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Results for “Intracellular quantification”

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At least 595 records · Page 33Linked to original sources

PubMed1999

Metabolic approaches for the optimisation of recombinant fermentation processes.

The aim of this work was the establishment of a novel method to determine the metabolic load on host-cell metabolism resulting from recombinant protein production in Escherichia coli. This tool can be used to develop strategies to optimise recombinant fermentation processes through adjustment of recombinant-protein expression to the biosynthetic capacity of the host-cell. The signal molecule of the stringent-response network, guanosine tetraphosphate (ppGpp), and its precursor nucleotides were selected for the estimation of the metabolic load relating to recombinant-protein production. An improved analytical method for the quantification of nucleotides by ion-pair, high-performance liquid chromatography was established. The host-cell response upon overexpression of recombinant protein in fed-batch fermentations was investigated using the production of human superoxide dismutase (rhSOD) as a model system. E. coli strains with different recombinant systems (the T7 and pKK promoter system) exerting different loads on host-cell metabolism were analysed with regard to intracellular nucleotide concentration, rate of product formation and plasmid copy number.

Chromatography, High Pressure Liquid↗

PubMed2004

Assessment of 35mer amino-modified oligonucleotide based microarray with bacterial samples.

Parallel quantification of a large number of messenger RNA transcripts, using microarray technology, promises to provide unsuspected information about many cellular processes. Although experimental protocols on microarray applications are available, only limited methodological information on glass-slide manufacturing and signal interpretation has been published. The aim of this paper is to provide new insights into the practical aspects of the construction and hybridization of oligonucleotide-based microarrays. The intracellular symbiotic bacterium of aphids, Buchnera aphidicola, is used here as a model organism. The first part of the work is devoted to the optimization of procedures for printing slides, labeling of cDNA targets and hybridization. In the second part, based on a statistical analysis of the results, we discuss the influence of the probe attachment chemistry, of the labeling method, of the oligonucleotide position and of the concentration of a spotted oligonucleotide on signal intensity. The problem of signal specificity is also addressed, based on the calculation of the fluorescent ratio for each probe to its corresponding mismatch control probe. Lastly, the selection of internal spiked RNAs appropriate to our bacterial samples and useful for the data normalization step is presented.

Base Pair Mismatch↗

PubMed2000

Equilibrium and kinetic measurements of muscarinic receptor antagonism on living cells using bead injection spectroscopy.

Bead injection spectroscopy (BIS) techniques are introduced for automated measurement of pharmacological antagonism by functional assay. Chinese hamster ovary cells that express the rat type 1 muscarinic receptor are cultured on microbeads and used as a renewable biological target for muscarinic receptor antagonist ligands. A flow injection instrument is used to reproducibly sample and capture the cells in a jet ring chamber. The effect of the antagonist pirenzepine on the carbachol-induced intracellular calcium response of the cells is measured with a fluorescence microscope photometry system. The BIS functional assay is used to quantify both equilibrium and kinetic pharmacological values for pirenzepine. In addition, two muscarinic receptor antagonists (pirenzepine and atropine) are assayed to compare their relative efficacy at diminishing the calcium response. Due to the precision of the automated fluid/bead handling protocols, and reproducibility of the measured calcium response, the quantification of useful pharmacological information from living cells by BIS techniques is demonstrated.

Animals↗

PubMed1992

Expression of CRABP-I and -II in human epidermal cells. Alteration of relative protein amounts is linked to the state of differentiation.

