kn-62 and calmidazolium

The P2X7 receptor is a trimeric ligand-gated ion channel activated by ATP. It is implicated in the cellular response to trauma/disease and considered to have significant therapeutic potential. Using chimeras and point mutants we have mapped the binding site of the P2X7R-selective antagonist AZ11645373 to the known allosteric binding pocket at the interface between two subunits, in proximity to, but separated from the ATP binding site. Our structural model of AZ11645373 binding is consistent with effects of mutations on antagonist sensitivity, and the proposed binding mode explains variation in antagonist sensitivity between the human and rat P2X7 receptors. We have also determined the site of action for the P2X7R-selective antagonists ZINC58368839, brilliant blue G, KN-62, and calmidazolium. The effect of intersubunit allosteric pocket "signature mutants" F88A, T90V, D92A, F103A, and V312A on antagonist sensitivity suggests that ZINC58368839 comprises a binding mode similar to AZ11645373 and other previously characterized antagonists. For the larger antagonists, brilliant blue G, KN-62, and calmidazolium, our data imply an overlapping but distinct binding mode involving the central upper vestibule of the receptor in addition to the intersubunit allosteric pocket. Our work explains the site of action for a series of P2X7R antagonists and establishes "signature mutants" for P2X7R binding-mode characterization.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenosine Triphosphate; Allosteric Site; Amides; Binding Sites; Humans; Imidazoles; Indoles; Models, Molecular; Molecular Docking Simulation; Point Mutation; Purinergic P2X Receptor Antagonists; Receptors, Purinergic P2X7; Rosaniline Dyes; Thiazoles

Intracellular calcium ([Ca(2+)](i)) is the most relevant modulator of parathyroid hormone (PTH) secretion. Uniquely, an increase in [Ca(2+)](i) results in an inhibition of PTH secretion, and it probably exerts its function via calcium-binding protein pathways. The ubiquitous calcium-binding proteins, calmodulin and calmodulin-dependent protein kinase II (CaMKII), have well-established roles in regulated exocytosis in neurons and neuroendocrine cells. However, their roles in parathyroid cells and PTH secretion are still unclear. Using reverse transcription-PCR and western blot analysis, we have demonstrated the expression of calmodulin and CaMKII in human normal parathyroid and parathyroid chief cell adenomas. Blocking of calmodulin and CaMKII activity by the specific antagonists calmidazolium and KN-62 respectively caused a rise in PTH secretion from parathyroid adenoma cells in spite of increased [Ca(2+)](i). The inhibitory effect of Ca(2+) calmodulin on PTH secretion may be due to the absence of synaptotagmin 1 protein in parathyroid adenomas, as demonstrated by western blot analysis. An increased extracellular calcium level acutely lowered the amount of active phosphorylated CaMKII (pCaMKII) in adenoma cells in vitro, indicating the physiological importance of this pathway. Moreover, a negative correlation between the levels of pCaMKII in parathyroid adenomas and serum calcium was found in 20 patients with primary hyperparathyroidism. Taken together, these results show that calmodulin negatively contributes to the regulation of PTH secretion in parathyroid adenoma, at least partially via a CaMKII pathway.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenoma; Blotting, Western; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calmodulin; Enzyme Inhibitors; Humans; Imidazoles; Parathyroid Glands; Parathyroid Hormone; Parathyroid Neoplasms; Phosphorylation; Reverse Transcriptase Polymerase Chain Reaction; Synaptotagmin I

Nifedipine, a blocker of L-type Ca(2+)-channels, increased quantal content of endplate potentials in newly formed nerve-muscle synapses, while R 24571 (calmodulin inhibitor) and KN 62 (inhibitor of calmodulin-dependent kinase II) did not change it. KN 62 suppressed the increase in quantal content of endplate potentials evoked by nifedipine. Similar to nifedipine, chelerythrine and bisindolylmaleimide I (blockers of protein kinase C) increased quantal content of endplate potentials. In the presence of chelerythrine, nifedipine lost its ability to facilitate secretion of neurotransmitter. 4-Aminopyridine, a blocker of voltage-gated potassium channels, increased quantal content of endplate potentials. In the presence of chelerythrine, 4-aminopyridine induced no additional increase in the quantal content of endplate potentials. In its turn, chelerythrine induced no extra facilitation of secretion in the presence of 4-aminopyridine. It is hypothesized that Ca(2+)-dependent inhibition of secretion results from suppression of calmodulin-dependent kinase II and activation of protein kinase C, which potentiates the work of voltage-gated K(+)-channels.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; 4-Aminopyridine; Acetylcholine; Animals; Benzophenanthridines; Calcium Channels, L-Type; Calcium-Calmodulin-Dependent Protein Kinases; Excitatory Postsynaptic Potentials; Imidazoles; Indoles; Maleimides; Mice; Muscle, Skeletal; Nifedipine; Potassium Channels, Voltage-Gated; Protein Kinase C; Synapses

Spiral ganglion neuron (SGN) survival in vitro can be maintained by neurotrophins, permeant cAMP analogs, and depolarization in an additive manner, with depolarization being the most efficacious. Therefore, we used cultured SGNs to determine the mechanism by which depolarization promotes neuronal survival. Our data implicate Ca(2+)/calmodulin-dependent protein kinase (CaMK) activity by showing that it is induced by depolarization, that CaMK activity is necessary for at least part of the survival-promoting effect of depolarization, and that CaMKII or CamKIV activity suffices to support neuronal survival in the absence of other trophic stimuli. First, that depolarization of SGNs activates CaMKs is evidenced by observation of increased CaMKII phosphorylation and of CaMK-dependent CREB phosphorylation. Second, the requirement for CaMKs is shown by a reduction of SGN survival under depolarizing conditions in the presence of CaMK inhibitors. Third, transfection of COOH-terminal-truncated (lacking regulatory domain), constitutively active CaMKII or CaMKIV, but not of normal, full-length CAMKs, promotes SGN survival in the absence of other trophic stimuli, indicating that CaMK activity is sufficient to promote survival. The survival-promoting effect of truncated CaMKs is additive with that of depolarization, neurotrophins, or cyclic AMP. Although both CaMKII and CaMKIV activities converge in promoting survival, their actions on axon growth are markedly different: Transfection of truncated CaMKII, but not of truncated CaMKIV, into SGNs prevents axon outgrowth.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Amino Acid Sequence; Animals; Axons; Calcium Signaling; Calcium-Calmodulin-Dependent Protein Kinase Kinase; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Cell Survival; Cells, Cultured; Cyclic AMP Response Element-Binding Protein; Enzyme Inhibitors; Imidazoles; Membrane Potentials; Neurons; Peptide Fragments; Phosphorylation; Protein Isoforms; Protein Serine-Threonine Kinases; Rats; Sequence Deletion; Signal Transduction; Spiral Ganglion

