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1.
Neuroscience ; 310: 541-8, 2015 Dec 03.
Article in English | MEDLINE | ID: mdl-26415767

ABSTRACT

Phoenixin (PNX) is a 14-amino acid amidated peptide (PNX-14) or an N-terminal extended 20-residue amidated peptide (PNX-20) recently identified in neural and non-neural tissue. Mass spectrometry analysis identified a major peak corresponding to PNX-14, with negligible PNX-20, in mouse spinal cord extracts. Using a previously characterized antiserum that recognized both PNX-14 and PNX-20, PNX-immunoreactivity (irPNX) was detected in a population of dorsal root ganglion (DRG) cells and in cell processes densely distributed to the superficial layers of the dorsal horn; irPNX cell processes were also detected in the skin. The retrograde tracer, Fluorogold, injected subcutaneously (s.c.) to the back of the cervical and thoracic spinal cord of mice, labeled a population of DRG, some of which were also irPNX. PNX-14 (2, 4 and 8 mg/kg) injected s.c.to the nape of the neck provoked dose-dependent repetitive scratching bouts directed to the back of the neck with the hindpaws. The number of scratching bouts varied from 16 to 95 in 30 min, commencing within 5 min post-injection and lasted 10-15 min. Pretreatment of mice at -20 min with nalfurafine (20 µg/kg, s.c.), the kappa opioid receptor agonist, significantly reduced the number of bouts induced by PNX-14 (4 mg/kg) compared with that of saline-pretreated mice. Our results suggest that the peptide, PNX-14, serves as one of the endogenous signal molecules transducing itch sensation in the mouse.


Subject(s)
Ganglia, Spinal/metabolism , Peptides/metabolism , Pruritus/chemically induced , Spinal Cord/metabolism , Amino Acids/chemistry , Animals , Behavior, Animal/drug effects , Cervical Cord/metabolism , Male , Mice , Neck/innervation , Peptides/chemistry , Peptides/pharmacology , Peptides/physiology , Skin/metabolism
2.
Neuroscience ; 250: 622-31, 2013 Oct 10.
Article in English | MEDLINE | ID: mdl-23912037

ABSTRACT

Phoenixin-14 amide, herein referred to as phoenixin, is a newly identified peptide from the rat brain. Using a previously characterized rabbit polyclonal antiserum against phoenixin, enzyme-immunoassay detected a high level (>4.5 ng/g tissue) of phoenixin-immunoreactivity (irPNX) in the rat spinal cords. Immunohistochemical studies revealed irPNX in networks of cell processes in the superficial dorsal horn, spinal trigeminal tract and nucleus of the solitary tract; and in a population of dorsal root, trigeminal and nodose ganglion cells. The pattern of distribution of irPNX in the superficial layers of the dorsal horn was similar to that of substance P immunoreactivity (irSP). Double-labeling the dorsal root ganglion sections showed that irPNX and irSP express in different populations of ganglion cells. In awake mice, intrathecal injection of phoenixin (1 or 5 µg) did not significantly affect the tail-flick latency as compared to that in animals injected with artificial cerebrospinal fluid (aCSF). Intrathecal administration of phoenixin (0.5, 1.25 or 2.5 µg) significantly reduced the number of writhes elicited by intraperitoneal injection of acetic acid (0.6%, 0.3 ml/30 g) as compared to that in mice injected with aCSF. While not affecting the tail-flick latency, phoenixin antiserum (1:100) injected intrathecally 10 min prior to the intraperitoneal injection of acetic acid significantly increased the number of writhes as compared to mice pre-treated with normal rabbit serum. Intrathecal injection of non-amidated phoenixin (2.5 µg) did not significantly alter the number of writhes evoked by acetic acid. Our result shows that phoenixin is expressed in sensory neurons of the dorsal root, nodose and trigeminal ganglia, the amidated peptide is bioactive, and exogenously administered phoenixin may preferentially suppress visceral as opposed to thermal pain.


