Hypoxia inhibits the synthesis of phosphoinositides in the rabbit carotid body
Departamento de Bioquímica y Biologia Molecular y Fisiología, Instituto de Biología y Génetica Molecular (IBGM) CSIC, Facultad de Medicina, Universidad de Valladolid, E-47005 Valladolid, Spain.Pflügers Archiv - European Journal of Physiology (Impact Factor: 4.1). 06/1999; 437(6):839-45. DOI: 10.1007/s004240050853
Hypoxic transduction in the carotid body (CB) is regulated by several systems of second messengers, but the role of the phospholipase C system has not been studied. The aim of the present study was to characterize the turnover rate of inositol phosphates (InsPs) and phosphoinositides (PIs) and their modifications by hypoxia in the rabbit CB in vitro. In CBs, in which the PIs had been labelled previously with 3H-myo-inositol, hypoxia in the presence of LiCl did not modify the accumulation of 3H-InsPs, whilst exposure to hypoxia during the loading period in the presence of LiCl reduced the accumulation of 3H-InsPs by more than 50%. Endogenous levels of inositol 1,4,5-trisphosphate were unaltered by hypoxia. Synthesis of 3H-PIs from 3H-myo-inositol was markedly inhibited by hypoxia in the CB, but not in the rat superior cervical ganglion used as control tissue. Levels of 3H-phosphatidylinositol (3H-PtdIns), 3H-phosphatidylinositol 4-monophosphate and 3H-phosphatidylinositol 4,5-bisphosphate were similarly decreased, indicating that inhibition occurs at a step prior to PtdIns synthesis. It is concluded that the phospholipase C system of second messengers does not play a significant role in the short-term regulation of hypoxic transduction cascade. It can be speculated that the decrease in PI availability produced by hypoxia might be involved in the functional changes observed in the CB on chronic hypoxic exposure.
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ABSTRACT: The present study evaluated the effects of endothelin (ET) peptides on carotid sinus nerve (CSN) activity, catecholamine (CA) release, and second messenger signaling pathways in rabbit carotid bodies superfused in vitro, and in dissociated chemosensory type I cells. ET-1 (1.0 microM) and ET-3 (1.0 microM) did not alter basal CSN activity and CA release, but they potentiated nerve activity (P<0. 05) and CA release (P<0.05) evoked by hypoxia. Under basal conditions, ET-1 and ET-3 (1.0 microM each) elevated tissue cyclic AMP (cAMP) levels nearly 3-fold (P<0.001, ET-1; P<0.05, ET-3) and inositol phosphate (IP(n)) levels nearly 4-fold (P<0.01, ET-1). Hypoxia evoked an increase in carotid body cAMP, and this response was also potentiated in the presence of 1.0 microM ET-1 (P<0.01) or 1.0 microM ET-3 (P<0.001). Patch-clamp studies of isolated type I cells showed that 100 nM ET-1 elevated the peak amplitude of voltage-sensitive (L-type) Ca(2+)-currents by an average of 37.6% (P<0.001). Fluorescent Ca(2+)-imaging revealed that 100 nM ET-1 did not alter [Ca(2+)](i) under basal conditions, but that [Ca(2+)](i)-responses evoked by hypoxia were potentiated by 87% (P<0. 01). Our data indicate that ET augments chemoreceptor responses by activating second messenger signaling pathways which promote the phosphorylation of Ca(2+)-channel protein, thereby enhancing stimulus-evoked intracellular Ca(2+) levels.
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ABSTRACT: In light of recent observations that receptor-ligand binding and coupling are physiologically regulated, the present study examined the hypothesis that the direct effects of hypoxia on vascular contractility involve modulation of pharmacomechanical coupling via changes in agonist affinity and/or receptor density. Because the direct effects of hypoxia on vascular smooth muscle contractility can vary with age, we carried out these experiments using both fetal and adult arteries. In common carotid arteries from near-term fetal and adult sheep, hypoxia (PO(2) = 9-12 Torr for 30 min) reduced the maximum responses to potassium by 17.8 +/- 3.5% (fetus) and 20.5 +/- 2.2% (adult), significantly reduced the pD(2) for 5-HT in the fetus (7.01 +/- 0.1 to 6.3 +/- 0.2) but not the adult (6.1 +/- 0.1 to 6.0 +/- 0.1), and significantly reduced 5-HT-induced maximum contractions (as % maximum response to 120 mM K(+)) not in the fetus (from 114 +/- 7 to 70 +/- 10%, not significant) but only in the adult (from 83 +/- 15 to 25 +/- 7%, P < 0.05) arteries. Hypoxia significantly attenuated 5-HT binding affinity (pK(A), determined by partial irreversible blockade with phenoxybenzamine) in both fetal (from 6.5 +/- 0.2 to 6.0 +/- 0.2) and adult arteries (from 6.2 +/- 0. 2 to 5.7 +/- 0.1) and also decreased receptor density (fmol/mg protein, determined by competitive binding with ketanserin and mesulergine) in adult (from 18.3 +/- 1.1 to 10.9 +/- 1.0) but not in fetal (21.0 +/- 1.0 to 23.2 +/- 1.4) arteries. These results suggest that acute hypoxia modulates receptor-ligand binding via age-dependent modulation of agonist affinity and receptor density. These effects may contribute to hypoxic vasodilatation and help explain why the effects of hypoxia on vascular contractility differ between fetuses and adults.
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ABSTRACT: The presence, subcellular distribution, species specificity and possible hypoxic stimulus-induced translocation of classical protein kinase C (cPKC) isozymes were examined in the carotid body. Carotid bodies were dissected from cats exposed in vivo to normoxic or acute hypoxic conditions and from normoxic rats. For immunohistochemistry isoform-specific monoclonal antisera to PKCalpha, PKCbetaI, PKCbetaII and PKCgamma were used. The immunoreactivity was visualized by fluorescein isothiocyanate (FITC) labelling. FITC/Texas red double-labelled specimens for the cPKC isozymes/tyrosine hydroxylase were used to demonstrate the chemoreceptor cell localization of cPKC isozymes. The immunofluorescence was detected using laser scanning confocal image technology. The results showed expression of the PKCalpha and PKCgamma but not PKCbeta isoforms in the cytoplasm of carotid body chemoreceptor cells. The double labelling provided evidence for the chemoreceptor cell localization of the cPKC isoforms detected. The immunostaining was most intense in the periphery of the perikarya, the nuclear envelope and, occasionally, the nucleoplasm. No major differences were found in the immunolocalization of PKCalpha and PKCgamma under normoxic and hypoxic conditions or between species. However, the immunoreactivity tended to accumulate more in the peripheral cytoplasm and away from the nucleus in the hypoxic chemoreceptor cell. This study demonstrates the presence of classical protein kinase C enzymes in chemoreceptor cells. The intensity of the immunoreactivity may suggest a role for the classical protein kinase C signalling pathway in shaping the hypoxic response at the carotid body. However, this study failed to provide firm evidence of this.
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