monitoring are mainly based on fluorescent probes, which are commonly used for intracellular calcium ion detection (calcium imaging) and cannot achieve long-term monitoring. In this work, an all-solid-state silicone-rubber ion-sensitive membrane was fabricated on light-addressable potentiometric sensors to establish a program-controlled field-effect-based ion-sensitive light-addressable potentiometric sensor (LAPS) platform for extracellular calcium ion detection. L-type calcium channels blocker verapamil and calcium channel agonist BayK8644 were chosen to explore the effect of ion channel drugs on extracellular calcium ion concentration in HL-1 cell lines. Simultaneously, microelectrode array (MEA) chips were employed to probe the HL-1 extracellular field potential (EFP) signals. The Ca2+ concentration and EFP parameters were studied to comprehensively evaluate the efficacy of cardiovascular drugs. This platform provides more dimensional information on cardiovascular drug efficacy that can be utilized for accurate drug screening.

Hydroxychloroquine and azithromycin alter the contractility of living porcine heart slices.

The cardiotoxicity risk of hydroxychloroquine (HCQ) and azithromycin (AZM) has been the subject of intensive research triggered by safety concerns in COVID-19 patients. HCQ and AZM have been associated with QT interval prolongation and drug-induced arrhythmias, however other cardiotoxicity mechanisms remain largely unexplored. Our group has pioneered the living heart slice preparation, an ex-vivo platform that maintains native cardiac tissue architecture and physiological electrical and contractile properties. Here, we evaluated the cardiotoxic effect of HCQ and AZM applied alone or in combination on cardiac contractility by measuring contractile force and contraction kinetics in heart slices prepared from porcine hearts. Our results show that clinically relevant concentrations of HCQ monotherapy (1-10µM) reduced contractile force and contraction kinetics in porcine slices in a dose-dependent manner. However, AZM monotherapy decreased contractile force and contraction kinetics only at higher concentrations (30µM). Combination of HCQ and AZM induced a dose-dependent effect similar to HCQ alone. Furthermore, pre-treating porcine heart slices with the L-type calcium channel agonist Bay K8644 prevented the effect of both drugs, while administration of Bay K8644 after drugs interventions largely reversed the effects, suggesting a mechanism involving inhibition of L-type calcium channels. These findings indicate that HCQ and AZM alter cardiac function beyond QT prolongation with significant contractile dysfunction in intact cardiac tissue. Our porcine heart slices provide a powerful platform to investigate mechanisms of drug cardiotoxicity.

Calcium-Prolactin Secretion Coupling in Rat Pituitary Lactotrophs Is Controlled by PI4-Kinase Alpha

The role of calcium, but not of other intracellular signaling molecules, in the release of pituitary hormones by exocytosis is well established. Here, we analyzed the contribution of phosphatidylinositol kinases (PIKs) to calcium-driven prolactin (PRL) release in pituitary lactotrophs: PI4Ks - which control PI4P production, PIP5Ks - which synthesize PI(4, 5)P2 by phosphorylating the D-5 position of the inositol ring of PI4P, and PI3KCs – which phosphorylate PI(4, 5)P2 to generate PI(3, 4, 5)P3. We used common and PIK-specific inhibitors to evaluate the strength of calcium-secretion coupling in rat lactotrophs. Gene expression was analyzed by single-cell RNA sequencing and qRT-PCR analysis; intracellular and released hormones were assessed by radioimmunoassay and ELISA; and single-cell calcium signaling was recorded by Fura 2 imaging. Single-cell RNA sequencing revealed the expression of Pi4ka, Pi4kb, Pi4k2a, Pi4k2b, Pip5k1a, Pip5k1c, and Pik3ca, as well as Pikfyve and Pip4k2c, in lactotrophs. Wortmannin, a PI3K and PI4K inhibitor, but not LY294002, a PI3K inhibitor, blocked spontaneous action potential driven PRL release with a half-time of ~20 min when applied in 10 µM concentration, leading to accumulation of intracellular PRL content. Wortmannin also inhibited increase in PRL release by high potassium, the calcium channel agonist Bay K8644, and calcium mobilizing thyrotropin-releasing hormone without affecting accompanying calcium signaling. GSK-A1, a specific inhibitor of PI4KA, also inhibited calcium-driven PRL secretion without affecting calcium signaling and Prl expression. In contrast, PIK93, a specific inhibitor of PI4KB, and ISA2011B and UNC3230, specific inhibitors of PIP5K1A and PIP5K1C, respectively, did not affect PRL release. These experiments revealed a key role of PI4KA in calcium-secretion coupling in pituitary lactotrophs downstream of voltage-gated and PI(4, 5)P2-dependent calcium signaling.