Because the activity of the sodium pump (Na-K-ATPase) influences the secretion of aldosterone, we determined how extracellular potassium (K(o)) and calcium affect sodium pump activity in rat adrenal glomerulosa cells. Sodium pump activity was measured as ouabain-sensitive (86)Rb uptake in freshly dispersed cells containing 20 mM sodium as measured with sodium-binding benzofluran isophthalate. Increasing K(o) from 4 to 10 mM in the presence of 1.8 mM extracellular calcium (Ca(o)) stimulated sodium pump activity up to 165% and increased intracellular free calcium as measured with fura 2. Increasing K(o) from 4 to 10 mM in the absence of Ca(o) stimulated the sodium pump approximately 30% and did not increase intracellular free calcium concentration ([Ca(2+)](i)). In some experiments, addition of 1.8 mM Ca(o) in the presence of 4 mM K(o) increased [Ca(2+)](i) above the levels observed in the absence of Ca(o) and stimulated the sodium pump up to 100%. Ca-dependent stimulation of the sodium pump by K(o) and Ca(o) was inhibited by isradipine (10 microM), a blocker of L- and T-type calcium channels, by compound 48/80 (40 microg/ml) and calmidizolium (10 microM), which inhibits calmodulin (CaM), and by KN-62 (10 microM), which blocks some forms of Ca/CaM kinase II (CaMKII). Staurosporine (1 microM), which effectively blocks most forms of protein kinase C, had no effect. In the presence of A-23187, a calcium ionophore, the addition of 0.1 mM Ca(o) increased [Ca(2+)](i) to the level observed in the presence of 10 mM K(o) and 1.8 mM Ca(o) and stimulated the sodium pump 100%. Ca-dependent stimulation by A-23187 and 0.1 mM Ca(o) was not reduced by isradipine but was blocked by KN-62. Thus, under the conditions that K(o) stimulates aldosterone secretion, it stimulates the sodium pump by two mechanisms: direct binding to the pump and by increasing calcium influx, which is dependent on Ca(o). The resulting increase in [Ca(2+)](i) may stimulate the sodium pump by activating CaM and/or CaMKII.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Aldosterone; Animals; Calcimycin; Calcium; Calcium Channels; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin; Cells, Cultured; Enzyme Inhibitors; Extracellular Space; Female; Imidazoles; Intracellular Fluid; Ionophores; Isradipine; Potassium; Rats; Rats, Sprague-Dawley; Rubidium Radioisotopes; Sodium-Potassium-Exchanging ATPase; Staurosporine; Zona Glomerulosa

We have examined the interaction of P2 antagonists with the human P2X(7) receptor by studying their effect on 2' and 3'-O-benzoyl-benzoyl-ATP (DbATP) stimulated cellular accumulation of the fluorescent, DNA binding dye, YO-PRO-1 (MW=375Da). In suspensions of HEK293 cells expressing human recombinant P2X(7) receptors, DbATP produced time and concentration-dependent increases in YO-PRO-1 fluorescence. This response presumably reflects YO-PRO-1 entry through P2X(7) receptor channels and binding to nucleic acids. When studies were performed in a NaCl-free, sucrose-containing buffer, full concentration-effect curves to DbATP could be constructed. The P2 antagonists, pyridoxalphosphate-6-azophenyl-2', 4'-disulphonic acid (PPADS) and periodate oxidized ATP (oATP), reduced the potency of DbATP and decreased its maximum response. 1-[N,O-bis(1, 5-isoquinolinesulphonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN62) and its analogue, KN04, reduced the potency of DbATP. Schild slopes for KN62 and KN04 were shallow and exhibited a plateau at concentrations of compound greater than 1 microM, indicating that these compounds were not competitive antagonists. Calmidazolium and a monoclonal antibody to human P2X(7) receptors attenuated DbATP-stimulated YO-PRO-1 accumulation but they were not competitive antagonists and only produced 2 - 3 fold decreases in the potency of DbATP. The effects of PPADS and KN62 were partially reversible whereas those of oATP were not. PPADS protected cells against the irreversible antagonist effects of oATP suggesting a common site of action. In contrast KN62 was not effective suggesting that it may bind at a different site to oATP and PPADS. This study has demonstrated that P2X(7) receptor function can be quantified by measuring DbATP stimulated YO-PRO-1 accumulation and has provided additional information about the interaction of P2 receptor antagonists with the human P2X(7) receptor.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenosine Triphosphate; Antibodies, Blocking; Benzoxazoles; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line; Enzyme Inhibitors; Fluorescent Dyes; Humans; Imidazoles; In Vitro Techniques; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Quinolinium Compounds; Receptors, Purinergic P2X7; Stimulation, Chemical

