NG-Nitro-L-arginine Methyl Ester-Induced Potentiaton of the Effect of Aminophylline on Rat Diaphragm: the Role of Extracellular Calcium

We have undertaken total synthesis of the Saccharomyces cerevisiae a-factor (NH2-YIIKGVFWDPAC[S-farnesyl]-COOCH3) and several Cys-12 analogs to determine the significance of S-farnesylation and carboxy-terminal methyl esterification to the biological activity of this lipopeptide mating pheromone. Replacement of either the farnesyl group or the carboxy-terminal methyl ester by a hydrogen atom resulted in marked reduction but not total loss of bioactivity as measured by a variety of assays. Moreover, both the farnesyl and methyl ester groups could be replaced by other substituents to produce biologically active analogs. The bioactivity of a-factor decreased as the number of prenyl units on the cysteine sulfur decreased from three to one, and an a-factor analog having the S-farnesyl group replaced by an S-hexadecanyl group was more active than an S-methyl a-factor analog. Thus, with two types of modifications, a-factor activity increased as the S-alkyl group became bulkier and more hydrophobic. MATa cells having deletions of the a-factor structural genes (mfal1 mfa2 mutants) were capable of mating with either sst2 or wild-type MAT alpha cells in the presence of exogenous a-factor, indicating that it is not absolutely essential for MATa cells to actively produce a-factor in order to mate. Various a-factor analogs were found to partially restore mating to these strains as well, and their relative activities in the mating restoration assay were similar to their activities in the other assays used in this study. Mating was not restored by addition of exogenous a-factor to a cross of a wild-type MAT alpha strain and a MATaste6 mutant, indicating a role of the STE6 gene product in mating in addition to its secretion of a-factor.

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MO092THE ROLE OF CALCIUM IN UROMODULIN EXPRESSION AND SECRETION FROM RENAL MEDULLARY EPITHELIAL CELLS OF HYPERTENSIVE AND NORMOTENSIVE RATS

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfab106.001 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

Philipp Boder ◽

Sheon Mary ◽

Lesley Graham ◽

Christian Delles

Keyword(s):

Blood Pressure ◽

Epithelial Cells ◽

Mrna Level ◽

Extracellular Calcium ◽

Female Rats ◽

Thick Ascending Limb ◽

Molecular Signaling ◽

Extracellular Secretion ◽

Wistar Kyoto

Abstract Background and Aims Uromodulin (UMOD) is the most abundantly secreted protein found within the urine, primarily produced by medullary thick ascending limb (mTAL) epithelial cells of the kidneys. There is accruing genetic evidence implicating UMOD in blood pressure regulation and consequently hypertension. The molecular signaling induced by calcium in the kidney and its influence on blood pressure are not well understood. The aim of this study was to investigate the potential role of extracellular calcium and the calcium-sensing receptor (CaSR) in mTAL on UMOD production and secretion in TAL cells with the hope of defining novel clinical targets for the treatment of hypertension. Method Kidneys were harvested from normotensive Wistar-Kyoto (WKY) and stroke-prone spontaneously hypertensive (SHRSP) female rats. To determine the effect of extracellular calcium on UMOD secretion, mTAL tubules were incubated in media with and without 1mM calcium, nifedipine (10µM), NPS2143 (1 or 5 µM) and spermine (2mM). Extracellular and intracellular UMOD protein levels were detected by Western blot. Gene expression of Umod was determined by qRT-PCR. Results Calcium increased mTAL tubule UMOD secretion in WKY and SHRSP. Nifedipine slightly decreased UMOD secretion in WKY without calcium. In both strains, NPS2143 increased calcium-induced UMOD secretion, with an enhanced effect in SHRSP. Stimulation of CaSR with spermine decreased UMOD secretion in WKY. Analysis of intracellular UMOD levels in these conditions demonstrated increased accumulation when extracellular secretion was low, and vice versa. Incubation of primary mTAL cells with calcium confirmed increased localisation of UMOD at the membrane compared to the cytosol, without any major differences in cell morphology. The Umod mRNA level changes were not statistically significant among conditions. Conclusion Trafficking of UMOD in the mTAL is influenced by the type of CaSR ligand and the biased nature of G-protein coupled CaSR signalling. Unravelling the signalling events post-calcium will be necessary for identification of key regulators of UMOD secretion and provide new sites for therapeutic intervention in hypertension.

