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J C Stoclet

Publications and source records attributed to J C Stoclet.

At least 109 records · Page 6Linked to original sources
PubMed1989

Differential sensitivity to cardiotonic drugs of cyclic AMP phosphodiesterases isolated from canine ventricular and sinoatrial-enriched tissues.

A cardiac phosphodiesterase (PDE) which specifically hydrolyzes cAMP and is inhibited by cyclic GMP has been suggested to be the site of action of new cardiotonic drugs. To investigate the effect of inhibitors, canine cyclic nucleotide PDEs were isolated from left ventricle and from sinoatrial node-enriched tissue, using identical techniques. Four PDE forms could be chromatographically resolved from each tissue, including a peak I PDE (calmodulin-activated phosphodiesterase, CaM-PDE), a peak II PDE (cyclic GMP-stimulated phosphodiesterase, CGS-PDE) and a peak III PDE (specific for cyclic AMP). The latter was further fractionated into two forms: One was inhibited by cyclic GMP and by the platelet antiaggregant AAL 05 (CGI-PDE), and the second was insensitive to cyclic GMP and was inhibited by rolipram (ROI-PDE). Reference PDE inhibitors, isobutyl-1-methylxanthine (IBMX) and papaverine, nonselectively inhibited the four forms isolated from the two tissues. Cardiotonic drugs (CI 930, LY 181512, piroximone, enoximone, and SK&F 94120) selectively inhibited CGI-PDE from ventricular tissue but were poorly active on both CGI-PDE and ROI-PDE from the sinoatrial-enriched fraction. In contrast, milrinone inhibited CGI-PDEs and ROI-PDEs from both ventricular and sinoatrial tissues. These results are in good agreement with pharmacologic data in the literature on the positive chronotropic and inotropic effects of the studied drugs in the dog. They provide a possible basis for the dissociation of these two properties of PDE inhibitors.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1989

Intracellular Ca2+ and force determined simultaneously in isolated resistance arteries.

A method is described for the simultaneous measurement of intracellular Ca2+ ([Ca2+]i) and force generation in isolated resistance arteries using the fluorescent Ca2+ indicator fura-2. Branch II mesenteric resistance arteries were isolated from 12-wk-old Wistar-Kyoto rats and mounted in a wire myograph. The myograph was placed on the stage of a compound microscope interfaced with a dual excitation wavelength fluorometer, and the tissue was loaded with fura-2 by incubation over a 30-min period with the cell-permeable form of the dye. When stimulated with physiological salt solution containing 100 mM KCl and 10 microM norepinephrine, a rapid and transient increase in [Ca2+], was observed to precede active force development and plateau at approximately 60% of the maximal level after 50 s. Washout of the agonists induced relaxation of these small arteries, consisting of an 85% decline in active tension over 100 s and a fall in [Ca2+]i to 50% of prerelaxation level over the same time period. Forskolin (1 microM), which increases intracellular adenosine 3',5'-cyclic monophosphate, induced a 50% relaxation over a 150-s period that was preceded by a fall in [Ca2+]i. Nearly identical results were obtained with 100 microM sodium nitroprusside, which stimulates intracellular guanosine 3',5'-cyclic monophosphate production. These findings indicate that the initiating event of forskolin- and sodium nitroprusside-induced relaxation may be a reduction of [Ca2+]i. The method described is useful for examining basic physiological events and Ca2+-related mechanisms of action of vasoactive compounds in isolated resistance arteries.

Animals↗
PubMed1989

A comparison of cyclic AMP signaling system in rat aortic myocytes in primary culture and aorta.

Cyclic AMP (cAMP) metabolism has been studied in rat aortic myocytes grown in primary culture to characterize this second messenger system in vascular smooth muscle cells that retain responses to vasoactive drugs. For comparison, cAMP metabolism was also studied in the aorta from donor rats. Adenylate cyclase activity from myocytes and from the aorta was stimulated to a similar degree by GTP, NaF, or forskolin, and the enzyme activation produced by isoproterenol or vasoactive intestinal polypeptide was observed only in the presence of GTP. A cAMP phosphodiesterase activity was found in homogenates from cultured myocytes and aorta as well, and it was similarly stimulated by calmodulin in both cases. The rates of cAMP production and degradation were about seven-fold higher in cultured myocytes than in aorta. Basal levels of cAMP were also higher in the cultured cells than in the aorta. Hormones and drugs acting on adenylate cyclase or cAMP-phosphodiesterase in cell-free preparations altered the cAMP content of undisrupted cultured myocytes and aorta in the expected manner. Differences between cultured myocytes and aorta resided in the courses of drug-induced cAMP increases and in the magnitude of the cAMP response to isoproterenol, which was markedly increased in cultured myocytes compared with aorta. It is concluded that, despite some quantitative differences, the cAMP system of rat aortic myocytes grown in primary culture has characteristics similar to those displayed in rat isolated aorta. These cells are therefore suitable for studying the effects of drugs involving cAMP as a second messenger.