The physiological role of cellular retinoic acid-binding proteins (CRABPs) may be to influence the intracellular level of free retinoic acid in the cell. In the present study two isoforms of CRABP, CRABP-I and CRABP-II were partially characterized in various human Malpighian epithelia and in human cultured keratinocytes expressing various patterns of differentiation. We have developed a new sensitive radiobinding assay using a PAGE/autoradioblotting technique which effectively separates CRABP-I and CRABP-II. This method allows the simultaneous quantification of these proteins. We show that CRABP-I and -II have similar M(r) values (15,000), but differ in their dissociation constant towards retinoic acid (Kd of 16.6 nM and 50 nM respectively), in pI (4.86 and 5.13) and in their relative mobilities (RF) on PAGE under nondenaturating conditions (RF values 0.65 and 0.44). In addition, we show that CRABP-II is the major isoform expressed in human keratinocytes, in vivo as in vitro. Furthermore, we demonstrate that CRABP-II is actually the CRABP previously studied in epidermal cells by a PAGE assay (Siegenthaler & Saurat (1987) Eur. J Biochem. 166, 209-214) and whose levels are dramatically increased by retinoic acid and its analogues in human epidermis. Keratinocytes, in the absence of full terminal differentiation, as well as hyperplasia, such as cultured human differentiating keratinocytes, psoriatic plaques, and non-keratinized oral mucosa, contained high levels of CRABP-II. CRABP-I was not detected in cultured keratinocytes, whereas normal skin (at full terminal differentiation) expressed CRABP-I and CRABP-II at a ratio of approx. 1:1.4. This value was approx. 1:17 in lesional psoriatic skin and 1:8 in oral mucosa. These observations suggest that CRABP-I and -II are regulated differently in human keratinocytes. The sharp increases in CRABP-II levels are associated with an alteration in the differentiation programme, as well as with cell response to retinoic acid overload, whereas CRABP-I might be a marker for terminal differentiation.

Animals↗

PubMed1996

Muscarinic acetylcholine receptor expression in memory circuits: implications for treatment of Alzheimer disease.

Cholinergic transmission at muscarinic acetylcholine receptors (mAChR) has been implicated in higher brain functions such as learning and memory, and loss of synapses may contribute to the symptoms of Alzheimer disease. A heterogeneous family of five genetically distinct mAChR subtypes differentially modulate a variety of intracellular signaling systems as well as the processing of key molecules involved in the pathology of the disease. Although many muscarinic effects have been identified in memory circuits, including a diversity of pre- and post-synaptic actions in hippocampus, the identities of the molecular subtypes responsible for any given function remain elusive. All five mAChR genes are expressed in hippocampus, and subtype-specific antibodies have enabled identification, quantification, and localization of the encoded proteins. The m1, m2, and m4 mAChR proteins are most abundant in forebrain regions and they have distinct cellular and subcellular localizations suggestive of various pre- and postsynaptic functions in cholinergic circuits. The subtypes are also differentially altered in postmortem brain samples from Alzheimer disease cases. Further understanding of the molecular pharmacology of failing synapses in Alzheimer disease, together with the development of new subtype-selective drugs, may provide more specific and effective treatments for the disease.

Alzheimer Disease↗

PubMed1989

Ca2+-dependent and -independent secretagogue action on gastric mucus secretion in rabbit mucosal explants.

The secretion of high-molecular weight glycoprotein was studied in rabbit antral and fundic explants in response to acetylcholine (ACh), the calcium ionophore A23187, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), forskolin, histamine, pentagastrin, and prostaglandin E2 (PGE2). Glycoprotein secretion was measured in pulse-chase experiments by incorporation of [14C]N-acetyl-D-glucosamine into explants and quantification of the secreted high-molecular weight-labeled glycoprotein. All tested agents except histamine and pentagastrin stimulated glycoprotein secretion in a concentration-dependent manner. The response to A23187 was abolished by removal of extracellular Ca2+ and greatly reduced by the calmodulin antagonist N-(6-amino-hexyl)-l-naphthalenesulfonamide (W7). The response to ACh was abolished by equimolar concentrations of atropine and by depletion of intracellular Ca2+ stores, an effect which was reversible on addition of Ca2+, and reduced by W7. The response to forskolin and PGE2 was not significantly affected by either extracellular or intracellular Ca2+ depletion. The combination of ACh with forskolin and A23187 with TPA had a synergistic effect on secretion. The absolute dependence of cholinergic stimulation on the presence of intracellular Ca2+ and the Ca2+-independent stimulatory effect of forskolin and PGE2 suggest that gastric mucus secretion can be elicited by at least two distinct pathways.