alpha1-Adrenergic receptors mediate mitogenic responses and increase intracellular free Ca2+ ([Ca2+]i) in vascular smooth muscle cells. Induction of c-fos is a critical early event in cell growth; expression of this gene is regulated by a number of signaling pathways including Ca2+. We wondered whether Ca2+ signaling plays a critical role in the induction of c-fos gene by alpha1-adrenergic receptors. Using stably transfected rat-1 fibroblasts, we confirmed that PE induced c-fos mRNA expression in a time- and dose-dependent manner, and also increased [Ca2+]i (measured with Fura-2 AM). These responses were blocked by the alpha1-adrenergic receptor antagonist doxazosin. Both intracellular Ca2+ chelation (using BAPTA/AM) and extracellular Ca2+ depletion (using EGTA) significantly inhibited PE-induced c-fos expression by alpha1A and alpha1B receptors. Brief (1-min) stimulation of alpha1A and alpha1B receptors with PE did not maximally induce c-fos expression, suggesting that a sustained increase in [Ca2+]i due to Ca2+ influx is required. The calmodulin (CaM) antagonists, R24571, W7, and trifluoperazine, but not the CaM-dependent protein kinases inhibitor KN-62, significantly inhibited c-fos induction by alpha1A and alpha1B receptors. Neither inhibition of protein kinase C nor inhibition of adenylyl cyclase modified c-fos induction by PE. These results suggest that alpha1-adrenergic receptor-induced c-fos expression in rat-1 cells is dependent on a Ca2+/CaM-associated pathway.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adrenergic alpha-1 Receptor Antagonists; Animals; Calcium; Calmodulin; Doxazosin; Egtazic Acid; Enzyme Inhibitors; Fibroblasts; Gene Expression Regulation; Genes, fos; Imidazoles; Kinetics; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-fos; Rats; Receptors, Adrenergic, alpha-1; Recombinant Proteins; Sulfonamides; Transcription, Genetic; Trifluoperazine

Previous studies from our laboratory have demonstrated that stimulation of 5-hydroxytryptamine2A receptors in rat cerebellar granule cells produces an increase in the levels of 5-hydroxytryptamine2A receptor messenger RNA and binding sites, and that this up-regulation requires de novo RNA and protein synthesis. Here we showed that up-regulation of 5-hydroxytryptamine2A receptor binding sites induced by stimulation with the 5-hydroxytryptamine2A/2C receptor agonist, (+/-)-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), is associated with an increase in the 5-hydroxytryptamine2A receptor transcription rate. To examine the possible role of transcriptional activation in DOI-induced 5-hydroxytryptamine2A receptor up-regulation, we studied the effects of DOI on transcription factor binding to activator protein-1 and cyclic AMP-responsive element (CRE) DNA consensus sequences. We found that DOI induces a time-dependent increase in activator protein-1 and CRE transcription factor binding activity, which is blocked by 5-hydroxytryptamine2A receptor antagonists. Similar to 5-hydroxytryptamine2A receptor up-regulation, DOI-induced activator protein-1 binding is suppressed by inhibitors of calmodulin and Ca2+/calmodulin-dependent kinases. The increased activator protein-1 binding is effectively competed by excessive activator protein-1 and CRE sequences as well as endogenous activator protein-1-like sequences present in the rat 5-hydroxytryptamine2A receptor gene. Supershift assays revealed that cAMP-responsive element-binding protein (CREB) and Jun D are common components of both activator protein-1 and CRE binding complexes. DOI also increased the level of phospho-CREB in a time-dependent manner. The binding of phospho-CREB transcription factor to the activator protein-1 site suggests that CREB may modulate the transcription of genes that contain activator protein-1 but lack CRE site in their promoters, through interaction with the activator protein-1 site. The rat 5-hydroxytryptamine2A receptor up-regulation may involve such a mechanism.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Amphetamines; Animals; Animals, Newborn; Calcium-Calmodulin-Dependent Protein Kinases; Cells, Cultured; Cerebellum; Cyclic AMP Response Element-Binding Protein; Enzyme Inhibitors; Gene Expression Regulation; Imidazoles; Kinetics; Neurons; Proto-Oncogene Proteins c-jun; Rats; Rats, Sprague-Dawley; Receptor, Serotonin, 5-HT2A; Receptors, Serotonin; Serotonin Receptor Agonists; Transcription Factor AP-1; Transcription, Genetic; Up-Regulation

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Amino Acid Transport Systems, Neutral; Animals; Calcium; Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin; Carrier Proteins; Cell Line; Chick Embryo; Enzyme Inhibitors; Glycine Plasma Membrane Transport Proteins; Imidazoles; Kinetics; Sulfonamides; Trifluoperazine

The cholinergic agonist carbachol stimulates the apical H+-K+-ATPase and apical as well as basolateral K+ channels in the rat distal colon. The effect of carbachol was tested in the presence of different inhibitors of the Ca2+ signaling pathway in order to characterize the intracellular mechanisms involved. Both carbachol-stimulated Rb+-efflux as well as carbachol-stimulated mucosal Rb+-uptake were dependent on the presence of serosal Ca2+. The Ca2+-calmodulin antagonist calmidazolium (10(-7) mol l(-1)) inhibited the stimulation of mucosal and serosal Rb+ efflux by carbachol. A similar effect had KN-62 (10(-5) mol l(-1)), an inhibitor of the Ca2+-calmodulin-dependent kinase II, suggesting the regulation of basolateral and apical K+ channels by this kinase. Staurosporine (10(-6) mol l(-1)), which potently inhibits protein kinase C, did not alter the effect of carbachol on Rb+ efflux, although the stimulation of apical Rb+ efflux by carbachol seemed to be less prolonged, indicating that protein kinase C is not involved in the regulation of K+ permeability. In contrast, mucosal Rb+ uptake, which is determined by the ouabain- and vanadate-sensitive K+ transport via the apical H+-K+-ATPase, was decreased to nearly one third of control values in the presence of calmidazolium. Both calmidazolium and staurosporine, but not KN-62, prevented the stimulatory action of carbachol on the H+-K+-ATPase, suggesting a synergistic control of this ion pump by both Ca2+-calmodulin and protein kinase C.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Calcium; Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin; Carbachol; Cholinergic Agonists; Colon; Cyclooxygenase Inhibitors; Enzyme Inhibitors; Imidazoles; In Vitro Techniques; Indomethacin; Intestinal Mucosa; Ion Transport; Potassium; Protein Kinase C; Rats; Rats, Wistar; Rubidium Radioisotopes; Staurosporine

