UMLS:C0432222 - FACTA Search

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To study the cellular basis for FSH-stimulated dose-dependent graded increases in intracellular Ca2+ concentrations in populations of Sertoli cells, we investigated the effects of FSH on free Ca2+ ion concentrations ([Ca2+]i) in individual rat Sertoli cells using the Ca(2+)-sensitive dye fura-2/AM and digital fluorescent videomicroscopy. Ovine or rat FSH elicited a hormone-specific rise in [Ca2+]i within 20-140 sec, with a peak level 2.7 +/- 0.9-fold greater than the basal value (mean +/- SEM; n = 8) lasting for 4-16 min. The amplitude and kinetics of the FSH-induced [Ca2+]i signal were not dose dependent. Instead, increasing doses of FSH recruited a higher percentage of responding cells. Chelation of extracellular Ca2+ or cotreatment with verapamil or cobalt abolished FSH-induced [Ca2+]i increases. Furthermore, in the presence of extracellular Mn2+, direct evidence for FSH-mediated Ca2+ influx was obtained from the quench of fura-2 fluorescence. Induced Ca2+ increases were mimicked by forskolin or protein kinase-A type I activators [8-(6-amino-hexyl)amino-cAMP and N6-benzoyl-cAMP (N6B)]. However, the cAMP analogs, 8-bromo-cAMP, N6,2'-O-dibutyryl cAMP, or protein kinase-A type II activators (8-thiomethyl-cAMP and N6B), induced [Ca2+]i increases even in the absence of extracellular Ca2+, and the time course of the [Ca2+]i rise induced by cAMP analogs was more rapid than that induced by FSH. Similarly, the uninhibited rise in [Ca2+]i induced by FSH in pertussis toxin-pretreated Sertoli cells suggests that PT-sensitive G-proteins are not involved in the action of FSH on [Ca2+]i. In summary, we demonstrate that FSH evokes sustained [Ca2+]i increases in single Sertoli cells in a nongraded fashion and recruits increasing numbers of responding cells in a dose-dependent fashion. We also provide explicit evidence that FSH induces Ca2+ influx. Mimicry of the FSH-induced [Ca2+]i rise by certain cAMP analogs [8-(6-amino-hexyl)amino-cAMP and N6B; protein kinase-A type I activator] or forskolin suggests that Ca2+ may be part of a dual pathway of cAMP-initiated intracellular signaling.
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PMID:Cellular basis for follicle-stimulating hormone-stimulated calcium signaling in single rat Sertoli cells: possible dissociation from effects of adenosine 3',5'-monophosphate. 813 59

5-Hydroxytryptamine 5-HT1B/5-HT1D receptors are members of the same receptor subfamily, but display a different pharmacology (Hartig et al. (1992) Trends Pharmacol Sci 13: 152-159). Whereas several cell lines have been reported to contain 5-HT1B receptors, none has been described, however, that endogenously expresses well-characterized 5-HT1D receptors. The present study deals with the identification of 5-HT1D receptors inhibiting cyclic AMP accumulation in Madin-Darby canine kidney (MDCK) cells. 5-HT (1 nM-10 microM) induced a concentration-dependent inhibition of the cyclic AMP accumulation stimulated by prostaglandin E1 (1 microM) in MDCK cells. The maximal effect of 5-HT averaged 50% inhibition and was abolished after a pre-treatment of the cells with pertussis toxin. Other agonists mimicked the effects of 5-HT, with the following rank order of potency (pEC50 +/- SEM, n > or = 3): 5-carboxamidotryptamine (8.36 +/- 0.48) > PAPP (p-aminophenylethyl-m-trifluoromethylphenyl piperazine. 7.89 +/- 0.23) > 5-HT (7.35 +/- 0.05) > sumatriptan (6.65 +/- 0.27). PAPP behaved as a partial agonist. 8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)tetralin) was less potent, its maximal effect being not reached at 0.1 mM. Methiothepin. GR127935, (-)propranolol, rauwolscine and ketanserin were all devoid of intrinsic activity (up to 10 microM or 0.1 mM). Methiothepin (10 nM. 0.1 microM and 1 microM) antagonized 5-HT effect (pA2 8.57 +/- 0.44. Schild slope 1.17 +/- 0.21, n = 3).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:5-Hydroxytryptamine 5-HT1D receptors mediating inhibition of cyclic AMP accumulation in Madin-Darby canine kidney (MDCK) cells. 858 40