Taurine–magnesium coordination compound, a potential anti-arrhythmic complex, improves aconitine-induced arrhythmias through regulation of multiple ion channels

Taurine–magnesium coordination compound (TMCC) exhibits antiarrhythmic effects in cesium–chloride-and ouabain-induced arrhythmias; however, the mechanism underlying these effects on arrhythmia remains poorly understood. Here, we investigated the effects of TMCC on aconitine-induced arrhythmia in vivo and the electrophysiological effects of this compound in rat ventricular myocytes in vitro. Aconitine was used to induce arrhythmias in rats, and the dosages required to produce ventricular premature contraction (VPC), ventricular tachycardia (VT), ventricular fibrillation (VF), and cardiac arrest (CA) were recorded. Additionally, the sodium current (INa) and L-type calcium current (ICa,L) were analyzed in normal and aconitine-treated ventricular myocytes using whole-cell patch-clamp recording. In vivo, intravenous administration of TMCC produced marked antiarrhythmic effects, as indicated by the increased dose of aconitine required to induce VPC, VT, VF, and CA. Moreover, this effect was abolished by administration of sodium channel opener veratridine and calcium channel agonist Bay K8644. In vitro, TMCC inhibited aconitine-induced increases in INa and ICa,L. These results revealed that TMCC inhibited aconitine-induced arrhythmias through effects on INa and ICa,L.

Phenytoin inhibits contractions of rat gastrointestinal and portal vein smooth muscle by inhibiting calcium entry.

Phenytoin is widely used as a second-line treatment for status epilepticus. Besides its well-known cardiac pro-arrhythmogenicity, side effects on other organ systems have received less attention. This study investigates the effects of phenytoin on gastrointestinal tissue function using an invitro model of smooth muscle preparations from rats by combining registrations of pharmacological effects on mechanical contractions, electric field potentials, and dynamic intravital fluorescence microscopy. When added to the bathing solution at a concentration of 30μM, phenytoin reduced the frequency of spontaneous activity significantly in antrum and portal vein preparations to 72.2±36.5% (p=0.022) and 80.7±24.4% (p=0.037) of control values, respectively. At a concentration of 100μM, theheight of spontaneous contractions declined to 9.8±19.6% (p=0.005) (antrum), 15.7±28.2% (p=0.004) (portal vein), and 31.8±31.3% (p= 0.005) (colon) in comparison to the control conditions before the application of phenytoin. Depolarization triggered increases in calcium dependent fluorescence signals were reduced by 52.8±39.1% (p=0.012) The inhibition of spontaneous activity caused by phenytoin was reduced in the presence of the L-type calcium channel agonist BAY K8644(-). Phenytoin exerts strong inhibitory effects on the spontaneous and stimulated contractile activity of smooth muscles from both the upper and lower gastrointestinal tract. The mechanism underlying thiseffect is not related to the sodium channel blocking activity of phenytoin, but is rather causedby an inhibition of calcium entry through voltage dependent L-type calcium channels. The results of this study should raise vigilance to gastrointestinal complications in patients treated with phenytoin.

The calcium channel agonist BAY K8644 enhances the responsiveness of human airway muscle to KCl and histamine but not to carbachol.