To investigate the possible mechanisms involved in forskolin-induced c-jun mRNA decrease in rat C6 glioma cells, we examined effects of a PKA inhibitor (H-89), a L-type Ca2+ channel blocker (nimodipine), a calmodulin activation inhibitor (calmidazolium chloride) and a Ca2+/calmodulin-dependent protein kinase II inhibitor (KN-62) on forskolin-induced c-jun mRNA down-regulation. H-89 caused a reversal of forskolin-induced c-jun mRNA decrease. Furthermore, nimodipine, KN-62 and calmidazolium chloride partially blocked forskolin-induced c-jun mRNA down-regulation. Our results suggest that activation of adenylate cyclase appears to be involved in a down-regulation of c-jun mRNA expression through a PKA pathway. In addition, L-type calcium channels, calmodulin and Ca2+/calmodulin-dependent protein kinase II may be partially involved in c-jun mRNA down-regulation induced by forskolin.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenylyl Cyclases; Animals; Calcium Channel Blockers; Calmodulin; Colforsin; Cyclic AMP-Dependent Protein Kinases; Down-Regulation; Enzyme Activation; Enzyme Inhibitors; Glioma; Imidazoles; Isoquinolines; Nimodipine; Proto-Oncogene Proteins c-jun; Rats; RNA, Messenger; Sulfonamides; Tumor Cells, Cultured

The effect of prostaglandin E2 (PGE2) on proenkephalin (proENK) mRNA expression in primary cultured rat astrocytes was studied. The proENK mRNA level was significantly increased about 3.3-fold 4 h after PGE2 (10 microM) treatment and this increase was potentiated by the pre-treatment with cycloheximide (CHX; 15 microM) about 1.7-fold as much as PGE2 alone treated cells. The pretreatment with staurosporine (1 microM) completely inhibited the increase of PGE2-induced proENK mRNA level, although only a partial inhibition of PGE2-induced proENK mRNA level (approximately 1.5-fold) by H89 (10 microM) was observed. The increase of PGE2-induced proENK mRNA level was not affected by the pretreatment with PD98059 (1, 5, and 10 microM), omega-conotoxin GIVA (1 microM), nimodipine (1 microM), calmidazolium (1 microM), or KN-62 (1 microM). In addition to the proENK mRNA level, PGE2 also increased c-Fos (approximately 4.3-fold), Fra-1 ( approximately 3.8 fold), and Fra-2 (approximately 8.2-fold) protein levels at 4 h after drug treatment. However, c-Jun, JunB, and JunD protein levels were not affected by PGE2. Indeed, PGE2 failed to up-regulate c-jun mRNA expression as well as its protein product. Surprisingly, although three Jun proteins were not induced by PGE2, AP-1 and ENKCRE-2 DNA binding activities were increased by PGE2, (approximately 5 and approximately 2.8-fold, respectively) and which were effectively reduced by CHX (approximately 2.5 and 2-fold, respectively). In western blot analyses, PGE2 enhanced the phosphorylation of CREB (approximately 2.6-fold at 1 h), and CHX showed a potentiative effect on PGE2-induced CREB phosphorylation ( approximately 1.7 fold at 1 h) which is similar to the action on proENK mRNA regulation. Our results suggest that PGE2 increases proENK mRNA expression via activating serine/threonine protein kinase such as PKA, but not calcium/calmodulin dependent protein kinase and MAPK. In addition, phosphorylation of CREB rather than the increase of AP-1 may have a possible role at least early stage in PGE2-induced proENK mRNA level and CHX-evoked potentiation.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Animals, Newborn; Astrocytes; Cells, Cultured; Cerebral Cortex; Cyclic AMP Response Element-Binding Protein; Cycloheximide; Dinoprostone; Enkephalins; Enzyme Inhibitors; Flavonoids; Imidazoles; Isoquinolines; Kinetics; Nimodipine; omega-Conotoxin GVIA; Peptides; Phosphorylation; Protein Precursors; Proto-Oncogene Proteins c-jun; Rats; Rats, Sprague-Dawley; RNA, Messenger; Staurosporine; Sulfonamides; Transcription Factor AP-1; Transcription, Genetic

We investigated the involvement of calmodulin and Ca2+/calmodulin-dependent protein kinase II (CaMKII) in the photic entrainment of circadian rhythms using calmodulin inhibitors such as calmidazolium (CMZ) and trifluoperazine (TFP), and a CaMKII inhibitor, KN-62, in rats. Fos expression in the suprachiasmatic nucleus (SCN) of rats induced by photic stimulation (300 lux, 1 h) during the early subjective night of the rats was inhibited by treatment with CMZ (10 mg/kg i.p.) or TFP (20 mg/kg i.p.) 30 min before photic stimulation. With respect to the neuronal firing rate in the rat SCN slice, KN-62 and CMZ application during the early subjective night attenuated the glutamate (10 microM)-induced phase shift. The present results suggest that calmodulin and CaMKII are involved in the photic entrainment mechanism in the rodent SCN.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Analysis of Variance; Animals; Biomarkers; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin; Circadian Rhythm; Drug Evaluation, Preclinical; Enzyme Inhibitors; Imidazoles; In Vitro Techniques; Nerve Tissue Proteins; Proto-Oncogene Proteins c-fos; Rats; Rats, Wistar; Suprachiasmatic Nucleus; Trifluoperazine

