Receptor-mediated modulation of avian caecal muscle contraction by melatonin: role of tyrosine protein kinase.

Abstract: Melatonin receptors in the quail caecum were studied by 2[125I]iodomelatonin binding assay and the involvement of tyrosine protein kinase in the melatonin-induced contraction was explored. The binding of 2[125I]iodomelatonin in the quail caecum membrane preparations was saturable, reversible and of high affinity with an equilibrium dissociation constant (Kd) of 24.6 +/- 1.1 pm (n = 7) and a maximum number of binding sites (Bmax) of 1.95 +/- 0.09 fmol (mg/protein) (n = 7). The relative order of potency of indoles in competing for 2[125I]iodomelatonin binding was: 2-iodomelatonin > melatonin > 2-phenylmelatonin > 6-chloromelatonin > 6-hydroxymelatonin > N-acetylserotonin, indicating that ML(1) receptors are involved. The binding was inhibited by Mel1b melatonin receptor antagonists, luzindole and 4-phenyl-2-propionamidotetralin (4-P-PDOT) as well as by non-hydrolyzable analogs of GTP like GTPgammaS and Gpp(NH)p but not by adenosine nucleotides. The latter suggests that the action of melatonin on the caecum is G-protein linked. Cumulative addition of melatonin (1-300 nM) potentiated both the amplitude and frequency of spontaneous contractions in the quail caecum. The potentiation of rhythmic contractions was blocked by both luzindole and 4-P-PDOT. Antagonists of tyrosine kinase, genistein(2 microM) and erbstatin(4 microM) suppressed the modulation of spontaneous contractions by melatonin, but not inhibitors of protein kinase C (PKC) or protein kinase A (PKA). Melatonin-induced increment in spontaneous contraction was blocked by nifedipine (0.4 nM). Thus, we suggest that melatonin potentiates spontaneous contraction in the quail caecum via interacting with G-protein-coupled Mel(1b) receptor which may activate L-type Ca2+ channels by mobilizing tyrosine kinases.

L-NAME inhibits Mg(2+)-induced rat aortic relaxation in the absence of endothelium.

1 L-NG-nitro-arginine methyl ester (L-NAME; 100 microM), a nitric oxide synthase (NOS) inhibitor, reversed the relaxation induced by 3 microM acetylcholine (ACh) and 2-10 mM Mg2+ in endothelium-intact (+E) rat aortic rings precontracted with 1 microM phenylephrine (PE). In PE-precontracted endothelium-denuded (-E) rat aorta, 3 microM ACh did not, but Mg2+ caused relaxation which was reversed by L-NAME, but not by D-NAME. 2 The concentration response profiles of L-NAME in reversing the equipotent relaxation induced by 5 mM Mg2+ and 0.2 microM ACh were not significantly different. 3 L-NAME (100 microM) also reversed Mg(2+)-relaxation of -E aorta pre-contracted with 20 mM KCl or 10 microM prostaglandin F2alpha (PGF2alpha). L-NG-monomethyl-arginine (L-NMMA; 100 microM) was also effective in reversing the Mg(2+)-relaxation. 4 Addition of 0.2 mM Ni2+, like Mg2+, caused relaxation of PE-pre-contracted -E aorta, which was subsequently reversed by 100 microM L-NAME. 5 Reversal of the Mg(2+)-relaxation by 100 microM L-NAME in PE-precontracted -E aorta persisted following pre-incubation with 1 microM dexamethasone or 300 microM aminoguanidine (to inhibit the inducible form of NOS, iNOS). 6 Pretreatment of either +E or -E aortic rings with 100 microM L-NAME caused elevation of contractile responses to Ca2+ in the presence of 1 microM PE. 7 Our results suggest that L-NAME exerts a direct action on, as yet, unidentified vascular smooth muscle plasma membrane protein(s), thus affecting its reactivity to divalent cations leading to the reversal of relaxation. Such an effect of L-NAME is unrelated to the inhibition of endothelial NOS or the inducible NOS.