The role of calcium channel antagonists in asthma remains controversial. A new compound BAY K8644 is an agonist at the calcium voltage-dependent channel (VDC). Therefore, we studied the effect of BAY K8644 on contraction to KCl, histamine, and carbachol in human bronchus in vitro to assess the role of the VDC in this tissue. Cumulative concentration response curves were obtained to the agonists in bronchial spiral strips in the absence and in the presence of BAY K8644. Contractile responses to histamine were significantly (p less than 0.05) enhanced by BAY K8644 (10(-6) M) at concentrations between 10(-7) M and 3 X 10(-6) M and there was a significant decrease in the histamine EC50 (the concentration producing half the maximal response). Responses to low concentrations of KCl (10(-4) M to 3 X 10(-3) M) were also potentiated. BAY K8644 did not affect carbachol-induced contractions. We conclude that calcium influx through VDC occurs in response to KCl and histamine but not to carbachol. Such findings could explain the variability in efficacy of calcium channel antagonists in inhibiting various forms of bronchospasm.

Antispasmodic effects of myrrh due to calcium antagonistic effects in inflamed rat small intestinal preparations.

Myrrh is the oleo-gum resin of mainly Commiphora molmol and as a powdered substance, one compound in the traditional medicinal product Myrrhinil-Intest®, which has been used for the treatment of unspecific, inflammatory intestinal disorders. The aim of the present study was to evaluate the antispasmodic effect of myrrh under healthy and inflamed conditions, and to evaluate a calcium-antagonistic effect as a possible mode of action. Therefore, an ethanolic myrrh extract was tested for its effects on muscle tone and acetylcholine-induced contractions in untreated and inflamed rat ileum/jejunum preparations. Inflammation was experimentally induced by 2,4,6-trinitrobenzene sulfonic acid (10 mM, 30 min). Additionally, the effect of the calcium channel agonist Bay K8644 in the presence of varying myrrh extract concentrations was examined. Myrrh extract (0.99 mg/mL) suppressed the acetylcholine-induced contraction down to 25.8 % in untreated and 15.2 % in inflamed preparations. Myrrh extract (0.15; 0.25 and 0.35 mg/mL) induced a concentration-dependent rightward shift of the Bay K8644 concentration-response curve in untreated and inflamed preparations with a significant EC50 shift. Schild analysis resulted in a pA2 value of 0.93 for untreated preparations. Increasing myrrh extract concentrations induced a concentration-dependent decrease of the agonistic maximum effect in untreated and inflamed preparations down to 15.8 % and 25.8 %, respectively, for the highest concentration leading to a pD2 value of 0.58. Myrrh extract reduced intestinal muscle tone and acetylcholine-induced contraction of untreated and inflamed ileum/jejunum preparations based on dual calcium antagonism characterized by a right shift of the agonistic dose-response curve and a depression of the maximum effect. The resulting reduction of intestinal motility and spasmolytic effects provide a rationale for the symptom treatment of intestinal disorders such as irritable bowel syndrome.

Protective effect of calcium channel agonist BayK8644 on lungs against ischemia-reperfusion injury in rabbits

To explore the protective effect of calcium channel agonist BayK8644 preconditioning on the lungs against ischemia-reperfusion injury (I/R) and its mechanism in rabbits. Forty rabbits were randomly divided into 4 groups (10 in each group): a sham-operated group (Sham), an I/R group (I/R), an ischemic preconditioning (IP) group (IP), and a BayK8644 preconditioning group(BayK8644).The wet to dry weight (W/D) ratio, superoxide dismutase (SOD) activity, myleoperoxidase (MPO), and malondialdehyde (MDA) contents of the lung tissues were measured after the operation. Morphological and ultrastructural changes of the lung tissue were observed by light and electron microscope. The expression of pulmonary surfactant-associated protein-A (SP-A) was examined with immunohistochemistry. The W/D ratio, MPO and MDA contents of the lung tissue in the BayK8644 group and IP group were significantly lower than those in the I/R group (P<0.05), while the SOD activity and expression of SP-A of the lung tissue in the BayK8644 group and IP group were significantly higher than those in the I/R group(P<0.05), The morphological changes and ultrastructural changes were less notable in the BayK8644 group and IP group, compared with the I/R group. There was no significant difference in SOD activity, MPO and MDA contents between the BayK8644 group and IP group (P>0.05). Appropriate BayK8644 preconditioning can induce transient Ca²+ influx, and elicit strong protection against the lung ischemia-reperfusion injury, which can simulate the endogenous protective effect of ischemic preconditioning.