Acetylcholine↗

PubMed2006

CD8+HLA-DR+ T lymphocytes are increased in common variable immunodeficiency patients with impaired memory B-cell differentiation.

The recently proposed Piqueras classification of common variable immunodeficiency (CVID) patients is based on flow cytometric quantification of IgD class-switched and CD27 membrane-expressing mature blood B cells. But, many patients also have circulating T cells with immunophenotypic abnormalities, often associated with clinical complications, such as splenomegaly, autoimmune disease, lymphoid proliferation and/or granulomatosis. In 50 unselected CIVD patients, classified according to CD27 and IgD B-cell expression, we analyzed T-lymphocyte subsets according to their expression of HLA-DR and intracellular perforin and/or granzyme B in CD8+ T lymphocytes, CCR7 and CD45RA. CD3+DR+ T-lymphocyte percentages, predominantly CD8+DR+, were significantly higher in patients with clinical complications. MB0 classified patients, characterized by fewer CD27+ B cells, had higher percentages of CD8+DR+ T lymphocytes expressing perforin and/or granzyme with a differentiated effector (CCR7- and CD45RA+) phenotype. In contrast, MB2 patients (with normal CD27+ and IgD- B cells) were free of clinical complications and showed no signs of T-cell activation. MB1 patients (normal CD27+ numbers but fewer IgD- B cells) were either clinically normal or had complications. Combining the set of markers described herein might better define homogeneous groups of patients for etiological studies and clearly segregate patients with clinical complications.

Adolescent↗

PubMed2000

Cholesterol oxidation switches the internalization pathway of endothelin receptor type A from caveolae to clathrin-coated pits in Chinese hamster ovary cells.

We investigated the mechanism of endothelin receptor type A (ETA) internalization in Chinese hamster ovary cells using two assays; flow cytometric quantification of cell surface myc-ETA and in situ localization of Cy5-labeled ET-1. In both assays, agonist-dependent internalization of myc-ETA was inhibited by nystatin and filipin, both of which disrupt internalization via caveolae, whereas it was barely affected by chlorpromazine and hypertonic sucrose, both of which disrupt internalization via clathrin-coated pits. In addition to myc-ETA, ET-1 caused intracellular translocation of caveolin-1 and this translocation was also blocked by nystatin but not by chlorpromazine. These results strongly argue that ETA is internalized via caveolae but not clathrin-coated pits. Treatment of the cells with cholesterol oxidase reduced cellular cholesterol and caused intracellular translocation of caveolin-1 but did not affect cell surface localization of myc-ETA. In cholesterol oxidase-treated cells, however, both chlorpromazine and hypertonic sucrose effectively blocked ET-1-induced myc-ETA internalization and nystatin was less effective than in untreated cells. Accordingly, expression of a dominant negative form of beta-arrestin blocked myc-ETA internalization in cholesterol oxidase-treated cells but not in untreated cells. These results suggest that, in Chinese hamster ovary cells, 1) agonist-occupied ETA can be internalized either via caveolae or clathrin-coated pits; 2) of the two, the former is the default pathway; and 3) the oxidative state of cell surface cholesterol is one of the factors involved in the pathway selection.

Animals↗

PubMed2004

Cryopreservation of alginate-encapsulated recombinant cells for antiangiogenic therapy.