Effect of red blood cells and hemoglobin on spontaneously hypertensive and normotensive rat aortas.

Experiments were designed to compare the contractile effect of red blood cells (RBC) on aortic rings with and without endothelium from normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. Red blood cells of 4 week old WKY and SHR rats induced a negligible increase in tension of aortic rings, either with or without endothelium, being slightly more effective in SHR rats. However, red blood cells of 16 week old rats increased tension of WKY and SHR aortic rings, with endothelium at this age being more pronounced then red blood cells in 4 week old animals. The contractions induced by WKY and SHR red blood cells both in WKY and SHR aortic rings without endothelium at this age are significantly greater compared to the effect on aortic rings with endothelium. Red blood cell ghosts of rats of both strains increased the tension of the rings without endothelium of SHR aorta to near 50% of those induced by red blood cells, whereas they were ineffective in aortic rings without endothelium of WKY rats. Oxyhemoglobin increased the tension of 16 week SHR aortic rings both with and without endothelium, whereas the effect on the rings of WKY rats was negligible. This increase in tension was inhibited by BM 13505, nordihydroguaiaretic acid, and indomethacin in SHR rings both with and without endothelium, demonstrating an eicosanoid involvement in oxyhemoglobin-induced contractions. Hemoglobin or its metabolites may be involved in development or in maintenance of spontaneous hypertension.

Protein kinase C plays no role in KCl-induced vascular contraction in Ca(2+)-free medium.

AIM: To examine the role of protein kinase C (PKC) on the sustained contractile responses of rat aorta to high KCl in isotonic Ca(2+)- and Mg(2+)-free solutions. METHODS: The effects of phorbol 12-myristate 13-acetate (PMA, a PKC activator) and Calphostin C (a selective PKC inhibitor) were observed on the sustained contraction of rat aorta induced by K+ 136 mmol.L-1. EGTA (100 mumol.L-1) was added to prepare the Ca(2+)-free medium and EDTA (100 mumol.L-1) was added to reduce or remove the Mg2+. RESULTS: Aortic contraction to KCl was prominent in low Mg2+ medium and was enhanced by EDTA (K-EDTA contraction). Such contraction was concentration-dependently inhibited by Mg2+, but was not affected by Calphostin C 1 mumol.L-1. Pretreatment of the aortic preparations with PMA (0.8 mumol.L-1) potentiated the contraction to KCl in Ca(2+)-free, low Mg2+ medium and higher concentration of Mg2+ was required to cause relaxation. Such a reduced sensitivity to Mg2+ in the presence of PMA was partially reversed by Calphostin C and was accompanied by an increased sensitivity to Ca2+, which concentration-dependently caused contraction following Mg(2+)-induced relaxation. However, in the presence of EDTA 100 mumol.L-1 (eg, Mg(2+)-free medium), the maximal contraction to KCl in Ca(2+)-free medium was not affected by PMA or Calphostin C. CONCLUSION: KCl-induced contraction in Ca(2+)-free and Mg(2+)-free + EDTA 100 mmol.L-1 medium was not affected by PMA or Calphostoin C, indicating that PKC plays no role in such contractile responses.

A revisitation on the mechanism of action of KCl-induced vascular smooth muscle contraction: a key role of cation binding to the plasma membrane.