Subject(s)
Ganglia, Sensory/physiology , Hypothalamic Hormones/physiology , Peptide Hormones/physiology , Acetic Acid , Animals , Data Interpretation, Statistical , Ganglia, Sensory/metabolism , Hypothalamic Hormones/metabolism , Hypothalamic Hormones/pharmacology , Immunoenzyme Techniques , Immunohistochemistry , Male , Medulla Oblongata/drug effects , Medulla Oblongata/metabolism , Pain Measurement/drug effects , Peptide Hormones/metabolism , Peptide Hormones/pharmacology , Rats , Rats, Sprague-Dawley , Reaction Time/drug effects , Reaction Time/physiology , Spinal Cord/metabolism , Spinal Cord/physiology
3.
Neuroscience ; 240: 155-62, 2013 Jun 14.
Article in English | MEDLINE | ID: mdl-23470775

ABSTRACT

Irisin is a recently identified myokine secreted from the muscle in response to exercise. In the rats and mice, immunohistochemical studies with an antiserum against irisin peptide fragment (42-112), revealed that irisin-immunoreactivity (irIRN) was detected in three types of cells; namely, skeletal muscle cells, cardiomyocytes, and Purkinje cells of the cerebellum. Tissue sections processed with irisin antiserum pre-absorbed with the irisin peptide (42-112) (1 µg/ml) showed no immunoreactivity. Cerebellar Purkinje cells were also immunolabeled with an antiserum against fibronectin type II domain containing 5 (FNDC5), the precursor protein of irisin. Double-labeling of cerebellar sections with irisin antiserum and glutamate decarboxylase (GAD) antibody showed that nearly all irIRN Purkinje cells were GAD-positive. Injection of the fluorescence tracer Fluorogold into the vestibular nucleus of the rat medulla retrogradely labeled a population of Purkinje cells, some of which were also irIRN. Our results provide the first evidence of expression of irIRN in the rodent skeletal and cardiac muscle, and in the brain where it is present in GAD-positive Purkinje cells of the cerebellum. Our findings together with reports by others led us to hypothesize a novel neural pathway, which originates from cerebellum Purkinje cells, via several intermediary synapses in the medulla and spinal cord, and regulates adipocyte metabolism.


Subject(s)
Fibronectins/metabolism , Neuroglia/metabolism , Neurons/metabolism , Animals , Brain/cytology , Cell Count , Glutamate Decarboxylase/metabolism , Male , Mice , Mice, Inbred ICR , Myocardium/metabolism , Myocardium/ultrastructure , Neural Pathways/physiology , Rats , Rats, Sprague-Dawley , Stilbamidines/metabolism
4.
J Neuroendocrinol ; 25(2): 206-15, 2013 Feb.
Article in English | MEDLINE | ID: mdl-22963497

ABSTRACT

Normal anterior pituitary function is essential for fertility. Release from the gland of the reproductive hormones luteinising hormone and follicle-stimulating hormone is regulated primarily by hypothalamically-derived gonadotrophin-releasing hormone (GnRH), although other releasing factors (RF) have been postulated to exist. Using a bioinformatic approach, we have identified a novel peptide, phoenixin, that regulates pituitary gonadotrophin secretion by modulating the expression of the GnRH receptor, an action with physiologically relevant consequences. Compromise of phoenixin in vivo using small interfering RNA resulted in the delayed appearance of oestrus and a reduction in GnRH receptor expression in the pituitary. Phoenixin may represent a new class of hypothalamically-derived pituitary priming factors that sensitise the pituitary to the action of other RFs, rather than directly stimulating the fusion of secretary vesicles to pituitary membranes.


Subject(s)
Hypothalamic Hormones/metabolism , Peptide Hormones/metabolism , Pituitary Hormones/isolation & purification , Reproduction/genetics , Amino Acid Sequence , Animals , Cells, Cultured , Cloning, Molecular , Dose-Response Relationship, Drug , Female , Fertility Agents/chemistry , Fertility Agents/isolation & purification , Fertility Agents/metabolism , Fertility Agents/pharmacology , Gonadotropin-Releasing Hormone/genetics , Gonadotropin-Releasing Hormone/metabolism , Hypothalamic Hormones/genetics , Hypothalamic Hormones/isolation & purification , Hypothalamic Hormones/pharmacology , Luteinizing Hormone/blood , Male , Molecular Sequence Data , Peptide Hormones/genetics , Peptide Hormones/isolation & purification , Peptide Hormones/pharmacology , Pituitary Hormones/genetics , Pituitary Hormones/metabolism , Pituitary Hormones/pharmacology , Rats , Rats, Sprague-Dawley , Reproduction/drug effects , Reproduction/physiology , Sequence Homology, Amino Acid
5.
J Biol Chem ; 275(46): 36134-42, 2000 Nov 17.
Article in English | MEDLINE | ID: mdl-10908567