The potential benefit of continuous local administration of antiangiogenic proteins to CNS tumors in vivo has recently been demonstrated using endostatin-producing recombinant cells encapsulated in alginate beads. Due to the treatment potential of transplanted alginate-encapsulated cells producing therapeutic proteins, we describe a successful method of cryopreservation (CP) of such beads, in which cellular viability, alginate structure, and protein secretion were maintained. Alginate beads containing human embryonic kidney cells (HEK 293 cells) stably transfected with the gene encoding for endostatin were cryopreserved in dimethyl sulfoxide (DMSO) using a slow freezing procedure. Briefly, the DMSO concentration was gradually increased prior to the freezing procedure. The cryotubes were further supercooled to -7.5 degrees C and nucleated. Thereafter, the samples were cooled at a rate of 0.25 degrees C/min and stored in liquid nitrogen. The viability of the encapsulated cells was assessed using confocal microscopy quantification (CLSM) technique and a MTS assay. The cell cycle distribution inside the beads was assessed by DNA flow cytometry and endostatin production was determined by an endostatin-specific ELISA assay, both prior to and after CP. CLSM measurements showed sustained esterase activity in the beads after thawing, with only a slight transient decrease 24 h after CP. The MTS assay verified these findings by displaying similar variations of intracellular dehydrogenase activity. Flow cytometric analyses revealed no cryorelated disturbances in cellular ploidy. Furthermore, ELISA measurements showed a well-preserved endostatin production after CP. In conclusion, this work describes the successful CP of alginate-encapsulated recombinant cells secreting a therapeutic protein. Together with previous published reports, these results further substantiate the feasibility and potential of cell encapsulation therapy in the treatment of malignant tumors.

Alginates↗

PubMed1997

LDL-mediated interaction of Lp[a] with HepG2 cells: a novel fluorescence microscopy approach.

We studied the topography of Lp[a]-LDL-cell interactions by means of fluorescence microscopy, using fluorescence-labeled lipoproteins. In contrast to known methods which are based on noncovalent labeling of lipoproteins by positively charged amphiphiles, the protein moiety of LDL and Lp[a] was covalently labeled with either BODIP-succinimide-ester (green) or rhodamine X iodoacetamide (red). The interaction of the fluorescent lipoproteins with cultured HepG2 cells was studied using a confocal laser scanning fluorescence microscope. LDL and Lp[a], each labeled with a different dye, could be examined separately within a mixture of both lipoproteins during their interaction with HepG2 cells. At 4 degrees C, the majority of both fluorescent particles co-localized and only a few separate LDL- or Lp[a]-binding domains could be observed. Quantification of the amount of fluorescent lipoprotein associated with the cell surface at 4 degrees C showed that binding of Lp[a] was increased in the presence of LDL under these conditions, probably via formation of an Lp[a]-LDL complex. At 37 degrees C, LDL and Lp[a] were taken up by the cells within 10 min. Again the majority of LDL and Lp[a] particles co-localized intracellularly. Only minor amounts of LDL and Lp[a] could be observed separately. As the entire fluorescence of labeled Lp[a] co-localized with excess of LDL in cells, and taking into account the high tendency of LDL-Lp[a] association in solution and on cell surfaces, it is concluded that a significant portion of the internalized Lp[a] is taken up into the cells by the LDL receptor via LDL by a hitchhiking-like process.

Animals↗

PubMed2002

Observation and quantification of ultraviolet-induced reactive oxygen species in ex vivo human skin.

Two-photon fluorescence imaging is used to detect UV-induced reactive oxygen species (ROS) in ex vivo human skin in this study. ROS (potentially H202, singlet oxygen or peroxynitrite [or all]) are detected after reaction with nonfluorescent dihydrorhodamine-123 (DHR) and the consequent formation of fluorescent rhodamine-123 (R123). The cellular regions at each epidermal stratum that generate ROS are identified. R-123 fluorescence is detected predominately in the lipid matrix of the stratum corneum. In contrast, the strongest R123 fluorescence signal is detected in the intracellular cytoplasm of the viable epidermal keratinocytes. A simple bimolecular one-step kinetic model is used for estimating the upper bound of the number of ROS that are generated in the skin and that react with DHR. After ultraviolet-B radiation (280-320 nm) (UVB) equivalent to 2 h of noonday summer North American solar exposure (1600 J m(-2) UVB), the model finds that 14.70 x 10(-3) mol of ROS that react with DHR are generated in the stratum corneum of an average adult-size face (258 cm(-2)). Approximately 10(-4) mol are potentially generated in the lower epidermal strata. The data show that two-photon fluorescence imaging can be used to detect ROS in UV-irradiated skin.