Aortic rings of Sprague-Dawley rats developed reproducible contractions in Ca-free, nominally Mg-free (2 microM), Na-free, K solution, but not in Ca-free, nominally Mg-free, K-free, Na solution. Further reduction of the residual Mg by 0.5 mM EDTA in the above solutions potentiated this K-induced contraction and allowed the development of a relatively small contraction in Na-rich medium, respectively. The amplitude of EDTA-enhanced, K-induced contraction was almost the same as the amplitude of contraction induced in K-rich ([K] = 142 mM), Ca-containing (2.5 mM) solution. Such K-induced contraction was completely inhibited by 38 mM Na and by 3 mM of Mg (and Ni or Cd). Nifedipine (1 microM) also inhibited aortic smooth muscle contraction produced in K-EDTA, Ca-free solution. Modulators of Ca in sarcoplasmic reticulum (ryanodine, caffeine and norephinephrine), Ca-ionophore (A-23187) and protein kinase C inhibitor (Calphostin C) had no effect on EDTA-enhanced, K-induced contraction. It was suggested that K-induced contraction in rat aorta is not dependent on the increase in cytosolic Ca following membrane depolarization, is not a result of the release of Ca from intracellular stores, and is not due to change of Ca sensitivity upon the activation of protein kinase C. We propose that the competition of K for Mg and Na at external binding sites on the plasma membranes of the smooth muscle cells is primarily responsible for the development of vascular contraction.

Regulation of vascular contraction by ionic matrix surfaces of the smooth muscle cell: changes in SHR?

1. KCl-induced contractions in aortic rings of spontaneously hypertensive rats (SHR) and two normotensive strains, Sprague-Dawley (SD) and Wistar-Kyoto (WKY) rats, were studied. 2. The contraction elicited by isotonic Na-free, KCl solution containing 2.5 mmol/L Ca and 1 mmol/L Mg was concentration-dependently inhibited by Na and K. Maximal inhibition was induced by 20 mmol/L NaCl in WKY and SHR (65 and 85%, respectively) and by 60 mmol/L NaCl in SD (50%). KCl (10-40 mmol/L) had no effect on SD but produced almost full relaxation in WKY and about 50% relaxation in SHR. 3. Isotonic Ca-free, Na-free high Mg (2 mmol/L), K-solution caused strong phasic contractions in WKY and SHR, but weak responses in SD. 4. Aortic rings of SD, WKY and SHR developed strong, sustained and reproducible contractions in isotonic Ca-free, Na-free, K-solution containing EDTA, which were fully relaxed by 1.2, 1.6 and 8 mmol/L Na, respectively. 5. We suggest that the primary mechanism of KCl-contraction is potential-independent competition between monovalent and divalent cations for binding sites on both surfaces of the plasma membrane (e.g. K with Mg and Na with Ca) and that differences in aortic contractility in SHR, WKY and SD merely reflect strain differences in the composition of the ionic matrix, independent of hypertension.

[Effects of La3+ on calcium binding to erythrocyte cytoskeleton]. / Vliianie La3+ na sviazyvanie kal'tsiia s tsitoskeletom éritrotsitov.

When the whole erythrocytes were exposed to LaCl3, A--23187, ionomycin, orthovanadate and saponin, there was Ca2+ binding only following La3+ treatment of the cells. The binding was evident at a wide range (0.1 microM--1.OmM) of La3+ concentrations. Iodoacetamide-induced (incubation for 3 hours, 37 degrees C) decrease in erythrocyte ATP levels was found to result in a 3-fold reduction in Ca2+ binding to the cytoskeleton. La(3+)-induced Ca2+ binding enhanced the incorporation of 14C-glucose and/or its metabolites into the red cell skeleton. Thus, the detected new type of Ca2+ binding to the cytoskeleton of human and rat erythrocytes is likely to be due to the cumulative process: direct binding of La3+ to the outer surface of a membrane and the metal-induced trigger of nucleotide--dependent intracellular process.

[Calcium binding to the erythrocyte cytoskeleton in spontaneously hypertensive rats]. / Sviazyvanie kal'tsiia c tsitoskeletom éritrotsitov krys so spontannoi gipertenziei.