We previously reported that beta-endorphin and morphine administered supraspinally produce antinociception by activating different descending pain-inhibitory systems. To determine the role of spinal calcium channels, calmodulin and calcium/calmodulin-dependent protein kinase II in the production of antinociception induced by morphine, [D-Ala2,N-MePhe4,Gly-ol5]-enkephalin (DAMGO) or beta-endorphin administered supraspinally, the effects of nimodipine (an L-type calcium channel blocker), omega-conotoxin GVIA (an N-type voltage-dependent calcium channel blocker), calmidazolium (a calmodulin antagonist) or KN-62 (a calcium/calmodulin-dependent protein kinase II inhibitor) injected intrathecally (i.t.) on the antinociception induced by morphine, DAMGO or beta-endorphin administered intracerebroventricularly (i.c.v.) were examined in the present study. Antinociception was assessed by the mouse tail-flick test. The i.t. injection of nimodipine (from 0.024 to 2.4 pmol), omega-conotoxin GVIA (from 0.0033 to 0.33 pmol), calmidazolium (from 0.0015 to 0.15 pmol) or KN-62 (from 0.0014 to 0.14 pmol) alone did not affect the basal tail-flick latencies. The i.t. pretreatment of mice with nimodipine, omega-conotoxin GVIA, calmidazolium or KN-62 dose dependently attenuated the inhibition of the tail-flick response induced by beta-endorphin administered i.c.v. However, the inhibition of the tail-flick response induced by morphine or DAMGO administered i.c.v. was not changed by i.t. pretreatment with nimodipine, omega-conotoxin GVIA, calmidazolium or KN-62. The results suggest that spinally located L- and N-type calcium channels, calmodulin and calcium/calmodulin-dependent protein kinase II may be involved in the modulation of antinociception induced by beta-endorphin, but not morphine and DAMGO, administered supraspinally.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; beta-Endorphin; Calcium Channel Blockers; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Enzyme Inhibitors; Imidazoles; Injections, Intraventricular; Injections, Spinal; Male; Mice; Mice, Inbred ICR; Morphine; Nimodipine; omega-Conotoxin GVIA; Peptides; Spinal Cord

The present study was designed to determine if spinal calcium channels, calmodulin, and calcium/calmodulin-dependent protein kinase II were involved in the production of antinociception induced by cold water swimming stress (CWSS). The effects of intrathecal (i.t.) injection of nimodipine, omega-conotoxin GVIA, calmidazolium, or (S)-5-isoquinolinesulfonic acid, 4-[2-[(5-isoquinolinyl-sulfonyl)methylamino]-3-oxo-3-(4-phenyl-1-piperaz inyl)-propyl]phenyl ester (KN-62) on CWSS-induced antinociception were studied in ICR mice. The antinociception was assessed by the tail-flick test. CWSS produced inhibition of the tail-flick response. Various doses of nimodipine (10-40 ng), omega-conotoxin GVIA (5-40 ng), calmidazolium (10-40 ng), or KN-62 (5-40 ng) injected i.t. alone did not show any antinociceptive effect in the tail-flick test. I.t. pretreatment with omega-conotoxin GVIA, calmidazolium, or KN-62 dose dependently attenuated the CWSS-induced inhibition of the tail-flick response. However, i.t. pretreatment with nimodipine did not affect the inhibition of the tail-flick response induced by CWSS. Our results suggest that spinal N-type calcium channel, calmodulin and calcium/calmodulin-dependent protein kinase II may be involved in the production of antinociception induced by CWSS. On the other hand, CWSS-induced antinociception appears not to be mediated via the spinal L-type calcium channel.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Analgesia; Animals; Calcium Channel Blockers; Cold Temperature; Enzyme Inhibitors; Imidazoles; Injections, Spinal; Male; Mice; Mice, Inbred ICR; Mollusk Venoms; Nimodipine; omega-Conotoxin GVIA; Pain Measurement; Peptides; Stress, Physiological; Swimming

Calmodulin and calmodulin-dependent mechanisms are probably important in regulating thyroid cell function. However, calmodulin antagonists may directly modify calcium fluxes in cells. In the present investigation the effects of several calmodulin inhibitors and of KN-62, a specific calcium/calmodulin kinase II inhibitor, on the ATP- and thapsigargin-evoked changes in intracellular free calcium ([Ca2+]i) were investigated in Fura-2 loaded thyroid FRTL-5 cells. All of the inhibitors tested attenuated agonist-evoked calcium entry. The inhibitor calmidazolium per se potently released sequestered calcium followed by enhanced calcium entry. Pretreatment of the cells with calmidazolium inhibited both the thapsigargin-and the ATP-evoked calcium entry. Our results show that calmodulin antagonists are potent inhibitors of calcium entry in thyroid cells, possibly by directly inhibiting the calcium entry pathway. This inhibition may explain, in part, the results obtained with calmodulin inhibitors in previous studies.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenosine Triphosphate; Animals; Barium; Biological Transport; Calcium; Calcium-Calmodulin-Dependent Protein Kinases; Calcium-Transporting ATPases; Calmodulin; Cell Line; Depression, Chemical; Fluphenazine; Imidazoles; Isoquinolines; Membrane Potentials; Phenoxybenzamine; Piperazines; Rats; Sulfonamides; Terpenes; Thapsigargin; Thyroid Gland