ABSTRACT

The pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 (PAC1) receptor is a G protein-coupled receptor and class II receptor member. The receptor domains critical for signaling are unknown. To explore the role of the C terminus, truncations of 63 residues (Tr406), 53 residues (Tr416), 49 residues (Tr420), 44 residues (Tr424), and 37 residues (Tr433) were constructed and expressed in NIH/3T3 cells, and immunofluorescence, radioligand binding, adenylyl cyclase (AC) and phospholipase C (PLC) assays were performed. (125)I-PACAP-27 binding (K(d) = 0.6-1.5 nm) for the Tr406 and Tr433 were similar to wild type Hop and Null splice variants (K(d) = approximately 1.1 nm). Although internalization of ligand for both the Tr406 and Tr433 mutants was reduced to 50-60% at 60 min compared with 76-87% for WT, loss of G protein coupling did not account for differences in internalization. Despite similar binding properties Tr406 and Tr416 mutants showed no AC or PLC response. Addition of 14 amino acids distal to HopTr406 resulted in normal AC and PLC responses. Site-directed mutagenesis indicated that Arg(416) and Ser(417) are essential for G protein activation. The proximal C terminus mediates signal transduction, and the distal is involved with internalization. Two residues within the C terminus, Arg(416) and Ser(417) conserved among class II receptors are the likely sites for G protein coupling.


Subject(s)
Down-Regulation , Endocytosis , Receptors, Pituitary Hormone/chemistry , Receptors, Pituitary Hormone/metabolism , Signal Transduction , 3T3 Cells , Adenylyl Cyclases/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , Binding, Competitive , Cyclic AMP/metabolism , Fluorescent Antibody Technique , Half-Life , Humans , Inositol Phosphates/metabolism , Iodine Radioisotopes , Mice , Molecular Sequence Data , Mutation , Pituitary Gland/enzymology , Pituitary Gland/metabolism , Protein Binding , Protein Structure, Tertiary , Protein Transport , Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide , Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide , Receptors, Pituitary Hormone/genetics , Sequence Alignment , Transfection
6.
Ann N Y Acad Sci ; 921: 195-201, 2000.
Article in English | MEDLINE | ID: mdl-11193823

ABSTRACT

The objectives of the study reported here were to identify amino acid residues of the C-terminus that are critical for intracellular signaling. A total of nine amino acid substitution and truncation mutants were constructed by PCR and confirmed by sequencing. Mutant and wildtype receptors were stably transfected into NIH/3T3 fibroblasts and studied for their ability to bind PACAP-27 and activate phospholipase C (PLC) and adenylyl cyclase (AC). Receptor affinity of 125I-PACAP-27 for the wildtype and mutants were similar (Kd = 0.6-1.5 nM). However, truncation of the entire 63 amino acids of the hPAC1 resulted in no signaling to either AC or IP. Addition of the proximal 10 amino acids of the C-terminus failed to restore AC or IP signaling, whereas addition of the proximal 27 amino acids of the C-terminus resulted in reconstitution of complete AC and IP responses, identical to the WT. Point mutations within this 17 amino acid region identified specific amino acids involved in PAC1 signaling. These results indicate that a structural motif within the proximal region of the carboxyl terminus is critical for G protein coupling.


Subject(s)
Receptors, Pituitary Hormone/chemistry , Receptors, Pituitary Hormone/metabolism , 3T3 Cells , Adenylyl Cyclases/metabolism , Amino Acid Motifs , Animals , GTP-Binding Proteins/metabolism , Humans , In Vitro Techniques , Inositol Phosphates/metabolism , Mice , Neuropeptides/metabolism , Pituitary Adenylate Cyclase-Activating Polypeptide , Point Mutation , Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide , Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide , Receptors, Pituitary Hormone/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Deletion , Signal Transduction , Transfection , Type C Phospholipases/metabolism
7.
J Clin Invest ; 104(10): 1383-91, 1999 Nov.
Article in English | MEDLINE | ID: mdl-10562300

ABSTRACT

Pituitary adenylate cyclase activating polypeptide (PACAP) is present in gastric nerves, and PACAP receptors (PAC1) are found on gastric enterochromaffin-like (ECL) cells. Expression of PAC1 splice variants in purified ECL cells was determined by RT-PCR. PACAP effects on ECL cells were analyzed by video imaging of [Ca(2+)](i) and histamine release; its effects on gastric glands were examined by confocal microscopy of [Ca(2+)](i) in ECL and parietal cells. PACAP action on D cells was measured by [Ca(2+)](i) and radioimmunoassay. PACAP effects on acid secretion were determined in fistula rats with or without neutralizing anti-somatostatin antibodies. All splice variants of PAC1 were found, but vasoactive intestinal polypeptide (VIP) receptor (VPAC) products were absent. PACAP-27 and -38 dose-dependently raise [Ca(2+)](i) in ECL cells, and stimulated histamine release. VIP had a much lower affinity, which demonstrates the presence of PAC1 but not VPAC. PACAP elevated [Ca(2+)](i) in ECL and parietal cells of superfused gastric glands, but only the parietal cell signal was inhibited by ranitidine, showing the absence of PAC1 on parietal cells, and demonstrating functional coupling between the cell types. PACAP and VIP stimulated calcium signaling and somatostatin release from D cells with almost equal efficacy. Acid secretion was stimulated after intravenous injection of PACAP into rats treated with somatostatin antibody. PACAP is a candidate as a mediator of neural regulation of acid secretion.