The paper outlines a new La(3+)-induced mechanism of Ca2+ binding to rat red blood cells. Other compounds elevating intracellular Ca2+ concentrations, such as A-23187, ionomycin, and orthovanadate, and some activators of phosphotransferases (TPA, dibutyryl cAMP) fail to initiate the binding. Inhibitors of calmodulin-dependent processes, such as calmidazolium and trifluoroperazine, diminish La-dependent Ca2+ binding. La(3+)-induced Ca2+ binding is followed by an increased cytoskeleton incorporation rate of 14C metabolites of glucose. The rate of Ca2+ binding and 14C metabolite incorporation was 30% and 80% higher, respectively, but Ca+ binding capacity was increased by 45% in SHR as compared to WKY rats. Due to the fact that La3+ enhances the phosphorylation of proteins with molecular masses of 14, 36, and 58 kD, it is suggested that Ca2+ binding alterations should be determined by changes in phosphorylation and glucose metabolic disturbances in spontaneously hypertensive rats.

[Protein phosphorylation during mast cell secretion in radioprophylaxis]. / Fosforilirovanie belkov v protsesse sekretsii tuchnykh kletok v usloviiakh radioprofilaktiki.

A study was made of the role of protein phosphorylation of mast cells and their cytoskeleton upon secretion induced by biogenic amines (histamine and serotonin) and bradykinin, a possible mediator of the effect of MEA, a sulfur-containing radioprotector. The data obtained indicate that the incorporation of phosphate in some proteins of mast cells is an important stage in the process of exocytosis during radioprophylaxis. Cytoskeletal proteins were shown to be involved in mast cell secretion.

Protein kinase C activity in erythrocytes in primary hypertension: regulation of cell shape and cation transport.

Protein kinase C activity in the lysate of erythrocytes of patients with essential hypertension (EH) and spontaneously hypertensive rats (SHR) was found to be increased by 1.6-2.0 times as compared with normotensive controls. Membrane cytoskeleton alterations observed in the erythrocytes of patients with EH and SHR were revealed in decreased average erythrocyte volume, increase of cup-shaped cell formation, and increase of basal phosphorylation of band 4.9 protein. In addition, the rate of Na(+)-H+ exchange in erythrocytes of EH patients and SHR was increased by 1.9-fold. In vitro treatment of erythrocytes of healthy donors and Wistar-Kyoto rats (WKY) with protein kinase C activator (12-O-tetradecanoylphorbol-13-acetate) leads to similar changes of cell shape, cell volume, band 4.9 protein phosphorylation and Na(+)-H+ exchange, as well as to an increase of diS-C3-(5) fluorescence. It may be assumed that alterations of these parameters revealed in primary hypertension are caused by increased activity of protein kinase C.

[The role of protein phosphorylation in mast cell secretion, stimulated by concanavalin A. Connection to the cytoskeleton]. / Rol' fosforilirovaniia belkov v sekretsii tuchnykh kletok, stimulirovannykh konkavalinom A. sviaz' s tsitoskeletom.

The role of cytoskeleton and protein phosphorylation in concanavalin A and phorbol ester (PMA) induced mast cell secretion was investigated. It was shown that the receptor coupled with lectin interacts with the cytoskeleton. When the ligand-receptor complex is formed, an increased phosphorylation of some proteins is induced. The same proteins are phosphorylated under the influence of PMA, a protein kinase C activator, thus suggesting that protein kinase C is involved in the regulation of mast cell exocytosis. The results obtained testify to the effect that the mechanism of mast cell degranulation induced by concanavalin A is due to modification of the cytoskeleton.

[Calcium transport in brain synaptosomes during depolarization. The role of potential-dependent channels and Na+/Ca2+ metabolism]. / Transport kal'tsiia v sinaptosomakh mozga pri depoliarizatsii. Rol' potentsialzavisimykh kanalov i Na+/Ca2+-obmena.