The possible involvement of extracellular Ca2+ ([Ca2+]o) in mediating the acute gonadotropin (GtH) response to salmon gonadotropin-releasing hormone (sGnRH) and chicken gonadotropin-releasing hormone-II (cGnRH-II) in goldfish was examined using dispersed pituitary cells in perifusion. Perifusion with Ca(2+)-deficient medium reduced the GtH responses to 5-min pulses of either GnRH, indicating the participation of [Ca2+]o in acute GnRH action. Using a 10-min GnRH pulse application protocol, the dependence of the acute GtH responses to the two GnRHs on [Ca2+]o entry through voltage-sensitive Ca2+ channels (VSCC) was examined using the dihydropyridine VSCC blocker nifedipine and the cation Co2+. Treatment with nifedipine consistently reduced the acute GtH response to either sGnRH or cGnRH-II. Similarly, perifusion with CoCl2 reduced the sGnRH-induced GtH release. In contrast to its effects on sGnRH, CoCl2 abolished the cGnRH-II-induced GtH release. These results indicate that [Ca2+]o entry through VSCC participates in the acute GtH response to both native GnRHs; however, the cGnRH-II-stimulated acute release is relatively more dependent on [Ca2+]o and VSCC functions than sGnRH-induced secretion. The involvement of calmodulin (CaM) in mediating GnRH action was also examined. Treatment with a CaM antagonist, calmidazolium, or with a Ca2+/CaM-dependent protein kinase II inhibitor, KN62, reduced GtH responses to sGnRH and cGnRH-II in 2-hr static incubation, but not in perifusion studies with dispersed goldfish pituitary cells using 5- or 10-min GnRH pulses. These results suggest that CaM-dependent mechanisms participate in mediating the long-term, but not the acute, GtH response to GnRH. Compared to sGnRH, cGnRH-II-induced GtH release was more sensitive to inhibition by KN62, indicating a higher degree of dependence of cGnRH-II action on CaM. These results extend our understanding of the differential involvement of [Ca2+]o and CaM in mediating the short-term and long-term actions of the two native GnRH peptides on GtH release in goldfish.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Calcium; Calcium Channel Blockers; Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin; Cells, Cultured; Cobalt; Culture Media; Enzyme Inhibitors; Female; Goldfish; Gonadotropin-Releasing Hormone; Gonadotropins; Imidazoles; Isoquinolines; Male; Nifedipine; Piperazines; Pituitary Gland

We have previously demonstrated that neuropeptide Y (NPY) inhibits depolarization-stimulated catecholamine synthesis in rat pheochromocytoma (PC12) cells differentiated to a sympathetic neuronal phenotype with nerve growth factor (NGF). The present study uses multiple selective Ca2+ channel and protein kinase agonists and antagonists to elucidate the mechanisms by which NPY modulates catecholamine synthesis as determined by in situ measurement of DOPA production in the presence of the decarboxylase inhibitor m-hydroxybenzylhydrazine (NSD-1015). The L-type Ca2+ channel blocker nifedipine inhibited the depolarization-induced stimulation of DOPA production by approximately 90% and attenuated the inhibitory effect of NPY. In contrast, the N-type Ca2+ channel blocker omega-conotoxin GVIA inhibited neither the stimulation of DOPA production nor the effect of NPY. Antagonism of Ca2+/calmodulin-dependent protein kinase (CaM kinase) greatly inhibited the stimulation of DOPA production by depolarization and prevented the inhibitory effect of NPY, whereas alterations in the cyclic AMP-dependent protein kinase pathway modulated DOPA production but did not prevent the effect of NPY. Stimulation of Ca2+/phospholipid-dependent protein kinase (PKC) with phorbol 12-myristate 13-acetate (PMA) did not affect the basal rate of DOPA production in NGF-differentiated PC12 cells but did produce a concentration-dependent inhibition of depolarization-stimulated DOPA production. In addition, NPY did not produce further inhibition of DOPA production in the presence of PMA, and the inhibition by both PMA and NPY was attenuated by the specific PKC inhibitor chelerythrine. These results indicate that NPY inhibits Ca2+ influx through L-type voltage-gated Ca2+ channels, possibly through a PKC-mediated pathway, resulting in attenuation of the activation of CaM kinase and inhibition of depolarization-stimulated catecholamine synthesis.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Adenine; Animals; Calcium Channel Blockers; Calcium Channels; Calcium-Calmodulin-Dependent Protein Kinases; Carcinogens; Catecholamines; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Egtazic Acid; Enzyme Inhibitors; Imidazoles; Ion Channel Gating; Isoquinolines; Neuropeptide Y; Nifedipine; omega-Conotoxin GVIA; PC12 Cells; Peptides; Piperazines; Protein Kinase C; Protein Kinases; Protein Synthesis Inhibitors; Rats; Tetradecanoylphorbol Acetate; Thionucleotides

In cerebellar Purkinje neurons, gamma-aminobutyric acid (GABA)-mediated inhibitory synaptic transmission undergoes a long-lasting "rebound potentiation" after the activation of excitatory climbing fiber inputs. Rebound potentiation is triggered by the climbing-fiber-induced transient elevation of intracellular Ca2+ concentration and is expressed as a long-lasting increase of postsynaptic GABAA receptor sensitivity. Herein we show that inhibitors of the Ca2+/calmodulin-dependent protein kinase II (CaM-KII) signal transduction pathway effectively block the induction of rebound potentiation. These inhibitors have no effect on the once established rebound potentiation, on voltage-gated Ca2+ channel currents, or on the basal inhibitory transmission itself. Furthermore, a protein phosphatase inhibitor and the intracellularly applied CaM-KII markedly enhanced GABA-mediated currents in Purkinje neurons. Our results demonstrate that CaM-KII activation and the following phosphorylation are key steps for rebound potentiation.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Cerebellum; Drug Synergism; Enzyme Activation; Enzyme Inhibitors; gamma-Aminobutyric Acid; Imidazoles; In Vitro Techniques; Kinetics; Marine Toxins; Membrane Potentials; Nerve Fibers; Oxazoles; Purkinje Cells; Rats; Receptors, GABA-A; Signal Transduction; Staurosporine