Subject(s)
Alternative Splicing , Enterochromaffin-like Cells/physiology , Gastric Acid/metabolism , Gastric Mucosa/physiology , Genetic Variation , Receptors, Pituitary Hormone/genetics , Animals , Calcium/metabolism , Cells, Cultured , Enterochromaffin-like Cells/cytology , Enterochromaffin-like Cells/drug effects , Gastric Mucosa/cytology , Gastric Mucosa/drug effects , Histamine Release/drug effects , Microscopy, Video , Neuropeptides/pharmacology , Neurotransmitter Agents/pharmacology , Pituitary Adenylate Cyclase-Activating Polypeptide , Rats , Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide , Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide , Receptors, Pituitary Hormone/physiology , Reverse Transcriptase Polymerase Chain Reaction
8.
Ann N Y Acad Sci ; 865: 147-56, 1998 Dec 11.
Article in English | MEDLINE | ID: mdl-9928007

ABSTRACT

The current study was undertaken to determine the presence and distribution of PAC1-Rs within the gastric mucosa. Polyclonal antibodies to the carboxyl terminus of the rat PAC1-R were generated and shown to be specific against the PAC1-R expressed in NIH 3T3 cells. Western blot analysis using isolated (approximately 85% pure) ECL cell membranes identified a 48 kD protein consistent with the calculated molecular mass of the cloned PAC1-R. RT/PCR performed using specific primers for the PAC1-R confirmed the presence of splice variants of the rat PAC1-R, but not VPAC1-R or VPAC2-R. These data provide the first direct evidence for the existence of functional PACAP Type I receptors on ECL cells of the gastric mucosa and suggest a potential role for PACAP in the stimulation of gastric acid secretion and in the regulation of the growth of ECL cells.


Subject(s)
Enterochromaffin Cells/metabolism , Gastric Mucosa/metabolism , Receptors, Pituitary Hormone/genetics , 3T3 Cells , Animals , Blotting, Western , Cloning, Molecular , Enterochromaffin Cells/cytology , Gastric Mucosa/cytology , Humans , Immunohistochemistry/methods , Mice , Molecular Weight , Rats , Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide , Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide , Receptors, Pituitary Hormone/analysis , Receptors, Vasoactive Intestinal Peptide, Type II , Receptors, Vasoactive Intestinal Polypeptide, Type I , Reverse Transcriptase Polymerase Chain Reaction/methods , Transfection
9.
Am J Physiol ; 272(5 Pt 1): C1717-25, 1997 May.
Article in English | MEDLINE | ID: mdl-9176164

ABSTRACT

Na(+)-K(+)-ATPase is inhibited by cardiac glycosides and is insensitive to Sch-28080, an inhibitor of gastric H(+)-K(+)-ATPase. Gastric H(+)-K(+)-ATPase is not inhibited by cardiac glycosides. Both ouabain and, Sch-28080 binding are inhibited by K+, and it has been suggested that the inhibitors bind to corresponding regions on the alpha-subunit of each ion pump. For identification of regions of each pump that interact with the specific inhibitors, chimeric alpha-subunits consisting of selected regions from Na(+)-K(+)-ATPase and gastric H(+)-K(+)-ATPase have been prepared. One chimera (gM1/2) has been constructed from cDNA of the sheep alpha1-subunit of Na(+)-K(+)-ATPase by replacement of the last 12 amino acids of the first predicted transmembrane region (Ile99-Ile110) with corresponding amino acids from rat gastric H(+)-K(+)-ATPase. gM1/2 was expressed in yeast cells together with either the rat Na(+)-K(+)-ATPase beta 1-subunit (NK beta 1) or rat gastric H(+)-K(+)-ATPase beta-subunit (HK beta). Western blots show that the expression level of the chimeric alpha-subunit was comparable to the Na(+)-K(+)-ATPase alpha 1. Ouabain binds with high affinity to gM1/2+NK beta 1 [ouabain binding affinity (Kd) = 9.5 nM] but not to gM1/2+HK beta. The Kd for ouabain binding to Na(+)-K(+)-ATPase was 7.8 nM. Na(+)-K(+)-ATPase activity of gM1/2+NK beta 1 was inhibited both by ouabain and Sch-28080. The 50% inhibition concentration for Sch-28080 was 20-60 nM. Sch-28080 at 10 microM did not inhibit Mg(2+)- and Pi-dependent ouabain binding to gM1/2+NK beta 1. Ouabain (0.75 mM) inhibited palytoxin-induced K+ efflux from yeast cells expressing either gM1/2+NK beta 1 or gM1/2+NK beta, and Sch-28080 increased the palytoxin-induced K+ efflux from yeast cells expressing gM1/2+NK beta 1 or gM1/2+HK beta. These results implicate a small number of amino acids in the first transmembrane part of gastric H(+)-K(+)-ATPase as partial determinants of the sensitivity to Sch-28080. The data also suggest that ouabain and Sch-28080 do not bind to the same site on the chimera.