The contribution of Ca2+ channels and Na+/Ca2+ exchange to Ca2+ uptake in rat brain synaptosomes upon long- (t greater than or equal to 30 s) and short-term (t less than 30 s) depolarization by high K+ was studied by measuring the 45Ca content and free Ca2+ concentration (from Quin-2 fluorescence). At 37 degrees C, the system responsible for the K+-stimulated uptake of 45Ca (t greater than or equal to 30 s) and the Na+/Ca+ exchanger are characterized by a similar concentration dependence of external Ca2+ (Ca0(2+] and K0+ as well as by an equal sensitivity to verapamil (Ki = approximately 20-40 microM) and La2+ (Ki = approximately 50 microM). These data and the results from predepolarization suggest that the 45Ca entry into synaptosomes at t greater than or equal to 30 s is due to the activation of Na+/Ca+ exchange caused by its electrogenic component, while the insignificant contribution of Ca2+ channels can be accounted for by their inactivation. At low temperatures (2-4 degrees C) which decelerate the inactivation, the initial phase of 45Ca uptake is fully provided for by Ca2+ channels, showing a lower (as compared to the exchanger) affinity for Ca0(2+) (K0.5 greater than 1 mM)m a greater sensitivity to La3+ (Ki = approximately 0.2-0.3 microM) and verapamil (Ki = approximately 2-3 microM); these channels are fully inactivated by predepolarization with K0+, ouabain and batrachotoxin. The Ca2+ channels can be related to T-type channels, since they are not blocked by nicardipine and niphedipine.

[Protein kinase C activity in the brain tissue of spontaneously hypertensive rats]. / Aktivnost' proteinkinazy C v tkani golovnogo mozga krys so spontannoigipertenziei.

Protein kinase C activities in the brain tissue of spontaneously hypertensive rats (SHR) and normotensive control rats (WKY) were studied. Protein kinase C activity in SHR was found to be 35% higher than that in normotensive control rats. It is suggested that the increase in protein kinase C activity is involved in the mechanism of membrane alterations in primary hypertension.

Activity of protein kinase C in erythrocytes in primary hypertension.

The activity of protein kinase C and A was studied in the erythrocytes of patients with essential hypertension (EH) and in spontaneously hypertensive rats (SHR, Okamoto-Aoki strain). Protein kinase C activity was also studied in the erythrocytes of patients with hypertension of renal origin. Protein kinase C activity in the lysate of erythrocytes of patients with EH and in SHR was found to be increased 1.6-2.0-fold as compared to that in normotensive controls. No notable differences in protein kinase A activity were observed between hypertensive and normotensive groups. In erythrocytes of patients with renal hypertension, no notable changes in protein kinase C activity were revealed.

Effect of protein kinase C activation on cytoskeleton and cation transport in human erythrocytes. Reproduction of some membrane abnormalities revealed in essential hypertension.

Certain manifestations of alterations of membrane cytoskeleton, protein kinase C activity, and ion transport were revealed in erythrocytes of patients with essential hypertension: 1) the average volume of erythrocytes is reduced by 4%; 2) about 7% of the total number of erythrocytes is represented by cup-shaped forms compared with 1.5 to 3.0% in the control group; 3) basal phosphorylation of Band 4.9 protein is increased 1.6-fold to 1.8-fold; 4) activity of protein kinase C is increased by 60 to 70%; 5) the rate of proton electrochemical gradient (delta mu H+)-induced Na+-H+ exchange is increased twofold. Treatment of erythrocytes of healthy donors with protein kinase C activator (12-O-tetradecanoylphorbol-13-acetate) leads to similar but more marked changes in cell shape (17% of cup-shaped forms), volume reduction (by 7%), an increase of Band 4.9 protein phosphorylation (threefold), and an increase in the rate of Na+-H+ exchange (fourfold). Protein kinase activation does not modify Na+-Li+ exchange and slightly increases (by 20-50%) Na+-K+ pump activity, Na+-K+ cotransport, and the rate of 45Ca influx. It may be assumed that the increase of protein kinase C activity is one of the most probable molecular mechanisms conditioning abnormalities of the membrane skeleton and Na+-H+ exchange in primary hypertension.