1-Methyl-3-isobutylxanthine↗
PubMed1988

Comparison of cyclic nucleotide phosphodiesterase isoforms from rat heart and bovine aorta. Separation and inhibition by selective reference phosphodiesterase inhibitors.

The resolution as well as the biochemical properties of the multiple molecular forms of cyclic nucleotide phosphodiesterase, in a given tissue, may be strongly dependent upon experimental conditions of preparation (extraction of crude enzyme from tissues and fractionation procedures). In the present study, we compare the different molecular forms of cardiac (rat heart ventricle) and vascular (bovine aorta) phosphodiesterase isolated from crude extracts prepared either in sucrose medium or in hypotonic medium (in the presence of protease inhibitors and ion chelators) using two different fractionation procedures: isoelectric focusing on flat gel bed and DEAE-Trisacryl anion exchange chromatography. Both the calmodulin-dependent and the cAMP-specific forms exhibited close IEF and chromatographic patterns and showed similar sensitivities towards reference inhibitors regardless of the tissue of origin. In marked contrast, the cGMP-specific isoform notably differed from one to another tissue with respect to its biochemical properties (only the cardiac tissue being capable of stimulation by cGMP) and sensitivities to xenobiotics. Thus the possibility exists that pharmacological agents may modulate phosphodiesterase activity differently in cardiac and vascular target tissues.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1988

Further evidence for the existence of a homogenous beta-endorphin-sensitive receptor population in the rat tail artery.

Isolated rat tail arteries were perfused and vasoconstriction was evoked by electrical field stimulation (2 pulses at 1 Hz every 2 min). The vasoconstriction was depressed by DAGO (IC50 = 611 nM) and beta-endorphin (IC50 = 37 nM). Structuraly analogues and shorter fragments of beta-endorphin were also tested. beta-Endorphin and beta-endorphin-(1-26) were about equipotent whereas the beta-endorphin fragments 1-17, 1-16 and 6-31 were inactive. The potencies of beta-endorphin, beta-endorphin-(1-26), -(1-17) and -(1-16) were not changed in the presence of peptidase inhibitors. Structural analogues such as [D-Ala2]beta-endorphin or [Leu5]beta-endorphin had a somewhat lower potency than beta-endorphin itself. Naloxone 30 nM antagonized the effects of DAGO and beta-endorphin to a similar extent with dissociation constants 3.8 and 3.7 nM, respectively for the antagonist against the agonists. The results support the existence in the rat tail artery of a homogenous population of beta-endorphin-sensitive receptors which may belong to the epsilon-type.

Animals↗
PubMed1988

Interaction of calmodulin and calcium antagonists with [3H]diltiazem and [3H]nitrendipine binding sites.

The interaction of calmodulin antagonists and hydrophobic calcium antagonists with calmodulin and calcium antagonist ( [3H]nitrendipine and [3H]diltiazem) binding sites was investigated. The classical calmodulin antagonists calmidazolium, trifluorperazine, and W-7 were active at similar concentrations in the three experimental systems. The hydrophobic calcium antagonists prenylamine and bepridil, however, interacted with [3H]diltiazem binding at concentrations up to 50 times lower than their calmodulin inhibiting concentrations. The structural requirements for binding to calmodulin and to calcium channels are thus not identical for these hydrophobic drugs, suggesting that the calcium channel interacting properties of these antagonists are not a direct consequence of their calmodulin binding properties.

Animals↗
PubMed1988

Effects of inaktin on calcium metabolism in the rat.