Subject(s)
Enzyme Inhibitors/pharmacology , Gastric Mucosa/metabolism , H(+)-K(+)-Exchanging ATPase/genetics , H(+)-K(+)-Exchanging ATPase/metabolism , Imidazoles/pharmacology , Acrylamides/pharmacology , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Animals , Cell Membrane/metabolism , Chimera , Cnidarian Venoms/metabolism , Hydrolysis/drug effects , Ouabain/metabolism , Potassium/metabolism , Rats , Sheep , Yeasts/cytology , Yeasts/metabolism
10.
J Biol Chem ; 270(23): 13937-47, 1995 Jun 09.
Article in English | MEDLINE | ID: mdl-7775454

ABSTRACT

High-affinity ouabain binding to Na+/K(+)-ATPase (sodium- and potassium-transport adenosine triphosphatase (EC 3.6.1.37)) requires phosphorylation of the alpha subunit of the enzyme either by ATP or by inorganic phosphate. For the native enzyme (alpha/beta 1), the ATP-dependent reaction proceeds about 4-fold more slowly in the absence of Na+ than when saturating concentrations of Na+ are present. Hybrid pumps were formed from either the alpha 1 or the alpha 3 subunit isoforms of Na+/K(+)-ATPase and a chimeric beta subunit containing the transmembrane segment of the Na+/K(+)-ATPase beta 1 isoform and the external domain of the gastric H+/K(+)-ATPase beta subunit (alpha/NH beta 1 complexes). In the absence of Na+, these complexes show a rate of ATP-dependent ouabain binding from approximately 75-100% of the rate seen in the presence of Na+ depending on buffer conditions. Nonhydrolyzable nucleotides or treatment of ATP with apyrase abolishes ouabain binding, demonstrating that ouabain binding to alpha/NH beta 1 complexes requires phosphorylation of the protein. Buffer ions inhibit ouabain binding by alpha/NH beta 1 in the absence of Na+ rather than promote ouabain binding, indicating that they are not substituting for sodium ions in the phosphorylation reaction. The pH dependence of ATP-dependent ouabain binding in the presence or absence of Na+ is similar, suggesting that protons are probably not substituting for Na+. Hybrid alpha/NH beta 1 pumps also show slightly higher apparent affinities (2-3-fold) for ATP, Na+, and ouabain; however, these are not sufficient to account for the increase in ouabain binding in the absence of Na+. In contrast to phosphoenzyme formation and ouabain binding by alpha/NH beta 1 complexes in the absence of Na+, ATPase activity, measured as release of phosphate from ATP, requires Na+. These data suggest that the transition from E1P to E2P during the catalytic cycle does not occur when the sodium binding sites are not occupied. Thus, the chimeric beta subunit reduces or eliminates the role of Na+ in phosphoenzyme formation from ATP, but Na+ binding or release by the enzyme is still required for ATP hydrolysis and release of phosphate.


Subject(s)
Adenosine Triphosphate/metabolism , Sodium-Potassium-Exchanging ATPase/metabolism , Sodium/metabolism , Hydrogen-Ion Concentration , Ouabain/metabolism , Phosphorylation , Recombinant Fusion Proteins/metabolism , Sodium-Potassium-Exchanging ATPase/chemistry
11.
Cell Biol Toxicol ; 9(2): 141-8, 1993.
Article in English | MEDLINE | ID: mdl-8242430

ABSTRACT

Cd2+ provokes inositol trisphosphate production and releases stored Ca2+, apparently by binding to a zinc site in the external domain of an orphan receptor. One microM Cd2+ evokes an immediate spike in cytosolic free Ca2+, which is similar to that evoked by bradykinin. Platelet-derived growth factor (PDGF) also increases free Ca2+ in human dermal fibroblasts, but there is a distinct lag before free Ca2+ rises in response to PDGF. Genistein, which selectively inhibits tyrosine kinases, markedly inhibited Ca2+ mobilization evoked by PDGF. Calcium mobilization triggered by cadmium or bradykinin was relatively insensitive to genistein. The PDGF receptor is known to be a tyrosine kinase, which phosphorylates and thereby activates phospholipase C gamma, whereas a G protein couples the bradykinin receptor to another phospholipase C isoform. These findings support the hypothesis that the orphan receptor triggered by cadmium is coupled to phospholipase C via a G protein.