We have described a thyroid hormone receptor in synaptosomes of the chick embryo brain. To understand how the hormones exert their actions at this level, we performed a series of studies to demonstrate that this receptor could be linked to G proteins. Guanosine 5'-[gamma-thio]triphosphate (GTP gamma S)(100 muM) lowered the binding capacity of the receptor high affinity site from 8.9 +/- 1.3 to 3.4 +/- 1.3 ng T3/mg protein, a finding consistent with the coupling of receptor to G proteins. Furthermore, ADP ribosylation with pertussis toxin showed that thyroid hormones induced a dose-dependent increase in the inactive alpha 0-subunit of the G0 protein. This effect was detected at 10 pM, with a maximal increase (mean +/- SEM, 50 +/- 3.6%) at 100 nM, and T4 was as effective as T3. Both hormones also decreased the intrinsic guanine triphosphatase activity of G proteins by lowering the binding of GTP to the alpha-subunit and their rate of hydrolysis. This inhibition was greater with T4 (25 +/- 5%) than with T3 (14 +/- 2%), suggesting that the former could be the more active hormone at the synaptosomal level. The effect on guanine triphosphatase activity confirms that the synaptosomal thyroid hormone receptor is coupled to a G(zero) protein. These results demonstrate that thyroid hormones increase or favor the ADP ribosylation of G alpha(zero) by pertussis toxin. Thus, they enhance the alpha(zero)-GDP form of the G(zero) protein, namely its inactive conformation. By decreasing the activity of this protein, these hormones may modulate the formation of second messengers in synaptosomes and intervene in the regulation of neuronal proliferation and differentiation induced by several factors. Therefore, thyroid hormones may exert their action on brain maturation at least in part by modulating G alpha(zero) through their synaptosomal receptor.
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PMID:Effect of thyroid hormones on G proteins in synaptosomes of chick embryo. 864 Dec 9

Orphanin FQ (OFQ) has recently been reported to be an endogenous ligand for the opioid-like LC132 receptor. The effect of OFQ on high voltage-gated calcium channels (VGCCs) was examined in freshly dissociated rat pyramidal neurons using the whole-cell configuration of the patch-clamp technique. High-threshold Ba2+ currents were reversibly inhibited by OFQ. The depression of the currents was associated with a slowed rate of activation and a change in the activation I-V relationship at step potentials higher than +30 mV. In concentration-response experiments, a mean (+/-SEM) pEC50 value of 7.0 +/- 0.07 and a Hill coefficient of 1.5 +/- 0.08 (n = 5) were obtained. The near-maximum inhibition of the Ba2+ currents by OFQ (1 microM) amounted to 31 +/- 2.2% of control (n = 15). Opioid receptors could not account for the effects of OFQ on VGCCs, because naloxone, a broad spectrum mu-, delta-, and kappa-receptor antagonist, did not reduce the effectiveness of OFQ. When GTP-gamma-S was included in the pipette, the depression of the currents by OFQ was irreversible, whereas currents from neurons preincubated with pertussis toxin were not inhibited by OFQ, consistent with the involvement of a PTX-sensitive G-protein. When selective blockers of VGCCs were used, it was demonstrated that all subtypes of VGCCs were affected by OFQ. In conclusion, the effect of OFQ on VGCCs expressed in hippocampal CA3 and CA1 neurons may play an important role in the regulation of hippocampal cell excitability and neurotransmitter release.
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PMID:Modulation of voltage-gated calcium channels by orphanin FQ in freshly dissociated hippocampal neurons. 882 6