Experiments were designed to investigate further the alterations in calcium metabolism caused by inaktin, a thiobarbiturate that impairs parathyroid hormone action in rats (1981). Treatment with an anesthetic dose of inaktin induced a drop in serum calcium without any variation in immunoreactive parathyroid hormone serum level and slowed body calcium turnover as studied with 45Ca, but was without effect on blood pH or partial pressures of oxygen and carbon dioxide. In contrast, calcium metabolism in rats was unchanged after treatment with an anesthetic dose of pentothal, another thiobarbiturate anesthetic. The effect of inaktin on body calcium turnover was dose-dependent and significant even at non-anesthetic levels. A marked slowing of the fast phenomena accounting for the initial dilution of the tracer in the animal was observed, accompanied by a corresponding decrease in rapid 45Ca uptake into bone, but not other tissues. These results show that inaktin slows calcium turnover, especially the fast renewal of calcium in bone. These effects were not correlated with anesthesia or immobilization, and we suggest that inaktin-induced changes in calcium metabolism involve an impairment of hypocalcemia-induced parathyroid hormone secretion, in addition to the impairment of parathyroid hormone effects previously reported. Furthermore, the present findings suggest that inaktin might be a useful tool for investigation of the rapid mobilization of bone calcium, which is as yet not well understood.

Anesthesia, General↗
PubMed1988

Potentiation by neuropeptide Y of vasoconstriction in rat resistance arteries.

1. The effects of neuropeptide Y (NPY) on resistance arteries were investigated on 3rd generation mesenteric arterioles of the rat. 2. Contractions were elicited by noradrenaline (NA), 5-hydroxytryptamine (5-HT), prostaglandin F2 alpha (PGF2 alpha), depolarization (KCl substituted for NaCl) and by the calcium agonist Bay K 8644, in the absence and in the presence of NPY (100 nM), a concentration which by itself did not induce vasoconstriction. 3. NPY produced a leftward shift of the concentration-response curves to the agonists and to KCl, without any alteration of maximal contractions. 4. NPY also potentiated contractions elicited by addition of CaCl2 to KCl-depolarized vessels, but its effect on calcium-induced contractions decreased with increasing KCl concentrations (from 20 to 100 mM). 5. Calcium-induced contractions were inhibited by the calcium channel blocker nitrendipine, both in the presence and absence of NPY (100 nM). NPY increased slightly (but significantly) the sensitivity to nitrendipine (the apparent KB increased from 2.9 x 10(-10) M to 1.6 x 10(-10) M). 6. The KCl concentration necessary for the maximal effect of Bay K 8644 was decreased in the presence of NPY, and the sensitivity to the calcium channel agonist was increased. 7. Elevating the KCl concentration in the bath from 5 to 20 mM (which gives the same displacement to the left of the KCl concentration-effect curve seen in the presence of NPY) induced a parallel leftward shift of NA and 5-HT concentration-response curves. This shift was identical to the one induced by NPY on 5-HT-evoked contractions, but it was significantly smaller (P less than 0.001) than the shift of the NA concentration-response curve observed in the presence of NPY. In the latter case, NPY enhanced more markedly the contractions induced by low NA concentrations (between 10(-9) and 3 x 10(-8 M) than those induced by high concentrations (up to 3 x 10(-7) M), thus giving a shallow concentration-response curve. 8. The results strongly suggest that NPY partially depolarizes the arterioles and induces an increase in calcium entry through voltage-dependent channels, thus enhancing contractions elicited by agonists or by KCl-depolarization. In addition, they support the view that another mechanism also plays a part in the potentiation by NPY of the effects of low concentrations of NA.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1988

Stereoselective blockade of alpha-adrenoceptors by berbine derivatives.

The effects of the two enantiomers of berbine (5,6,13,13a-tetrahydro-8H-dibenzo[a,g]quinolizine) and of derivatives obtained by introducing various substitutions on aromatic rings were investigated on alpha 1- or alpha 2-adrenoceptor subtypes. Binding studies carried out on rat cerebral cortex membranes using [3H]prazosin or [3H]yohimbine showed that the affinities of the (+) and (-)enantiomers for alpha 1 and alpha 2 binding sites were different and were differently modified by substitutions added to the berbine nucleus, leading to alpha 1- and alpha 2-selective compounds. Experiments performed on the isolated rat aorta and in pithed rats in vivo demonstrated the alpha-blocking property of berbine derivatives and confirmed the stereoselectivity of the effects of the (+) and (-)enantiomers on alpha 1- and alpha 2-adrenoceptor subtypes.

Adrenergic alpha-Antagonists↗
PubMed1987

Role of cyclic AMP- and cyclic GMP-phosphodiesterases in the control of cyclic nucleotide levels and smooth muscle tone in rat isolated aorta. A study with selective inhibitors.