The regulation of proenkephalin (proENK) mRNA levels by cAMP and protein kinase C (PKC) pathways was studied in cultured rat spinal cord cells in the present study. Spinal cord cells were cultured from 14 day (E 14) embryos of Sprague-Dawley rats. After 7 days in vitro, the spinal cord cells were incubated with either forskolin (5 microM) or phorbol-13-myristate acetate (PMA; 2.5 microM) for 1, 3, 6, 9, 12 or 24 h and total RNA and proteins were isolated for Northern and Western blot analyses. The proENK mRNA level began to increase within an hour, then reached and remained at a peak 3-12 h after stimulation by both forskolin and PMA. The increased proENK mRNA level in forskolin-treated cells was slightly decreased 24 h after the stimulation, whereas the level of proENK mRNA returned to basal levels in PMA-treated cells. A Western blot assay revealed that the intracellular level of proENK protein was not changed by treatment with either forskolin or PMA. Pretreatment of cells with cycloheximide (a protein synthesis inhibitor; 10 microM) did not affect the forskolin- or PMA-induced increase of proENK mRNA. However, pretreatment with nimodipine (an L-type Ca2+ channel blocker; 2 microM), omega-conotoxin (an N-type Ca2+ channel blocker; 1 microM), calmidazolium (a calmodulin antagonist; 1 microM) or KN-62 (a Ca2+/calmodulin-dependent protein kinase II inhibitor; 5 microM) attenuated the forskolin- or PMA-induced increase of proENK mRNA levels. Dexamethasone (1 microM) did not affect the forskolin-induced increase of proENK mRNA levels. Our results suggest that the elevation of proENK mRNA levels in the spinal cord is regulated by both cAMP and PKC pathways. Calcium influx through both L- and N-type calcium channels, calmodulin and Ca2+/calmodulin-dependent protein kinase II appear to be involved in the increase of proENK mRNA levels induced by either forskolin or PMA. Furthermore, ongoing protein synthesis is not required for forskolin- or PMA-induced alterations in proENK mRNA.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Calcium Channel Blockers; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Cells, Cultured; Colforsin; Cyclic AMP; Cycloheximide; Dexamethasone; Enkephalins; Enzyme Inhibitors; Gene Expression Regulation; Imidazoles; Mollusk Venoms; Nimodipine; Protein Kinase C; Protein Precursors; Protein Synthesis Inhibitors; Rats; Rats, Sprague-Dawley; RNA, Messenger; Second Messenger Systems; Signal Transduction; Spinal Cord; Tetradecanoylphorbol Acetate

Calcium/calmodulin-dependent protein kinase-II (CamK-II) is a major neuronal protein which plays a significant role in the cellular process of long-term potentiation (LTP), and vesicular release of neurotransmitters. Here, we show that KN-62, 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4- phenylpiperazine, a specific cell-permeable inhibitor of CamK-II substantially protected neurons from (1) acute NMDA toxicity and (2) hypoxia/hypoglycemia-induced neuronal injury in fetal rat cortical cultures. KN-62 did not directly inhibit glutamate, kainate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), glycine, or [piperidyl-3,4-(N)]-(N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine) (TCP) binding to rat brain membranes. Finally, KN-62 significantly reduced cellular calcium accumulation following either NMDA challenge or hypoxia/hypoglycemia insult. Our results show that CamK-II plays a key role in mediating some of the biochemical events leading to cell death following an acute excitotoxic insult.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Calcium; Calcium-Calmodulin-Dependent Protein Kinases; Carbazoles; Cell Death; Cell Hypoxia; Cell Survival; Cells, Cultured; Cerebral Cortex; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fetus; Genistein; Imidazoles; Indole Alkaloids; Isoflavones; Isoquinolines; Kinetics; N-Methylaspartate; Naphthalenes; Neurons; Piperazines; Polycyclic Compounds; Protein Kinase C; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Spectrin

The effects of nicotine on the transcriptional activity of the proENK gene, proenkephalin A (proENK) mRNA levels, and the secretion of [Met5]-enkephalin (ME) were studied in bovine adrenal medullary chromaffin (BAMC) cells. Nicotine (10 microM) caused an immediate secretion (within 1 hr) of ME followed by a delayed secretion (12-24 hr after treatment) into the medium. Posttreatment with the cholinergic antagonists, hexamethonium (1 mM) and atropine (1 microM), up to 6 hr after the nicotine treatment significantly inhibited the delayed secretion of ME induced by nicotine. However, nicotine-induced long-term secretion of ME was not affected when cholinergic antagonists were added 9 or 12 hr after the nicotine treatment. Long-term (24 hr) stimulation of BAMC cells with nicotine also increased proENK mRNA level. This nicotine-induced response was inhibited by posttreatment with cholinergic antagonists 0.5, 1, 3 and 6 hr after the nicotine treatment. As with the secretion experiments, these cholinergic antagonists did not affect the nicotine-induced responses when they were added at 9 and 12 hr. Posttreatment with nimodipine (1 microM), calmidazolium (1 microM) or KN-62 (5 microM) up to 6 hr after the nicotine treatment significantly inhibited the increases of the long-term secretion of ME and proENK mRNA level induced by nicotine. However, these agents were ineffective in blocking the long-term secretion of ME and proENK mRNA level induced by nicotine when BAMC cells were posttreated after 9 and 12 hr.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adrenal Medulla; Animals; Base Sequence; Calcium-Calmodulin-Dependent Protein Kinases; Cattle; Cholinergic Antagonists; DNA Probes; Enkephalin, Methionine; Enkephalins; Enzyme Inhibitors; Gene Expression Regulation; Imidazoles; Isoquinolines; Molecular Sequence Data; Nicotine; Nicotinic Agonists; Nimodipine; Piperazines; Protein Precursors; RNA, Messenger; Transcription, Genetic