Carbachol increased ventricular automaticity in a concentration-dependent fashion from a control rate of 72 +/- 5 (mean +/- SEM) to 86 +/- 4 beats per minute at 10(-4) M carbachol. Pirenzepine, an M1-selective antagonist, and AFDX 116, an M2-selective antagonist, both at 10(-7) M, did not block the carbachol-induced positive chronotropic response. In contrast, 10(-7) M HHSiD, an M3-selective antagonist, completely blocked the positive chronotropic effect of carbachol. Carbachol stimulated the accumulation of IP1 in a concentration-dependent manner at concentrations > or = 3 x 10(-6) M. AFDX 116 had no effect on carbachol-induced IP1 accumulation. HHSiD significantly inhibited IP1 accumulation at concentrations > or = 3 x 10(-8) M, while pirenzepine inhibited IP1 accumulation only at concentrations > or = 10(-5) M. McN A343 and methacholine, two muscarinic receptor agonists with minimal M2 activities, and carbachol did not alter basal cAMP concentration, but all three agonists significantly attenuated the increase in cAMP accumulation in response to isoproterenol. Carbachol inhibited isoproterenol-mediated cAMP accumulation at concentrations > or = 10(-7) M. AFDX 116, HHSiD, and pirenzepine blocked the carbachol-induced inhibition of isoproterenol-stimulated cAMP accumulation. At equimolar concentrations, the inhibitory effects of HHSiD and AFDX-116 were similar, while that of pirenzepine was much less. Pretreatment with pertussis toxin for 24 h did not prevent the carbachol-mediated positive chronotropic response or accumulation of IP1 but completely abolished the inhibition of isoproterenol-stimulated cAMP accumulation. These results indicate that (a) neonatal ventricular myocytes in culture have a heterogeneous population of muscarinic (M2 and M3) receptors, (b) the M3 receptor is coupled to pertussis toxin-sensitive and pertussis toxin-insensitive G proteins, (c) M3 receptor stimulation activates phosphoinositide hydrolysis and increases automaticity via a pertussis toxin-insensitive G protein-dependent pathway, and (d) both M2 and M3 receptors couple to pertussis toxin-sensitive G protein(s) to mediate the inhibition of intracellular cAMP accumulation in response to isoproterenol stimulation.
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PMID:Muscarinic receptor heterogeneity in neonatal rat ventricular myocytes in culture. 884 59

The expression of muscarinic acetylcholine receptor (mAChR) subtypes in freshly isolated adult rat ventricular myocytes was investigated by reverse transcription of cellular mRNA followed by amplification of cDNA using the polymerase chain reaction (PCR). After reverse-transcriptase PCR, bands were obtained corresponding to the expected sizes for the m1 and m2 but not for the m3 to m5 mAChRs. The identity of the m1 and m2 bands was confirmed by single-cell PCR, restriction digest mapping, and Southern blot analysis. The presence of m1 and m2, but not m3, mAChR protein in these cells was shown by indirect immunofluorescence studies using subtype-specific antibodies. It was further investigated whether the identified m1 mAChR was responsible for the stimulatory effects on Ca2+ transients by high concentrations of carbachol ( > 10 mumol/L) known to occur in these cells. In pertussis toxin-treated ventricular myocytes electrically stimulated at 1 Hz, carbachol (300 mumol/L) increased the basal Ca2+ level from 96 +/- 7 to 118 +/- 8 nmol/L and the peak Ca2+ transient level from 519 +/- 32 to 640 +/- 36 nmol/L (mean +/- SEM P < .05 for both, n = 8). These effects of carbachol on Ca2+ transients were antagonized by 10 nmol/L pirenzepine, an m1 mAChR-selective antagonist. In contrast, the m2 mAChR-selective antagonist methoctramine (up to 100 nmol/L) did not inhibit the response. These results are the first to use single-cell PCR to probe cardiomyocyte-specific gene expression and indicate that m1 mAChRs are expressed on adult rat ventricular myocytes in addition to m2 mAChRs. The results further suggest that m1 mAChRs mediate the stimulatory responses on Ca2+ transients to high concentrations of cholinergic agonists seen in these cells.
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PMID:Molecular and functional identification of m1 muscarinic acetylcholine receptors in rat ventricular myocytes. 892 73