TRPA1 channels: Novel targets of 1,4-dihydropyridines

Transient receptor potential type A1 (TRPA1) channels are cation permeable channels activated by irritant chemicals and pungent compounds. Their location in peptidergic sensory terminals innervating the skin and blood vessels makes them important effectors of vasodilator responses of neural origin. 1,4-dihydropyridines are a class of L-type calcium channel antagonists commonly used in the treatment of hypertension and ischemic heart disease. Here we show that four different 1,4-dihydropyridines (nifedipine, nimodipine, nicardipine and nitrendipine), and the structurally related L-type calcium channel agonist BayK8644, exert powerful excitatory effects on TRPA1 channels. The activation does not depend on elevated Ca2+ levels and cross-desensitizes with that produced by other TRPA1 agonists. The activation produced by nifedipine was reduced by camphor and the selective TRPA1 antagonist HC03001. In a subclass of mouse nociceptors expressing TRPA1 channels, assessed by responses to the TRPA1 agonist mustard oil, nifedipine also produced large elevations in [Ca2+]i. These responses were fully abrogated in TRPA1(-/-) mice. These findings identify TRPA1 channels as a new molecular target for the 1,4-dihydropyridine class of calcium channel modulators.

Using Dihydropyridine-Release Strategies to Enhance Load Effects in Engineered Human Bone Constructs

We report on the development of a novel biodegradable scaffold capable of enhancing mechanical signals for tissue-engineering applications. It has been shown that mechanotransduction enhances bone formation in vitro and in vivo; in tissue-engineering applications, this phenomenon is exploited through the use of mechanical bioreactors to generate bone tissue. The dihydropyridine agonist Bay K8644 (Bay) acts to increase the opening time of mechanosensitive voltage-operated calcium channels (VOCCs), specifi- cally L-type VOCCs, which are known to play a fundamental role in the early mediation of mechanotransduction. We have produced porous 3-dimensional, Bay-encapsulated biodegradable poly(L-lactide) acid scaffolds using a solvent-casting and salt-leaching technique. The effects of the released Bay on osteoid production and mineralization in human bone cell-seeded constructs following incubation in a perfusion-compression bioreactor in vitro was investigated using Western blotting techniques and a calcium assay protocol developed in our lab. Our newly developed scaffolds act by slowly releasing the calcium channel agonist Bay K8644 as observed using ultraviolet spectroscopy, maintaining the open state of mechanosensitive VOCCs responding to load, which augments the load signal at sites of strain across the scaffold. Our results demonstrate that, in the presence of physiological loading regimes in vitro, release of Bay enhances collagen I protein production and osteoid calcification more than non-Bay control constructs do. Osteopontin and alpha2delta1 VOCC subunit protein levels were also higher as a result of perfusion-compression conditioning. These results indicate that Bay-encapsulated scaffolds can be used in the presence of load to enhance the production of load-bearing engineered tissue.

Cellular basis for the electrocardiographic and arrhythmic manifestation of Timothy Syndrome

Background: Timothy Syndrome (TS), also referred to as syndactyly-associated long QT syndrome (LQTS), is characterized by multisystem disorders including prolongation of the QT interval and the development of ventricular cardiac arrhythmias. The syndrome has recently been linked to a missense mutation in the Cav1.2 gene resulting in a gain of function of the L-type calcium current (Splawski et al, Cell:119,19,2004). In the present study, we used the calcium channel agonist BayK8644 to mimic the gain of function of ICa in an attempt to create an experimental model of TS.