Fluorescent Dyes↗

PubMed2002

Brain-derived neurotrophic factor prevents axotomized retinal ganglion cell death through MAPK and PI3K signaling pathways.

PURPOSE: Brain-derived neurotrophic factor (BDNF) has a potential neuroprotective effect on axotomized retinal ganglion cells (RGCs); however, the mechanism, in regard to intracellular signaling, of BDNF-induced neuroprotection of RGCs is largely unknown. Intracellular signaling was investigated, by using axotomized RGCs and the relative contribution of the two major downstream signaling routes of TrkB determined--that is, mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3 kinase (PI3K)-Akt routes, mediated by BDNF. METHODS: Neuroprotective effects of BDNF were determined by quantifying the surviving RGCs after axotomy, by retrograde labeling. The MAPK and Akt levels were determined by Western blot analysis and activity assays. Quantification of the relative contribution of the two signaling pathways was performed by use of specific inhibitors for MAPK and PI3K (i.e., U0126 and LY294002, respectively). RESULTS: Intravitreous administration of BDNF had the most profound neuroprotective effects on axotomized RGCs among the neurotrophins. Burst phosphorylation of MAPK and Akt was induced by BDNF within 1 hour and was sustained over 2 weeks in the whole retina. Immunohistochemistry revealed that phosphorylated MAPK was detected in the RGCs and retinal Müller cells, and Akt was in the RGCs. BDNF-induced phosphorylation of MAPK and Akt was suppressed by their specific inhibitors. Moreover, administration of U0126 and LY294002 decreased significantly, but only partially, the neuroprotective effect of BDNF on the axotomized RGCs. CONCLUSIONS: BDNF-mediated signaling involves activation of both MAPK and Akt on the axotomized adult rat retina, and the collaboration of both MAPK and PI3K-Akt pathways seems to be necessary in neuroprotective signaling in axotomized RGCs.

Animals↗

PubMed1993

Opsonization and phagocytosis of bacteria during various middle ear infections.

Samples of middle ear effusions (MEE) obtained from 50 children suffering from acute purulent otitis media (AOM; 23 ears), otitis media with effusion (OME; 15 ears) or chronic suppurative otitis media (COM; 23 ears) were subjected to cytological (cellular differentiation, degree of phagocytosis) and bacteriological (species quantification and identification, degree of opsonization) analyses. Methods used were direct microscopy of acridine orange stained material, immunofluorescence assay using fluorescein-labelled antibodies to immunoglobulin G (IgG) and complement cleavage product C3b, and standard bacterial culturing on agar plates. The most intense opsonization and phagocytosis was evident in COM effusions culture-positive for Staphylococcus aureus. AOM and OME effusions showed rather poor opsonization and roughly 1% of the phagocytes harboured intracellular bacteria. COM effusions, culture-positive for Pseudomonas aeruginosa, showed no bacterial opsonization or phagocytosis whatsoever. Opsonization and phagocytosis of bacteria in the middle ear cleft is highly species-specific.

Adolescent↗

PubMed1990

Minocycline-treatment of diabetic rats normalizes skin collagen production and mass: possible causative mechanisms.