The purpose of this study was to visualize muscarinic receptors and their distribution on cardiomyocytes and to examine the effects of muscarinic cholinergic receptor (mACh-R) stimulation with carbachol on phosphatidylcholine hydrolysis. Cardiomyocytes were prepared as primary culture from 7-day-old chick embryo hearts. Cardiomyocytes, grown on cover slips, were labelled with BODIPY PZ, a fluorescent analog of the muscarinic receptor antagonist pirenzepine, and examined with a laser scanning confocal microscope, mACh-R clusters were visualized and were fairly homogeneous in size with diameters ranging from 0.5 to 1.0 micron. The number of receptor clusters per cell was 83.5 +/- 6.8 (mean +/- SEM) and clusters were found at the periphery of the cell. Cardiomyocytes, grown as a monolayer in dishes, were treated with the 10(-4) M carbachol, a mACh-R agonist, and the effects on phosphatidylcholine hydrolysis were ascertained in cells preincubated with [methyl-3H]choline for 18 h. Cells were washed, lysed, and subjected to thin-layer chromatography to separate [3H]choline in various metabolites of phosphatidylcholine. Carbachol significantly (p < 0.05) increased intracellular free choline and decreased cellular phospholipid consistent with phosphatidylcholine hydrolysis. Carbachol increased the amount of [3H]choline that effluxed out of the cardiomyocyte into the medium. Carbachol-induced choline efflux was not prevented by pretreatment with n-butanol, a phospholipase D inhibitor, suggesting that other lipases such as phospholipase C are the major enzyme involved in phosphatidylcholine hydrolysis. Pertussis toxin prevented carbachol-induced choline efflux and the changes in intracellular free choline and phospholipid. An action of carbachol through G proteins was supported by the ability of pertussis toxin to antagonize the carbachol-induced reduction in cAMP generation from isoproterenol. In summary, mACh-Rs, visualized in living cardiomyocytes, were peripheral to the nucleus. Phosphatidylcholine hydrolysis induced by mACh-R stimulation may be a signal transduction pathway for mACh-R in the cardiomyocyte, operating through inhibitory G proteins sensitive to pertussis toxin.
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PMID:Visualization of muscarinic cholinergic receptors on chick cardiomyocytes and their involvement in phosphatidylcholine hydrolysis. 925 Mar 60

The effects of ET-1 on contraction, Ca2+ transient and L-type Ca2+ current (ICa.L) were studied in single cells isolated from ventricles of guinea-pig hearts. The aim of our study was to elucidate the mechanism of the positive inotropic effect during endothelin receptor stimulation by focusing on the role of PKC. ET-1 at concentrations of 5 and 10 nM produced a biphasic pattern of inotropism: a first decrease in contraction by 34.4 +/- 2.5% of the control followed by a sustained increase in contraction by 66.6 +/- 8.4% (mean +/- SEM, n = 9). The Ca2+ transient decreased by 13.5 +/- 1.0% during the negative inotropic phase, while it increased by 58.1 +/- 8.4% (n = 10) during the positive inotropic phase. Using the whole-cell voltage-clamp technique with conventional microelectrodes, the application of ET-1 (5 nM) increased the ICa.L by 32.6 +/- 5.1% (n = 10), which was preceded by a short-lived decrease in ICa.L. Incubation of myocytes with pertussis toxin (PTX, at 2 micrograms/ml for > 3 h at 35 degrees C) failed to block the ET-1-induced enhancement of ICa.L. The increases in contraction, Ca2+ transient, and ICa.L by ET-1 were inhibited by pretreatment with 5-N-methyl-N-isobutyl amiloride (MIA; 10 microM), an amiloride analog, and a novel selective Na+/H+ exchange inhibitor HOE694 (10 microM). To determine whether activation of protein kinase C (PKC) is responsible for the enhancement of ICa.L by ET-1, we tested a PKC inhibitor, GF109203X, and found that it does exert an inhibitory effect on the ET-1-induced ICa.L increase. Our study suggests that during ET receptor stimulation an increase in ICa.L due to stimulation of Na+/H+ exchange via PKC activation causes an increase in Ca2+ transients and thereby in the contractile force of the ventricular myocytes.
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PMID:Effects of endothelin-1 on Ca2+ signaling in guinea-pig ventricular myocytes: role of protein kinase C. 1019 93