Mechanism of angiotensin II-induced phospholipase D activation in bovine adrenal glomerulosa cells

Based on previous data demonstrating activation of phospholipase D (PLD) in response to angiotensin II (AngII), we have hypothesized a role for PLD in mediating aldosterone secretion from bovine adrenal glomerulosa cells. In this study we demonstrate that a PLD-generated signal(s) is required for the AngII-elicited secretory response, since interfering with lipid second messenger formation using a primary alcohol inhibited AngII-induced aldosterone secretion, but not that elicited by incubation with a hydrophilic cholesterol analog, 22( R)-hydroxycholesterol, which bypasses signaling pathways. Three mechanisms for hormonal activation of PLD have been described in other systems: direct receptor coupling, activation through protein kinase C (PKC) and a combination of these two mechanisms. Our results indicate that the PKC activator, phorbol 12-myristic 13-acetate (PMA), is able to activate PLD, and that receptor engagement is apparently not necessary for PLD activation in response to this agent. Maximal doses of AngII and PMA produced no additive effect on PLD activation, suggesting that these two agents function through a common PKC pathway. This interpretation was confirmed by the ability of a PKC inhibitor, Gö 6976, to inhibit partially AngII-induced PLD activation. Finally, treatment with the calcium ionophores A23187 or ionomycin or the calcium channel agonist BAY K8644 had no effect on PLD activity. Likewise, inhibiting calcium influx with high-dose nitrendipine affected neither basal PLD activity nor that stimulated by AngII. Thus, our results suggest a role for PKC, independent of calcium influx, in mediating AngII-induced PLD activation in glomerulosa cells.

Effects of dextromethorphan on the seizures induced by kainate and the calcium channel agonist BAY k-8644: comparison with the effects of dextrorphan

BAY k-8644 (an L-type Ca 2+ channel agonist of the dihydropyridine class) is recognized as a potent convulsant agent. In this study, we used BAY k-8644 to explore the effects of dextromethorphan (DM) and its major metabolite, dextrorphan (DX), on the (pro)convulsant activity regulated by calcium channels. BAY k-8644 (2 mg/kg, s.c) potentiated seizures induced in rats by kainic acid (KA) (10 mg/kg, i.p.). DM appears more efficacious than DX in attenuation of KA-induced seizures. The anticonvulsant effect of a low dose (12.5 mg/kg, s.c.) of DM was reversed by BAY k-8644 (2 mg/kg) challenge. In contrast, BAY k-8644 (1 or 2 mg/kg) did not significantly affect an anticonvulsant effect from a higher dose (25 mg/kg) of either DM or DX. Intracerebroventricular injection of BAY k-8644 (37.5 μg) significantly induced seizures in mice. DM (12.5 or 25 mg/kg) pretreatment more significantly attenuated seizures evoked by BAY k-8644 than did DX (12.5 or 25 mg/kg). Furthermore, seizure activity induced by KA or BAY k-8644 was consistent with respective activator protein-1 DNA binding activity of the hippocampus. Therefore, our results suggest that the anticonvulsant effects of the morphinans involve, at least in part, the L-type calcium channel. They also suggest that DM is a more potent anticonvulsant than DX in the KA and BAY k-8644 seizure models.

Complex influence of the L-type calcium-channel agonist bayk8644(±) on N-methyl- d-aspartate responses and neuronal survival

Past studies have implicated calcium influx through the N-methyl- d-aspartate class of ionotropic glutamate receptors as a key factor in excitotoxicity. Here, primary cultures of hippocampal neurons were exposed to N-methyl- d-aspartate with or without the L-type calcium channel agonist BayK8644(±). Calcium influxes were monitored with Fura-2 microfluorescent imaging and 45Ca measurements, and survival was assayed through cell counts. While 100 μM BayK8644 alone evoked a moderate elevation of intraneuronal calcium concentrations ([Ca 2+] i), it dramatically attenuated the larger calcium influxes triggered by 500 μm N-methyl- d-aspartate. This attenuation was non-competitive and reversible; it was not inhibited by charybdotoxin or cyclosporin A. In spite of this attenuation of [Ca 2+] i responses, 5-min exposures to BayK8644 produced much greater neurotoxicity 24 h later than did doses of N-methyl- d-aspartate evoking larger [Ca 2+] i increases. This neurotoxicity was not observed with r potassium-mediated depolarization or cobalt; indeed, both reversed the neurotoxicity of BayK8644. The relevant conclusions are two-fold: BayK8644 inhibits influx of calcium through a ligand-gated glutamate receptor, and BayK8644 exhibits considerable neurotoxicity. The former effect does not appear to depend upon the major metabolic pathways that modulate N-methyl- d-aspartate channels and thus may involve a direct allosteric interaction with the N-methyl- d-aspartate receptor. The toxicity of BayK8644 depends, at least partially, upon its activation of voltage-gated (cobalt-sensitive) calcium channels. However, the reversal of this toxicity by depolarization suggests that depolarization can be beneficial to neuronal survival through mechanisms other than calcium influx through voltage-gated calcium channels.