Three isoforms of cyclic nucleotide phosphodiesterase (PDE) have been recently isolated from aortic tissue and two of them specifically hydrolyzed adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3':5'-cyclic monophosphate (cGMP), respectively (Lugnier et al., Biochem. Pharmac. 35, 1743, 1986). The role of these forms in controlling cyclic nucleotide levels and smooth muscle tone was investigated by the use of PDE inhibitors. The effects of selective inhibitors of the two forms specifically hydrolyzing cAMP or cGMP (cAMP-PDE and cGMP-PDE, respectively) were compared to those of non-selective inhibitors of the three aortic PDE forms, including the calmodulin-sensitive one (CaM-PDE). Relaxation responses and accumulation of tissue cAMP and cGMP induced by these drugs were studied in precontracted rat isolated aorta, and compared to the effects of isoprenaline and forskolin (stimulants of adenylate cyclase) or sodium nitroprusside (SNP) and sodium azide (stimulants of guanylate cyclase). The eight PDE inhibitors tested all relaxed aorta with potencies that correlated with their potencies as inhibitors of cAMP-PDE, but not of cGMP-PDE. At a concentration producing half-maximal relaxation, all PDE inhibitors induced a moderate but significant accumulation of cAMP, which was comparable to the accumulation of cAMP elicited by half-maximally relaxing concentrations of adenylate cyclase stimulating agents. At this concentration, some PDE inhibitors (M&B 22,948, dipyridamole and to a lesser extent, trequinsin) also induced a significant increase in cGMP levels, of the same order of magnitude as that caused by agents stimulating guanylate cyclase. However, the cGMP-increasing effect of these inhibitors was dissociated from their relaxing effect. In particular, the relaxing concentrations of M&B 22,948 (a selective inhibitor of cGMP-PDE) were clearly higher than the cGMP-increasing concentrations of the compound. At a concentration at which they elicited 10% relaxation by themselves, the selective cAMP-PDE inhibitor, rolipram, as well as the mixed inhibitor of cAMP- and cGMP-PDE, AAL 05 (a cilostamide analogue) enhanced both the cAMP-increasing and the relaxing effect of isoprenaline. Under the same conditions, no clear enhancement of the relaxation induced by SNP was observed. Only M&B 22,948 showed a slight potentiating effect on SNP-induced relaxation, but this effect was limited to low concentrations of SNP (less than 10 nM).(ABSTRACT TRUNCATED AT 400 WORDS)

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1987

Adrenomedullary and beta-adrenergic participation in enhanced sympathetic pressor responses of spontaneously hypertensive rats.

The beta-adrenergic and adrenomedullary components of pressor responses to sympathetic nerve stimulation were studied in spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY). The effects of electrical stimulation of the entire spinal cord of pithed rats pretreated with tubocurarine and atropine were studied on systolic blood pressure, heart rate and plasma cyclic AMP levels. The heart rate increase upon low frequency stimulation (1 Hz) and the blood pressure elevation upon stimulation at higher frequencies (3 and 5 Hz) were higher in SHR than in WKY whereas the increase in circulating cyclic AMP level was not different in the two strains. Pretreatment with propranolol (2.5 mg X kg-1) further enhanced the pressor responses in SHR but not in WKY, although it inhibited the heart rate acceleration and decreased the circulating level of cyclic AMP similarly in the two strains. After acute adrenalectomy, the elevations of blood pressure and circulating cyclic AMP levels were reduced to an identical level in SHR and WKY. These results show that the marked enhancement of the pressor response observed in SHR upon stimulation of the entire sympathetic outflow is mostly of adrenomedullary origin and includes a hypotensive component due to beta-adrenoceptor stimulation which is not present in WKY.

Adrenal Medulla↗
PubMed1987

Comparative effects of calmodulin inhibitors on calmodulin's hydrophobic sites and on the activation of cyclic nucleotide phosphodiesterase by calmodulin.

Experiments were designed to investigate the effect of inhibitors on calmodulin's hydrophobic sites and their consequences on the activation of a target enzyme, cyclic nucleotide phosphodiesterase. Two fluorescent probes, 2-(p-toluidinyl)-naphthalene-6-sulfonate (TNS) and 9-anthroylcholine (9AC) were used to study the interactions with calmodulin of inhibitors devoid of direct effect on the probes. Contrary to W-7, nicergoline, nicardipine and quercetin, which decreased the fluorescence of the two probes bound to calmodulin, bepridil only decreased 9AC fluorescence but increased the fluorescence intensity at the wavelength of the emission maximum of TNS. In spite of this difference, bepridil as well as W-7 and nicergoline competitively inhibited calmodulin activation of phosphodiesterase. In addition, nicergoline also inhibited phosphodiesterase activity competitively to cyclic GMP. These results show differences in the interactions of inhibitors with calmodulin; these differences are not detected in functional studies of the effect of inhibitors on phosphodiesterase activation.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1987

A 16 kDa protein substrate for protein kinase C and its phosphorylation upon stimulation of vasopressin receptors in rat aortic myocytes.