[Regulation of the shape and size of human erythrocytes by protein kinases A and C]. / Reguliatsiia formy i ob''ema éritrotsitov cheloveka proteinkinazami A i C.

The effect of 12-O-tetradecanoyl-phorbol-12-acetate (TPA) and dibutyryl-3,5-cAMP on the shape, volume and protein phosphorylation in human red blood cells (RBC) was studied. TPA (but not cAMP) reduced the average volume of RBC and their transformed definite pool in the cup-shaped form. The changes in the physical parameters were accompanied by an increase in the cytoskeleton protein phosphorylation. An additive effect of cAMP and TPA on the phosphorylation of bands 4.1 and 4.9 was established, thus indicating that distinct domains of amino acid residues were phosphorylated by these stimuli. It was concluded that protein kinase C regulates the shape and volume of human RBC. A model, in which the state of spectrin 4.1-actin and actin 4.9-myosin complexes define the shape and volume of RBC and thus influence ion transport, is proposed.

[Protein kinase A and C activity of erythrocytes in patients with essential hypertension]. / Aktivnost' proteinkinaz A i C v éritrotsitakh bol'nykh s éssentsial'noi gipertenziei.

Activities of protein kinases A and C in erythrocyte cytoplasmic fraction purified by CM-Sephadex and DEAE-cellulose have been measured. Protein kinase C activity is shown to be 1.6-1.8-fold higher, as compared to controls, in essential hypertension, but remain unchanged in renal hypertension. Protein kinase A activity is slightly elevated in patients with essential hypertension, but the difference is not significant. It is suggested that the increase of protein kinase C activity, and perhaps some other activities as well, in essential hypertension may be a result of altered expression of protooncogenes with protein kinase activities.

[Changes in erythrocyte volume in relation to characteristics of protein phosphorylation of the membrane cytoskeleton in hypertension: role of protein kinase C]. / Izmenenie ob'ema éritrotsitov v sviazi s osobennostiami fosforilirovaniia belkov membrannogo tsitoskeleta pri gipertonicheskoi bolezni: rol' proteinkinazy C.

Shape, volume and protein phosphorylation of RBC were studied in normal subjects and patients with essential hypertension. Mean volume of RBC of hypertensive patients was reduced by 3-4%, as compared to the control group. The level of band 4.9 phosphorylation was simultaneously increased in RBC of hypertensive patients. Activation of protein kinase C with phorbol ester produced volume reduction and shape changes in RBC. These changes are accompanied with phosphorylation of bands 4.1 and 4.9. It is suggested that RBC volume reduction in essential hypertension may be related to an alteration in the regulation of cytoskeleton proteins phosphorylation.

[Limited proteolysis of prolactin in secretory granules from the pituitary gland of estrogenized rats]. / Ogranichennyi proteoliz prolaktina v sekretornykh granulakh iz gipofiza éstrogenizirovannykh krys.

The LTH-converting proteolytic activity in LTH granules isolated from estrogenized rat hypophysis was studied. Suspensions of granules were incubated at different values of pH for 4 hours at 37 degrees C. The reaction was controlled by SDS electrophoresis. Intensive proteolysis of LTH was observed at pH 6.0 and 3.9, which was accompanied by the formation of fragments with Mr 10, 12 and 17 kD and probably of smaller peptides. An inhibitory analysis revealed that the formation of the 17 kD fragment at pH 3.9 was partly and selectively inhibited by chloroquine, phenanthroline and phenylmethylsulfonyl fluoride. Pepstatin A fully inhibited the proteolysis, whereas leupeptin had no inhibiting influence. The data obtained testify to the presence in the granular fraction of the endopeptidase LTH-converting activity which is sensitive to pepstatin A, an aspartyl proteinase inhibitor as well as to chelators and a serine proteinase inhibitor.