L-type calcium channels contribute to the tottering mouse dystonic episodes.

Tottering mice inherit a recessive mutation of the calcium channel alpha1A subunit that causes ataxia, polyspike discharges, and intermittent dystonic episodes. The calcium channel alpha1A subunit gene encodes the pore-forming protein of P/Q-type voltage-dependent calcium channels and is predominantly expressed in cerebellar granule and Purkinje neurons with moderate expression in hippocampus and inferior colliculus. Because calcium misregulation likely underlies the tottering mouse phenotype, calcium channel blockers were tested for their ability to block the motor episodes. Pharmacologic agents that specifically block L-type voltage-dependent calcium channels, but not P/Q-type calcium channels, prevented the inducible dystonia of tottering mutant mice. Specifically, the dihydropyridines nimodipine, nifedipine, and nitrendipine, the benzothiazepine diltiazem, and the phenylalkylamine verapamil all prevented restraint-induced tottering mouse motor episodes. Conversely, the L-type calcium channel agonist Bay K8644 induced stereotypic tottering mouse dystonic at concentrations significantly below those required to induce seizures in control mice. In situ hybridization demonstrated that L-type calcium channel alpha1C subunit mRNA expression was up-regulated in the Purkinje cells of tottering mice. Radioligand binding with [3H]nitrendipine also revealed a significant increase in the density of L-type calcium channels in tottering mouse cerebellum. These data suggest that although a P/Q-type calcium channel mutation is the primary defect in tottering mice, L-type calcium channels may contribute to the generation of the intermittent dystonia observed in these mice. The susceptibility of L-type calcium channels to voltage-dependent facilitation may promote this abnormal motor phenotype.

Protein kinase A, protein kinase C, and Ca(2+)-regulated expression of 21-hydroxylase cytochrome P450 in H295R human adrenocortical cells.

The physiological importance of adrenal 21-hydroxylase cytochrome P450 (CYP21) expression is clearly demonstrated by 21-hydroxylase deficiency, which results in adrenal hyperplasia and over-production of C19 steroids, leading to virilization. The mechanisms regulating normal expression of this key enzyme in human adrenocortical cells are ill defined. Herein we examine the role of the calcium, protein kinase C, and protein kinase A signaling pathways in the expression of CYP21 messenger ribonucleic acid (mRNA) using the H295R human adrenocortical cell model. Forskolin (10 mumol/L) treatment caused a progressive increase in CYP21 mRNA levels (maximum, 4-fold; P < 0.05) over 36 h of treatment, whereas angiotensin II (AII; 10 nmol/L) produced a smaller, biphasic rise (maximum, 1.8-fold at 12 h; P < 0.05). K+ (14 mmol/L) also induced a time-dependent (maximal, 1.5-fold at 12 h; P < 0.05) and dose-dependent (P < 0.05 12 mmol/L or above at 20 h) rise in CYP21 mRNA levels. The action of forskolin was reproduced by dibutyryl cAMP, confirming the involvement of cAMP in this response. The action of AII was greater than that of K+ or the calcium channel agonist BAYK8644, suggesting that AII action was not solely through the Ca2+ signaling pathway. The action of AII was reproduced and indeed exceeded by the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA; 10 nmol/L; 5.5-fold increase; P < 0.05). The actions of forskolin alone were not significantly increased by combined treatment with AII, suggesting neither synergy nor attenuation of the effects of protein kinase A activation. This was further demonstrated at the level of mRNA and 21-hydroxylase activity by the observation that the effect of forskolin and TPA in combination did not exceed that of TPA alone. Inhibition of protein synthesis with cycloheximide blocked induction of CYP21 as well as type II 3 beta-hydroxysteroid dehydrogenase (3 beta HSDII) mRNA expression in response to AII, forskolin, and dibutyryl cAMP, but had no effect on 17 alpha-hydroxylase cytochrome P450 (CYP17) or cholesterol side-chain cleavage cytochrome P450 (CYP11A) mRNA. Together, these findings were remarkably similar to those of our previous studies regarding mechanisms regulating 3 beta HSDII expression and underline the existence of a subset of steroidogenic enzymes regulated positively (CYP21 and 3 beta HSDII) as opposed to negatively (CYP17 and CYP11A) by the protein kinase C signaling pathway. The additional finding of a small induction of CYP21 expression in response to increased Ca2+, as previously reported for CYP17, but not 3 beta HSDII, expression, also demonstrates that the mechanisms of control of CYP21 and 3 beta HSDII are not identical. This latter finding may also relate to how CYP21 as well as CYP17 expression continues in the zona reticularis after adrenarche, whereas 3 beta HSD expression declines.