Subject(s)
Bradykinin/antagonists & inhibitors , Cadmium/antagonists & inhibitors , Calcium/metabolism , Fibroblasts/drug effects , Isoflavones/pharmacology , Platelet-Derived Growth Factor/antagonists & inhibitors , Protein-Tyrosine Kinases/antagonists & inhibitors , Fibroblasts/metabolism , Genistein , Humans , In Vitro Techniques , Isoenzymes/metabolism , Type C Phospholipases/metabolism
12.
Am J Physiol ; 263(3 Pt 1): C628-34, 1992 Sep.
Article in English | MEDLINE | ID: mdl-1415512

ABSTRACT

We have found that immortalized aortic myocytes have high Na(+)-Ca2+ exchange activity that is similar in amount to that of low-passage myocyte cultures. Replacing all external Na+ with K+ slightly increased 45Ca2+ uptake in cells with basal Na+ and greatly increased 45Ca2+ uptake in cells with increased intracellular Na+. External Na+ competitively inhibited 45Ca2+ uptake (inhibition constant approximately 10 mM). The dependence of exchange activity on intracellular Na+ was sigmoidal. The 50% maximum concentration (K0.5) for Na+ was 33 +/- 1 mmol/l cell water space. The Hill coefficient was 2.9 +/- 0.2, which is consistent with a stoichiometry of 3 Na+/1 Ca2+. Cytosolic free Na+ was estimated from the ratio of sodium-binding benzofuran isophthalate (SBFI) fluorescence and an in vivo calibration curve. Sulfinpyrazone, an inhibitor of anion transport, markedly increased the SBFI content of the cells without affecting cell Na+. Cytosolic free Na+ was 9 mM under basal conditions. Because free Na+ did not differ significantly from total Na+, little or no Na+ is bound or compartmentalized in these cells. The complete replacement of extracellular Na+ with K+ fails to evoke appreciable Ca2+ influx, at least in part, because of the low cell Na+ concentration relative to the K0.5 of the exchanger and its sigmoid dependence on Na+.


Subject(s)
Aorta/metabolism , Calcium/metabolism , Carrier Proteins/metabolism , Muscle, Smooth, Vascular/metabolism , Sodium/metabolism , Animals , Aorta/cytology , Benzofurans , Calibration , Carrier Proteins/antagonists & inhibitors , Cell Line, Transformed , Cytosol/metabolism , Drug Synergism , Ethers, Cyclic , Fluorescent Dyes , Intracellular Membranes/metabolism , Muscle, Smooth, Vascular/cytology , Osmolar Concentration , Ouabain/pharmacology , Potassium/metabolism , Potassium/pharmacology , Sodium/pharmacology , Sodium-Calcium Exchanger
13.
Am J Physiol ; 263(3 Pt 1): C635-41, 1992 Sep.
Article in English | MEDLINE | ID: mdl-1415513

ABSTRACT

Depolarization with 50 mM K+ evoked a spike in cytosolic free Ca2+ ([Ca2+]i) and increased 45Ca2+ uptake in immortalized aortic myocytes. The following evidence indicates that the electrogenic Na(+)-Ca2+ exchanger caused the Ca2+ influx that was evoked by K+ depolarization. First, K+ depolarization had no effect on [Ca2+]i and 45Ca2+ uptake in cells with basal Na+ but strikingly increased both in Na(+)-loaded cells. Second, the [Ca2+]i increases produced by K+ depolarization depended hyperbolically on external Ca2+ (50% maximum concentration = 1.5 mM). Third, the increases in [Ca2+]i and 45Ca2+ uptake were greater when external Na+ was replaced with K+ rather than with N-methyl-D-glucamine or choline. A series of K+ depolarizations elicited a sequence of [Ca2+]i spikes, provided there was a short incubation at 5 mM K+ between the depolarizations. A prior K+ depolarization almost abolished the 45Ca2+ uptake response to K+ depolarization. The inhibition of exchange activity by a prior K+ depolarization required external Ca2+ and was completely reversible. A prior incubation with angiotensin II, platelet-derived growth factor, or ionomycin also inhibited exchange activity. Moderate [Ca2+]i increases probably feedback inhibit Ca2+ influx via the exchanger by a kinetic mechanism. Inactivation of the exchanger, together with Ca2+ extrusion or sequestration, causes the rapid decrease in [Ca2+]i from the peak evoked by depolarization.