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Role of calcium channels and protein kinase C for release of norepinephrine and neuropeptide Y

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1990.259.5.r925 ◽

1990 ◽

Vol 259 (5) ◽

pp. R925-R930

Author(s):

M. Haass ◽

C. Forster ◽

G. Richardt ◽

R. Kranzhofer ◽

A. Schomig

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Calcium Channels ◽

Calcium Channel ◽

Neuropeptide Y ◽

Nerve Fibers ◽

Extracellular Calcium ◽

Minor Effect ◽

Kinase C

The role of calcium for the release of norepinephrine (NE, determined by high-pressure liquid chromatography) and neuropeptide Y (NPY, determined by radioimmunoassay) was investigated in guinea pig perfused hearts with intact sympathetic innervation. In the presence of extracellular calcium (1.85 mM), electrical stimulation of the left stellate ganglion (12 Hz, 1 min) induced a closely related release of NE and NPY with the molar ratio of approximately 400-600 (NE) to 1 (NPY). The stimulation-evoked overflow of both transmitters was dependent from the extracellular calcium concentration and was almost completely suppressed by calcium-free perfusion. The corelease of both transmitters was not affected by the L-type calcium channel blocker felodipine (1-10 microM). However, the overflow of NE and NPY was markedly attenuated by the unselective calcium antagonist flunarizine (1-10 microM) and completely prevented by the neuronal (N-type) calcium channel blockers omega-conotoxin (1-100 nM) and cadmium chloride (10-100 microM), indicating a key role for N-type calcium channels in the exocytotic release of transmitters from cardiac sympathetic nerve fibers. Possibly due to unspecific actions, such as interference with sodium channels or uptake1-blocking properties, the phenylalkylamines verapamil (0.01-10 microM) and gallopamil (1-10 microM) reduced NPY overflow with only a minor effect on NE overflow. The stimulation-induced transmitter release was increased up to twofold by activation of protein kinase C (phorbol 12-myristate 13-acetate, 3 nM-3 microM) and completely suppressed by inhibition of protein kinase C (polymyxin B, 100 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

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Free radicals in hypoxic rat diaphragm contractility: no role for xanthine oxidase

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.2001.281.6.l1402 ◽

2001 ◽

Vol 281 (6) ◽

pp. L1402-L1412 ◽

Cited By ~ 22

Author(s):

Leo M. A. Heunks ◽

Herwin A. Machiels ◽

Ronney de Abreu ◽

Xiao Ping Zhu ◽

Henricus F. M. van der Heijden ◽

...

Keyword(s):

Free Radicals ◽

Xanthine Oxidase ◽

Power Output ◽

Harmful Effect ◽

Force Generation ◽

Muscle Performance ◽

Rat Diaphragm ◽

Shortening Velocity ◽

Contraction Pattern

Recent evidence indicates that hypoxia enhances the generation of oxidants. Little is known about the role of free radicals in contractility of the rat diaphragm during hypoxia. We hypothesized that antioxidants improve contractility of the hypoxic rat diaphragm and that xanthine oxidase (XO) is an important source of free radicals in the hypoxic diaphragm. The effects of N-acetylcysteine (NAC; 18 mM), Tiron (10 mM), and the XO inhibitor allopurinol (250 μM) were studied on isometric and isotonic force generation during hypoxia (Po 2 ∼7 kPa). NAC and Tiron decreased maximal force generation, slowed the shortening velocity, and decreased the power output. Fatigue rate was decreased in the presence of either NAC or Tiron. Allopurinol did not alter the contractility or fatigability of the diaphragm. During hyperoxia (Po 2 ∼85 kPa), neither NAC nor allopurinol affected the contractility or fatigability of the diaphragm. Thus free radicals play a significant role in diaphragm contractility during hypoxia. Whether antioxidants exert a beneficial or harmful effect on muscle performance depends on the contraction pattern of the muscle. Free radicals generated by XO do not play a role in diaphragm contractility during either hypoxia or hyperoxia.