Low temperature signal transduction during cold acclimation: protein phosphatase 2A as an early target for cold‐inactivation

SummaryThe authors have previously shown that cold acclimation and cold acclimation‐specific (cas) gene expression in alfalfa require cold‐triggered calcium influx and phosphorylation of specific pre‐existing proteins. In this study, the authors used the expression of cas15 gene as an end‐point marker to examine the role of protein phosphorylation in low temperature signal transduction in alfalfa cells. Whereas the protein kinase inhibitor staurosporine prevented the cold induction of cas15, the protein phosphatase inhibitor okadaic acid induced the cas15 at 25°C. Upon exposure of cells to cold, total cellular protein phosphatase activity rapidly declined by 30% but all of this decline could be attributed to an almost complete inhibition of protein phosphatase 2A (PP2A). This cold‐inactivation of PP2A was found to be mediated by calcium influx and could be reproduced at 25°C by treating the cells with the calcium ionophore A23187 or with the calcium channel agonist Bay K8644. On exposure of cells to cold, the transcript and protein levels of the PP2A catalytic subunit (PP2Ac) did not decline, but the binding of anti‐PP2Ac antibody to native PP2Ac increased, indicating a demasking of the PP2Ac epitope. The possible mechanisms by which cold might inhibit PP2A are discussed.

Dynamic changes in prolactin promoter activation in individual living lactotrophic cells.

The firefly luciferase gene has become widely used as a convenient reporter for studies of gene promoter regulation. Very recently, the development of ultralow-light imaging cameras has enabled the quantitative digital imaging of light signals resulting from luciferase activation in the presence of luciferin substrate. We have applied this technology to the study of PRL promoter activation in individual pituitary tumor cells to study the temporal and spatial characteristics of the expression of a well-characterized pituitary hormone gene. Rat pituitary GH3 cells were transfected by lipofection with a luciferase reporter gene linked to 5000 bp from the human PRL gene 5'-flanking region. A series of stably transfected cell clones were generated, and one of these was chosen for detailed study on the basis of appropriate regulation of high-level luciferase expression by a series of known stimuli including TRH, forskolin, the calcium channel agonist Bay K8644, and basic fibroblast growth factor (bFGF). These cells were subjected to direct imaging of luciferase activity using a Hamamatsu photon-counting camera linked to a Zeiss Axiovert microscope with an Argus-50 image processor. Cells were exposed to 1 mM luciferin, and images were integrated over 30-min periods for up to 72 h. The total photon count over a given field settled to steady levels within 10 h and then remained constant for over 55 h. Addition of forskolin, TRH, or bFGF increased the total photon count of fields of 20-100 cells by 2- to 4-fold consistent with previous data from transient expression assays using the human PRL promoter. Individual cells, on the other hand, showed marked marked temporal and spatial heterogeneity and variability of luciferase expression when studied at 3-h intervals. Unstimulated cells showed variable luciferase expression with up to 40-fold excursions in photon counts per single cell area within 12-h periods. Stimulation of cells with either TRH, forskolin, or bFGF resulted in smooth increases in photon output over fields of 20-100 cells, but again individual cell responses differed widely, with some cells showing slow progressive rises in photon output, others showing phasic or transient responses, and yet others showing no response. In conclusion, we found a surprising degree of heterogeneity and temporal variability in the level of gene expression in individual living pituitary tumor cells over long periods of time, with markedly divergent responses to hormonal or intracellular stimulation. The use of stably transfected clonal cell lines with extended periods of reporter gene imaging offers a valuable insight into control of gene expression in living cells in real time.