Subject(s)
Aorta/metabolism , Calcium/metabolism , Carrier Proteins/metabolism , Muscle, Smooth, Vascular/metabolism , Animals , Aorta/cytology , Calcium/physiology , Carrier Proteins/antagonists & inhibitors , Cell Line, Transformed , Choline/pharmacology , Cytosol/metabolism , Electrophysiology , Feedback , Meglumine/pharmacology , Muscle, Smooth, Vascular/cytology , Osmolar Concentration , Ouabain/pharmacology , Potassium/metabolism , Potassium/pharmacology , Potassium/physiology , Sodium/metabolism , Sodium-Calcium Exchanger
14.
J Membr Biol ; 124(1): 73-83, 1991 Oct.
Article in English | MEDLINE | ID: mdl-1662727

ABSTRACT

Kinetic properties of Na(+)-Ca2+ exchange in a renal epithelial cell line (LLC-MK2) were assessed by measuring cytosolic free Ca2+ with fura-2 and 45Ca2+ influx. Replacing external Na+ with K+ produced relatively small increases in free Ca2+ and 45Ca2+ uptake unless the cells were incubated with ouabain. Ouabain markedly increased cell Na+ and strongly potentiated the effect of replacing external Na+ with K+ on free Ca2+ and 45Ca2+ uptake. 45Ca2+ influx in 140 mM K+ or N-methyl-D-glucamine minus influx in 140 mM Na+ was used to quantify Na(+)-Ca2+ exchange activity of Na(+)-loaded cells. The dependence of exchange on cell Na+ was sigmoidal; the K0.5 was 26 +/- 3 mmol/liter cell water space, and the Hill coefficient was 3.1 +/- 0.2. The kinetic features of the dependence of exchange on cell Na+ partly account for the small increase in Ca2+ influx when all external Na+ is replaced by K+. Besides raising cell Na+ ouabain appears to activate the exchanger. Magnesium competitively inhibited exchange activity. The potency of Mg2+ was 8.2-fold lower with potassium instead of N-methyl-D-glucamine or choline as the replacement for external Na+. Potassium also increased the Vmax of exchange by 86% and had no effect on the Km for Ca2+. The exchanger does not cause detectable 22Na(+)-Mg2+ exchange and does not appear to require K+ or transport 86Rb+. Although exchange activity was plentiful in the epithelial cells from monkey kidney, others from amphibian, canine, opossum, and porcine kidney had no detectable exchange activity. All of the measured kinetic properties of Na(+)-Ca2+ exchange in the renal epithelial cells are very similar to those of the exchanger in rat aortic myocytes.


Subject(s)
Carrier Proteins/physiology , Kidney/cytology , Magnesium/pharmacology , Sodium/physiology , Animals , Biological Transport/drug effects , Biological Transport/physiology , Calcium/pharmacokinetics , Calcium/pharmacology , Calcium Channels/drug effects , Calcium Channels/physiology , Cells, Cultured , Epithelial Cells , Epithelium/chemistry , Epithelium/physiology , Kidney/chemistry , Kidney/physiology , Ouabain/pharmacology , Potassium/pharmacology , Sodium/analysis , Sodium/pharmacology , Sodium-Calcium Exchanger , Tetraethylammonium , Tetraethylammonium Compounds/pharmacology
15.
Biochim Biophys Acta ; 1068(1): 97-104, 1991 Sep 10.
Article in English | MEDLINE | ID: mdl-1892860

ABSTRACT

Plasma membrane vesicles were purified from rat aortic myocytes by centrifugation in a discontinuous sucrose gradient. Vesicles were prepared in the presence or absence of five proteinase inhibitors (aprotinin, benzamidine, leupeptin, pepstatin A and phenylmethylsulfonyl fluoride). The proteinase inhibitors decreased the Vmax by 3.4-fold and had no effect on the Km for Ca2+ of Na+ gradient-dependent 45Ca2+ influx. The proteinase inhibitors had no direct effect on exchange activity, and they had no effect on membrane purity as indicated by 5'-nucleotidase activity. Removing the proteinase inhibitors or adding trypsin or chymotrypsin increased exchange activity approx. 2-fold. The Vmax of exchange activity in intact aortic myocytes is approx. 10-fold higher than the Vmax in plasma membrane vesicles prepared in the presence of proteinase inhibitors. Exchange activity in plasma membrane vesicles is only a sixtieth of the expected value, because the vesicles have approx. 7-fold higher 5'-nucleotidase activity and approx. 6-fold higher specific exchange activity than the crude homogenate. The large loss of exchange activity may be caused by a change in a regulatory domain of the exchanger because endogenous proteolysis restores some of the activity lost during vesicle preparation.