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Significance of C-terminal cysteine modifications to the biological activity of the Saccharomyces cerevisiae a-factor mating pheromone.

Molecular and Cellular Biology ◽

10.1128/mcb.11.7.3603 ◽

1991 ◽

Vol 11 (7) ◽

pp. 3603-3612 ◽

Cited By ~ 74

Author(s):

S Marcus ◽

G A Caldwell ◽

D Miller ◽

C B Xue ◽

F Naider ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Biological Activity ◽

Methyl Ester ◽

Total Synthesis ◽

Biologically Active ◽

Structural Genes ◽

Wild Type ◽

Mating Pheromone ◽

Carboxy Terminal

We have undertaken total synthesis of the Saccharomyces cerevisiae a-factor (NH2-YIIKGVFWDPAC[S-farnesyl]-COOCH3) and several Cys-12 analogs to determine the significance of S-farnesylation and carboxy-terminal methyl esterification to the biological activity of this lipopeptide mating pheromone. Replacement of either the farnesyl group or the carboxy-terminal methyl ester by a hydrogen atom resulted in marked reduction but not total loss of bioactivity as measured by a variety of assays. Moreover, both the farnesyl and methyl ester groups could be replaced by other substituents to produce biologically active analogs. The bioactivity of a-factor decreased as the number of prenyl units on the cysteine sulfur decreased from three to one, and an a-factor analog having the S-farnesyl group replaced by an S-hexadecanyl group was more active than an S-methyl a-factor analog. Thus, with two types of modifications, a-factor activity increased as the S-alkyl group became bulkier and more hydrophobic. MATa cells having deletions of the a-factor structural genes (mfal1 mfa2 mutants) were capable of mating with either sst2 or wild-type MAT alpha cells in the presence of exogenous a-factor, indicating that it is not absolutely essential for MATa cells to actively produce a-factor in order to mate. Various a-factor analogs were found to partially restore mating to these strains as well, and their relative activities in the mating restoration assay were similar to their activities in the other assays used in this study. Mating was not restored by addition of exogenous a-factor to a cross of a wild-type MAT alpha strain and a MATaste6 mutant, indicating a role of the STE6 gene product in mating in addition to its secretion of a-factor.

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Role of NO in goat basal cerebral circulation and after vasodilatation to hypercapnia or brief ischemias

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1993.265.6.r1410 ◽

1993 ◽

Vol 265 (6) ◽

pp. R1410-R1415 ◽

Cited By ~ 1

Author(s):

G. Dieguez ◽

J. L. Garcia ◽

N. Fernandez ◽

A. L. Garcia-Villalon ◽

L. Monge ◽

...

Keyword(s):

Nitric Oxide ◽

Heart Rate ◽

Methyl Ester ◽

Cerebral Circulation ◽

Systemic Arterial Pressure ◽

Nitric Oxide Production ◽

Hemodynamic Effects ◽

Arterial Pco2 ◽

Different Levels

The role of nitric oxide (NO) in the cerebral circulation under basal conditions and after vasodilatation to hypercapnia or reactive hyperemias was studied in 17 anesthetized goats. The intravenous administration of NG-nitro-L-arginine methyl ester (L-NAME, 3-4 or 8-10 mg/kg), an inhibitor of nitric oxide production, reduced middle cerebral artery (MCA) flow (electromagnetically measured) by 19 and 30% and increased systemic arterial pressure by 21 and 26%, respectively, whereas heart rate did not significantly change; MCA resistance increased by 48 and 86%, respectively. These hemodynamic effects were reversed by L-arginine (200-300 mg/kg iv; 5 goats). Different levels of hypercapnia (PCO2 of 30-35, 40-45, and 55-65 mmHg) (12 goats) produced arterial PCO2-dependent increases in MCA flow that were similar under control and L-NAME treatment. Graded cerebral hyperemia occurred after 5, 10, and 20 s of MCA occlusion in 5 goats, but its magnitude was decreased during L-NAME treatment. It suggests that, in the cerebral circulation, nitric oxide 1) produces a basal vasodilator tone and 2) is probably not involved in the vasodilatation to hypercapnia but may mediate hyperemic responses after short brain ischemias.

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