Daily minocycline-treatment of streptozotocin-induced diabetic rats not only prevented a diabetes-caused atrophy of skin collagen mass (10-mos old rats), but also normalized skin collagen mass to match that of growing (ca. 1%/d) non-diabetic controls (4- and 5-mos old rats). The causative mechanism by which minocycline-treatment normalizes skin collagen mass must, in part, be related to a general anabolic effect on growth (body weight) because the effect on skin collagen mass correlates strongly to that on body weight. Consequently, a minocycline-stimulated increase of a systemic factor (such as insulin-like growth factor) is not unlikely. The anabolic effect of minocycline-treatment of diabetic rats is also expressed as a normalized cellular ribosome mass (an index of total protein synthetic capacity) and a normalized absolute rate of collagen production. (Calculation of an absolute rate was justified by an apparent maximum saturation of the prolyl-tRNA pool(s) of skin, maximum saturation obtained by the pool-flooding approach). The normalized skin ribosome amount does not, however, explain a selective effect of minocycline-treatment on collagen production as opposed to that for non-collagen protein, this selective effect measured as relative collagen production. To explain such selectivity, the inhibition of diabetes-induced excess skin collagenase activity seems unlikely. (This inference is based on results from a preliminary study indicating that recently [less than 2 h] synthesized collagen is not degraded by the excess collagenase in skin of diabetic rats). Thus, the principal collagen fraction acted on by pathologically excess collagenase might be collagen at a later stage (greater than 2 h after synthesis) in its life cycle. (Another possibility for the selective effect of minocycline on collagen production, as yet untested, is reduced intracellular procollagen degradation.) Overall, this is the first study aimed at discerning the mechanism(s) by which minocycline-treatment enhances the rate of collagen production in tissues of a diabetic rat. For future studies, the extent to which the positive effect on growth, ribosome mass, and rate of collagen production contributes to the change of collagen mass must, along with the known minocycline-inhibition of collagenase activity, be quantified. Such quantification is a prerequisite for evaluating the chemotherapeutic efficacy of minocycline-treatment on collagen-degradative diseases.

Animals↗

PubMed2000

Body composition assessed by neutron activation analysis in dialysis patients.

Malnutrition is prevalent in end-stage renal disease (ESRD) patients treated with hemodialysis (HD) and peritoneal dialysis (PD). In addition, there is increased incidence of morbidity in this group. Evaluation of nutritional status is important. Application of body composition in the ESRD population to evaluate body compartments and to assess nutritional health has become more common in clinical practice. Neutron activation analysis (NAA) may provide data on metabolically active tissue by quantification of total body potassium (TBK) for body cell mass and assessment of protein by total body nitrogen (TBN). This method may be able to detect changes in body composition before clinical signs of malnutrition are apparent. Ten HD (5 male and 5 female) and 10 PD patients (7 male and 3 female) were evaluated by NAA, TBK, and isotope dilution. Female PD patients had an increased total body water (TBW) and increased intracellular water compared to HD females. Albumin was lower in PD women. There was no significant difference between PD men and laboratory controls in TBW, extracellular water, and TBN. The clinical application of body composition methods for evaluation of dialysis patients by serial assessment and for development of a bedside tool needs further study.

Adult↗

PubMed1995

Evaluating the role of niacin in human carcinogenesis.

Our understanding of the role of ADP-ribose polymer metabolism in limiting carcinogenic events and the dependence of this metabolism on cellular NAD levels predicts that niacin deficiency leading to reduced NAD levels may enhance carcinogenesis. This prediction has led us to initiate studies to evaluate the potential of niacin as a preventive factor in human cancer. The first approach involves development of a method to assess biochemically niacin status in humans using intracellular NAD derived from whole blood, primarily erythrocytes, as the relevant marker of niacin status. We have shown that erythrocyte NAD content varies by as much as 12-fold within a population and can be modulated readily by supplementation. A second approach to testing this hypothesis involves understanding the relationship of dietary niacin, circulating levels of NAD precursors (nicotinamide and nicotinic acid) and NAD in target tissues for human cancer. Current analytical methods for quantification of plasma levels of nicotinic acid and nicotinamide following intake in the dietary range are not sufficient. Thus, we have developed a GC-MS method for the rapid, sensitive, and selective determination of both nicotinamide and nicotinic acid in plasma. These methods will now allow assessment of niacin metabolism in humans that could lead to a new understanding of niacin in prevention of cancer.