Phosphorylation induced by protein kinase C was examined in a plasma membrane fraction from rat aortic myocytes. Labelled phosphate incorporation produced by addition of kinase C to the membrane preparation allowed to identify a 16 kDa protein as the major substrate of the enzyme. This protein electrophoretically migrated with a protein phosphorylated by cAMP dependent protein kinase, but the two kinases produced phosphorylation of different sites since their effects were additive. Pretreatment of the myocytes with a kinase C activating phorbol ester or with vasopressin decreased further phosphate incorporation into the 16 kDa protein under the influence of exogenous kinase C. The results provide evidence that vasopressin produced in situ phosphorylation of the 16 kDa protein in rat aortic myocytes, with a time course and at concentrations consistent with a role of kinase C activation in the response of aortic myocytes to stimulation of V1 receptors.

Animals↗
PubMed1987

Differences in myocyte subpopulations from segments of the thoracic aorta and their modifications with age and hypertension in the rat.

Smooth muscle cells obtained from three distinct segments of the thoracic aorta of both Wistar Kyoto (WKY) and Spontaneously Hypertensive Rats (SHR) taken at different stages of development were studied in terms of their volume, DNA content in single cell suspensions, and doubling time in primary cultures. The proliferative activity and mean cell volume of myocytes from WKY rats increase along the thoracic aorta in a gradient from the aortic arch to the diaphragm. The slope of this gradient is increased in SHR because of an extension of the area that contains myocytes with low proliferative activity in primary cell culture and large cell volumes in suspension. Tetraploid myocytes are found in both strains and their proportions are larger in SHR than in WKY, specifically after the onset of hypertension. However, they appear to be evenly distributed along the thoracic aorta with a size distribution that is included in that of the diploid cells from the same area. It is suggested that changes in the structural properties of the aortic-cell compartment, associated with maturation and hypertension, reflect quantitative changes in the relative proportions of several myocyte subpopulations within the aorta of the rat.

Aging↗
PubMed1987

Cyclic AMP and cyclic GMP phosphodiesterase activities in Hodgkin's disease lymphocytes.

Cyclic nucleotide phosphodiesterase (PDE) activities were studied in peripheral blood monocyte-depleted lymphocytes and enriched T-lymphocyte suspensions from thirteen patients with previously untreated Hodgkin's disease (HD) and fourteen age and sex matched healthy volunteers. Monocyte-depleted lymphocytes from HD patients showed PDE-activities which were two times higher than in their normal counterpart cells. The mean cAMP-PDE activity present in enriched HD T-lymphocyte suspensions was four times higher than in control T-lymphocytes, and the mean cGMP-PDE associated with HD T-lymphocytes was three times higher than in the controls. The hydrolytic activities present in both monocyte-depleted and T-lymphocyte enriched cells suspensions remained unchanged in absence or in the presence of calmodulin and calcium. Since depressed cAMP and cGMP resting levels have been observed in HD lymphocytes and lymphocyte subpopulations, our results suggest that the elevated PDE activities are, at least in part, responsible for the alterations in lymphocyte cyclic nucleotide levels.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1987

Effect of parathyroid hormone and antagonist on aortic cAMP levels.

Experiments were designed to further investigate the vasoactive mechanisms of parathyroid hormone (PTH) on vascular smooth muscle cells. Time courses of the cAMP responses to the fragment (1-34) of bovine PTH (bPTH(1-34)) on cAMP levels have been studied in rat isolated aorta and in aortic myocytes in primary culture. In both aorta and myocytes bPTH (1-34) induced an increase in cAMP levels that was maximal and reached, respectively, 1.6- and 1.9-fold the basal level after 2 min of contact with bPTH (1-34). The effect of bPTH (1-34) on aortic cAMP content was concentration dependent in the range of 30-300 nM. (Nle8,18, Tyr34)-bPTH (3-34)amide, an antagonist of bPTH (1-34) with a stimulant effect on renal and vascular adenylate cyclase activity, inhibited the cAMP-increasing effect of bPTH (1-34). These results are in favour of a role for cAMP in the vasodilating effect of PTH.

Animals↗