Subject(s)
Carrier Proteins/metabolism , Cell Membrane/metabolism , Muscle, Smooth, Vascular/metabolism , 5'-Nucleotidase/metabolism , Animals , Aorta , Cell Membrane/enzymology , Cells, Cultured/metabolism , Kinetics , Muscle, Smooth, Vascular/enzymology , Protease Inhibitors/pharmacology , Rats , Sodium-Calcium Exchanger , Trypsin/pharmacology
16.
Biochem Pharmacol ; 41(4): 601-9, 1991 Feb 15.
Article in English | MEDLINE | ID: mdl-1847637

ABSTRACT

The inhibitory potencies of several amiloride congeners towards Na(+)-Ca2+ and Na(+)-H+ exchange were compared in rat aortic myocytes. N-(2,4-Dimethylbenzyl)amiloride (DMB) was 10 times more potent towards Na(+)-Ca2+ than Na(+)-H+ exchange. Amiloride and ethylisopropylamiloride were about 5,000 and 10,000 times more potent toward Na(+)-H+ than Na(+)-Ca2+ exchange respectively. N-(3,4-Dichlorobenzyl)amiloride was almost equipotent towards both exchangers. About 40 nM ethylisopropylamiloride inhibited Na(+)-H+ exchange by 50%. Ethylisopropylamiloride (10 microM) had no effect on basal or angiotensin-evoked 45Ca2+ efflux or net Ca2+ efflux. In contrast to ethylisopropylamiloride, 25-50 microM DMB, which strongly inhibits Na(+)-Ca2+ exchange, markedly decreased both 45Ca2+ efflux and net Ca2+ efflux produced by angiotensin. Replacing extracellular Na+ with N-methyl-D-glucamine inhibited angiotensin-evoked 45Ca2+ efflux similarly to DMB. Neither DMB nor Na+ placement had any effect on basal or angiotensin-evoked production of [3H]inositol phosphates. These findings suggest that Na(+)-H+ exchange has no major influence on short-term Ca2+ regulation and provide evidence that Na(+)-Ca2+ exchange is a major pathway of rapid Ca2+ efflux in stimulated arterial muscle cells.


Subject(s)
Amiloride/pharmacology , Calcium/metabolism , Muscle, Smooth, Vascular/metabolism , Sodium/metabolism , Amiloride/analogs & derivatives , Angiotensin II/pharmacology , Animals , In Vitro Techniques , Ionomycin/pharmacology , Protons , Rats
19.
Cell Calcium ; 10(3): 125-34, 1989 Apr.
Article in English | MEDLINE | ID: mdl-2720758

ABSTRACT

Ionomycin (1 microM) produced a large spike in cytosolic free Ca2+ [( Ca2+]i). The ionophore had no effect on [Ca2+]i if the sarcoplasmic reticulum had previously been Ca2+ depleted by stimulating neurohormone receptors. Ionomycin markedly increased 45Ca2+ efflux and decreased total cell Ca2+ by 60 to 70% in 1 min. Replacing extracellular Na+ [( Na+]o) with choline or N-methyl-D-glucamine strongly inhibited the effects of ionomycin on 45Ca2+ efflux and total Ca2+. Ionomycin caused similar peak increases in [Ca2+]i in the presence and absence of [Na+]o, but the exponential fall from the peak was faster in the presence of [Na+]o. Dimethylbenzamil, a potent blocker of Na+/Ca2+ exchange in these cells, strongly inhibited the effects of ionomycin on 45Ca2+ efflux and total cell Ca2+. We conclude that the increase in cytosolic free Ca2+ produced by ionomycin may be sufficient to activate the plasma membrane Na+/Ca2+ exchanger which removes Ca2+ from the cytosol and helps restore basal [Ca2+]i.


Subject(s)
Calcium/pharmacokinetics , Muscle, Smooth, Vascular/metabolism , Sarcoplasmic Reticulum/metabolism , Sodium/pharmacokinetics , Amiloride/analogs & derivatives , Amiloride/pharmacology , Animals , Calcium/physiology , Cells, Cultured , Cytosol/metabolism , Ethers/pharmacology , Ionomycin , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/drug effects , Rats , Sarcoplasmic Reticulum/drug effects , Sodium/physiology
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