Anticarcinogenic Agents↗

PubMed2006

Ferri-bacillibactin uptake and hydrolysis in Bacillus subtilis.

Upon iron limitation, Bacillus subtilis secretes the catecholic trilactone (2,3-dihydroxybenzoate-glycine-threonine)3 siderophore bacillibactin (BB) for ferric iron scavenging. Here, we show that ferri-BB uptake is mediated by the FeuABC transporter and that YuiI, a novel trilactone hydrolase, catalyses ferri-BB hydrolysis leading to cytosolic iron release. Among several Fur-regulated ABC transport mutants, only DeltafeuABC exhibited impaired growth during iron starvation. Quantification of intra- and extracellular (ferri)-BB in iron-depleted DeltafeuABC cultures revealed a fourfold increase of the extracellular siderophore concentration, confirming a blocked ferri-BB uptake in the absence of FeuABC. Ferri-BB was found to bind selectively to the periplasmic binding protein FeuA (Kd = 57 +/- 1 nM), proving high-affinity transport of the iron-charged siderophore. During iron starvation, a DeltayuiI mutant displayed impaired growth and strong intracellular (30-fold) and extracellular (6.5-fold) (ferri)-BB accumulation. Kinetic studies in vitro revealed that YuiI hydrolyses both BB and ferri-BB. While BB hydrolysis led to strong accumulation of the tri- and dimeric reaction intermediates, ferri-BB hydrolysis yielded exclusively the monomeric reaction product and occurred with a 25-fold higher catalytic efficiency than BB single hydrolysis. Thus, ferri-BB was the preferred substrate of the YuiI esterase whose gene locus was designated besA.

ATP-Binding Cassette Transporters↗

PubMed1993

Co-regulation of cAMP-activated Na+ current by Ca2+ in neurones of the mollusc Pleurobranchaea.

1. The cAMP-gated Na+ current (INa, cAMP) was studied in axotomized neurons of the pedal ganglion of the sea slug Pleurobranchaea. INa, cAMP responses were elicited by iontophoretic injection of cAMP and recorded in voltage clamp. 2. The current-voltage relation for INa, cAMP was flat between -90 and -50 mV, but declined steeply with depolarization from -50 to -30 mV. Depolarizing pulses also suppressed the INa, cAMP response, which recovered slowly over tens of seconds. 3. The inactivating effects of depolarization on the current were abolished both by blockade of Ca2+ current and intracellular injection of Ca2+ chelator. Thus, Ca2+ influx through voltage-dependent Ca2+ channels probably mediates inactivation of INa, cAMP within its normal physiological range of action. 4. Increasing intracellular cAMP levels antagonized the effects of Ca2+ influx on INa, cAMP. The mutual antagonism of the ions suggests that cAMP and Ca2+ act competitively in regulation of the INa, cAMP channel. 5. Measures of fractional inactivation of INa, cAMP provided evidence for the existence of an appreciable basal level of current, and hence cAMP, in the unstimulated neuron. Since INa, cAMP is a direct function of cAMP activity, measures of fractional inactivation permit quantification of cAMP levels in the living neuron. 6. Calcium inactivation of INa, cAMP completes a negative feedback loop that can contribute to endogenous burst activity. Over the burst cycle, depolarization and action potential activity driven by INa, cAMP would lead to Ca2+ influx, consequent inactivation of the inward current, and hyperpolarization. This mechanism of endogenous bursting resembles other in which the burst cycle has been found to be regulated by kinetics of Ca2+ influx and removal. However, INa, cAMP may vary in its Ca2+ sensitivity in different neurons and these variations may affect the functional expression of endogenous oscillatory activity.

Animals↗