Intercellular adhesion molecule-1 (ICAM-1) is an important cell surface adhesion receptor of the immune system. Its cell surface expression on a wide variety of cells, including cancer cells, is regulated by various proinflammatory cytokines. In the present study, we investigated the role of calcium (Ca2+) and calmodulin (CaM) in the retinoic acid and gamma-interferon (IFN-gamma) signaling in the human neuroblastoma cell line SK-N-SH for up-regulating ICAM-1 expression. A 24-h incubation in the presence of Ca(2+)-mobilizing agents (A23187 and thapsigargin) resulted in the induction of ICAM-1 expression. Both Ca(2+)-mobilizing agents stimulated ICAM-1 expression additively to IFN-gamma but not to retinoic acid, suggesting that IFN-gamma does not use Ca2+ to stimulate ICAM-1, whereas retinoic acid might use it in part. As a second messenger, Ca2+ can be coupled with calmodulin. Using calmodulin inhibitors (W7 and calmidazolium), we found that retinoic acid-stimulated, A23187-stimulated, and thapsigargin-stimulated but not FIN-gamma-stimulated ICAM-1 were inhibited. Calmodulin signaling elicited by retinoic acid was an early event occurring within the first h of retinoic acid treatment, providing evidence that they may both be coupled to regulate gene expression. Using a novel CaM kinase II inhibitor, KN-62, we demonstrated that retinoic acid stimulated ICAM-1 expression in a CaM kinase II-dependent fashion. The mechanisms whereby CaM kinase II mediates retinoic acid activity on ICAM-1 expression remain to be elucidated.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Calcimycin; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin; Cell Adhesion Molecules; Enzyme Activation; Humans; Imidazoles; Intercellular Adhesion Molecule-1; Interferon-gamma; Isoquinolines; Neuroblastoma; Piperazines; Protein Kinase C; Sulfonamides; Terpenes; Thapsigargin; Tretinoin; Tumor Cells, Cultured; Up-Regulation

A variety of experimental evidence suggests that calmodulin and protein kinases, especially protein kinase C, may participate in regulating neurite development in cultured neurons, particularly neurite initiation. However, the results are somewhat contradictory. Further, the roles of calmodulin and protein kinases on many aspects of neurite development, such as branching or elongation of axons vs dendrites, have not been extensively studied. Cultured embryonic rat hippocampal pyramidal neurons develop readily identifiable axons and dendrites. We used this culture system and the new generation of highly specific protein kinase inhibitors to investigate the roles of protein kinases and calmodulin in neurite development. Neurons were cultured for 2 days in the continuous presence of calphostin C (a specific inhibitor of protein kinase C), KT5720 (inhibitor of cyclic AMP-dependent protein kinase), KN62 (inhibitor of Ca(2+)-calmodulin-dependent protein kinase II), or calmidazolium (inhibitor of calmodulin), each at concentrations from approximately 1 to 10 times the concentration reported in the literature to inhibit each kinase by 50%. The effects of phorbol 12-myristate 13-acetate (an activator of protein kinase C) and 4 alpha-phorbol 12,13-didecanoate (an inactive phorbol ester) were also tested. At concentrations that had no effect on neuronal viability, calphostin C reduced neurite initiation and axon branching without significantly affecting the number of dendrites per neuron, dendrite branching, dendrite length, or axon length. Phorbol 12-myristate 13-acetate increased axon branching and the number of dendrites per cell, compared to the inactive 4 alpha-phorbol 12,13-didecanoate. KT5720 inhibited only axon branching. KN62 reduced axon length, the number of dendrites per neuron, and both axon and dendrite branching. At low concentrations, calmidazolium had no effect on any aspect of neurite development, but at high concentrations, calmidazolium inhibited every parameter that was measured (including viability). These results suggest that these three protein kinases selectively modulate different aspects of neurite development. The university of effects caused by calmodulin inhibition make it impossible to determine if there are specific targets of calmodulin action involved in neurite development. Finally, our data indicate that some superficially similar characteristics of neuronal differentiation, such as neurite initiation and branching, may be control

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Axons; Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin; Carbazoles; Cells, Cultured; Dendrites; Female; Hippocampus; Imidazoles; Indoles; Isoquinolines; Naphthalenes; Neurites; Piperazines; Polycyclic Compounds; Pregnancy; Protein Kinase C; Protein Kinase Inhibitors; Pyramidal Tracts; Pyrroles; Rats; Rats, Sprague-Dawley; Tetradecanoylphorbol Acetate

High levels of polyamines have been identified in the lumen of the intestines. Luminal polyamines are involved in normal mucosal growth and may be the primary source of extracellular polyamines for tumors grown in animals undergoing polyamine antimetabolite therapy. The vectorial movement of polyamines across an in vitro model of the gut was studied in epithelial cells grown in culture. IEC-6 cells were plated on either plastic or raised inserts. Cells grown on plastic were employed to define the kinetic constants for putrescine and spermidine uptake. Eadie-Hofstee plot analysis of putrescine uptake was characteristic of a single class of transporter with a Michaelis constant (Km) of 4.85 +/- 0.57 microM and a maximal velocity (Vmax) of 627 +/- 85 pmol x 15 min-1 x 10(6) cells-1. The plot for spermidine uptake was curvilinear and representative of the interaction of spermidine with two sites: Km 1 and 2 are 0.26 +/- 0.13 and 2.1 +/- 0.77 microM; Vmax 1 and 2 are 177 +/- 50 and 429.5 +/- 70 pmol x 15 min-1 x 10(6) cells-1, respectively. Calmodulin antagonism blocked the uptake of putrescine and the low-affinity but not high-affinity spermidine uptake system. Seven-day postconfluent cells grown on plastic inserts were used to study the vectorial movement of polyamines in a polarized epithelium. The apical membrane domain expressed two sites with similar kinetic constants to those observed when cells were grown on plastic. In contrast, however, the basolateral membrane did not transport polyamines. Spermidine uptake through this membrane was only a fraction of that in the apical membrane and was completely nonspecific.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Azepines; Biological Transport; Calmodulin; Cell Line; Duodenum; Epithelium; Humans; Imidazoles; Isoquinolines; Kinetics; Naphthalenes; Piperazines; Protein Kinase Inhibitors; Putrescine; Spermidine; Sulfonamides