A randomized, double-blind, placebo-controlled trial was conducted to evaluate the effect of simultaneous vitamin A supplementation and diphtheria, pertussis and tetanus (DPT) vaccination on the antibody levels. Infants aged 6-17 wk (n = 56) were randomly given 15 mg oral vitamin A or placebo at the time of their DPT immunization. Three such doses were given at monthly intervals. Immunoglobulin (Ig) G antibodies to diphtheria, pertussis and tetanus were assayed on enrollment and 1 mo after the third dose. Baseline antibody concentrations to diphtheria, pertussis and tetanus did not differ between the vitamin A-supplemented and placebo-treated groups. The postdose antibody to diphtheria level was significantly greater in the vitamin A than in the placebo-treated group. The geometric mean +/- SEM antibody levels (mg/L) were 22.9 +/- 1.2 and 11.0 +/- 1.3 in the vitamin A and placebo groups, respectively (P = 0.029). The postsupplementation concentrations of antibodies to pertussis and tetanus did not differ between the two groups. These results suggest that antibody response to diphtheria vaccination was potentiated by simultaneous vitamin A administration and DPT immunization.
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PMID:Simultaneous vitamin A administration at routine immunization contact enhances antibody response to diphtheria vaccine in infants younger than six months. 1057 48

The majority of the biological effects of pertussis toxin (PT) are the result of a toxin-catalyzed transfer of an adenosine diphosphate-ribose (ADP-ribose) moiety from NAD(+)to the alpha-subunits of a subset of signal-transducing guanine-nucleotide-binding proteins (G-proteins). This generally leads to an uncoupling of the modified G-protein from the corresponding receptor and the loss of effector regulation. This assay is based on the PT S1 subunit enzymatic transfer of ADP-ribose from NAD to the cysteine moiety of a fluorescent tagged synthetic peptide homologous to the 20 amino acid residue carboxyl-terminal sequence of the alpha-subunit of the G(i3)protein. The tagged peptide and the ADP-ribosylated product were characterized by HPLC/MS and MS/MS for structure confirmation. Quantitation of this characterized ADP-ribosylated fluorescently tagged peptide was by HPLC fluorescence using Standard Addition methodology. The assay was linear over a five hr incubation period at 20 degrees C at PT concentrations between 0.0625 and 4.0 microg/ml and the sensitivity of the assay could be increased several fold by increasing the incubation time to 24 h. Purified S1 subunit of PT exhibited 68.1+/-10.1% of the activity of the intact toxin on a molar basis, whereas the pertussis toxin B oligomer, the genetically engineered toxoid, (PT-9K/129G), and several of the other components of the Bordetella pertussis organism possessed little (<0.6%) or no detectable ribosylation activity. Commonly used pertussis vaccine reference materials, US PV Lot #11, BRP PV 66/303, and BRP PV 88/522, were assayed by this method against Bordetella pertussis Toxin Standard 90/518 and demonstrated to contain, respectively, 0.323+/-0.007, 0.682+/-0.045, and 0.757+/-0.006 microg PT/ml (Mean+/-SEM) or in terms of microg/vial: 3.63, 4.09 and 4.54, respectively. A survey of several multivalent pertussis vaccine products formulated with both whole cell as well as acellular components indicated that products possessed a wide range of ribosylation activities. The pertussis toxin S1 subunit catalyzed ADP- ribosylation of the FAC-Galpha(i3)C20 peptide substrate and its subsequent quantitation by HPLC was demonstrated to be a sensitive and quantitative method for measuring intrinsic pertussis toxin activity. This methodology not only has the potential to be an alternative physicochemical method to replace existing bioassay methodology, but has the added advantage of being a universal method applicable to the assay of pertussis toxin in both whole cell and acellular vaccines as well as bulk and final formulated vaccine products. Acceptance of this method by regulatory agencies and industry as a credible alternative to existing methods would, however, require validation in an international collaborative study against the widely accepted bioassay methods.
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PMID:A quantitative analysis for the ADP-ribosylation activity of pertussis toxin: an enzymatic-HPLC coupled assay applicable to formulated whole cell and acellular pertussis vaccine products. 1158 Feb 13

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