Dynamic Changes in Prolactin Promoter Activation in Individual Living Lactotrophic Cells

The firefly luciferase gene has become widely used as a convenient reporter for studies of gene promoter regulation. Very recently, the development of ultralow-light imaging cameras has enabled the quantitative digital imaging of light signals resulting from luciferase activation in the presence of luciferin substrate. We have applied this technology to the study of PRL promoter activation in individual pituitary tumor cells to study the temporal and spatial characteristics of the expression of a well-characterized pituitary hormone gene. Rat pituitary GH3 cells were transfected by lipofection with a luciferase reporter gene linked to 5000 bp from the human PRL gene 5′-flanking region. A series of stably transfected cell clones were generated, and one of these was chosen for detailed study on the basis of appropriate regulation of high-level luciferase expression by a series of known stimuli including TRH, forskolin, the calcium channel agonist Bay K8644, and basic fibroblast growth factor (bFGF). These cells were subjected to direct imaging of luciferase activity using a Hamamatsu photon-counting camera linked to a Zeiss Axiovert microscope with an Argus-50 image processor. Cells were exposed to 1 mm luciferin, and images were integrated over 30-min periods for up to 72 h. The total photon count over a given field settled to steady levels within 10 h and then remained constant for over 55 h. Addition of forskolin, TRH, or bFGF increased the total photon count of fields of 20–100 cells by 2- to 4-fold consistent with previous data from transient expression assays using the human PRL promoter. Individual cells, on the other hand, showed marked marked temporal and spatial heterogeneity and variability of luciferase expression when studied at 3-h intervals. Unstimulated cells showed variable luciferase expression with up to 40-fold excursions in photon counts per single cell area within 12-h periods. Stimulation of cells with either TRH, forskolin, or bFGF resulted in smooth increases in photon output over fields of 20–100 cells, but again individual cell responses differed widely, with some cells showing slow progressive rises in photon output, others showing phasic or transient responses, and yet others showing no response. In conclusion, we found a surprising degree of heterogeneity and temporal variability in the level of gene expression in individual living pituitary tumor cells over long periods of time, with markedly divergent responses to hormonal or intracellular stimulation. The use of stably transfected clonal cell lines with extended periods of reporter gene imaging offers a valuable insight into control of gene expression in living cells in real time.

Characterisation of the Role of Calcium in AlF4-Induced Long-Term Potentiation in Area CA1 of Rat Hippocampus

We investigated calcium influx in the long lasting potentiation induced in area CA1 of rat hippocampus by brief bath application of the G-protein activator A1F4-(NaF/AlCl3). Brief (10 min) bath application of A1F4 in standard saline (with 2 mM Ca2+) consistently induced a long lasting potentiation which was not observed if A1F4 was bath-applied in nominally calcium free saline. Increasing the potential calcium influx, either by raising extracellular calcium concentration to 3.5 mM or by addition of the voltage operated calcium channel (VOCC) agonist BayK8644, failed to increase the number of slices exhibiting potentiation or the mean level of potentiation. Bath application of AlF4 in the presence of the VOCC antagonist failed to block the potentiation and A1F4- readily induced a long lasting potentiation under voltage clamp conditions, strongly suggesting that the calcium influx required for A1F4-induced potentiation is not through NMDA receptors or VOCC channels. It is suggested that the calcium required may be provided by an ongoing recharging and emptying of IP3 sensitive intracellular Ca2+ stores.