Novel properties of peptides derived from the sequence coded by exon 26A of human elastin.

The exon 26A is a rarely expressed human elastin exon that codes for a hydrophilic and charged amino acid sequence. The functional role of elastin containing this additional sequence is unknown. The present investigation was aimed to determine the effect of synthetic peptides derived from this exon on the vascular tone of rat thoracic aorta. On phenilephrine-preconstricted rat thoracic aortic rings the peptides LSPELREGD and REGD cause dose-dependent relaxation in the concentration range from 10(-9) to 10(-5) M. omega-nitro-L-arginine methyl ester, a known inhibitor of the NO synthase, highly inhibits, although to a different extent, the relaxation induced by these peptides. Removal of endothelium and blocking of ATP-sensitive potassium channels by glibenclamide significantly inhibited the vasorelaxant activity of LSPELREGD but not that of REGD, suggesting a different mechanism of action and possibly a different receptor.

Specific binding of nicergoline on an alpha1-like adrenoreceptor in the rat retina.

Systemic treatment with nicergoline, an ergoline derivative showing alpha1-antagonist properties, causes vasodilatation in the eye without apparent untoward cardiovascular effects. In the present work we investigated the ability of nicergoline to inhibit the binding of radiolabelled prazosin in the rat retina and cortex. We found that nicergoline inhibited [3H]prazosin binding in both tissues, being more potent than unlabelled prazosin in the retinal tissue. The competition curves of the ergoline derivative were well fitted by a one-site model in the cortical tissue, with an IC50 (concentration of the drugs needed to inhibit the binding of labelled prazosin by 50%) of 2.54 x 10(-8) M, and by a two-site model in the retinal tissue, with IC50 values of 7.08 x 10(-12) M and 1.82 x 10(-5) M. 2-(2,6 dimetoxyphenoxyethyl) aminomethyl-1,4-benzodioxane hydrochloride (WB4101) and phentolamine, selective ligands for the high-affinity binding site for prazosin, in particular the alpha1A-site, fully inhibited prazosin binding in the cortex but only partially inhibited prazosin binding in the retina, being less potent in this tissue than either nicergoline or prazosin. Our results suggest that a binding component of alpha1-adrenoreceptors is expressed to a lesser extent in the retina than the cortex, leading to a reduced response of the retinal tissue to prazosin, and more particularly to WB4101 and phentolamine. The selective binding of the nicergoline on this retinal adrenoreceptor may explain the peculiar efficacy of the drug in ocular pathophysiology.

L-type calcium channels modulate the regression of left ventricular hypertrophy after ace-inhibition in genetic hypertension.

The aim of this study was to investigate the possible link between the regression of the left ventricular mass induced by ACE-inhibition and L-type calcium channels. For this purpose, an evaluation of both L-type calcium channels and AT1 receptor patterns in the left ventricular tissue of adult spontaneously hypertensive rats (SHR) was made before and after long-term treatment with ramipril. An abnormal density of both dihydropyridine and AT1 receptors was observed in SHR at 24 weeks, compared to age-matched control Wistar-Kyoto (WKY) rats (dihydropyridine receptor Bmax: 1. 30+/-0.09 vs 1.14+/-0.06 pmol mg-1 proteins, P<0.001; AT1 receptor Bmax: 1.35+/-0.07 vs 2.62+/-0.08, P<0.001 pmol mg-1 proteins). A treatment for 10 weeks with ramipril induced a significant decrease in the left ventricular mass index of SHR, as well as a significant decrease in dihydropyridine receptor density (Bmax: 0.96+/-0.01 vs 1. 39+/-0.08 pmol mg-1 proteins, P<0.001) and a significant increase in AT1 receptor density (Bmax: 3.08+/-0.26 vs 2.78+/-0.09 pmol mg-1 proteins, ramipril-treated SHR vs vehicle-treated SHR, P<0.001). These results suggest that the decrease in left ventricular mass after treatment with ramipril may be dependent on changes in L-type calcium channels other than the direct effect on circulating and tissue angiotensin II (ang II) levels: involvement of calcium channels and subsequent calcium influx into cardiac cells could be proposed as an additional mechanism for the regression of left ventricular mass after ACE-inhibition.

Identification of elastin peptides with vasorelaxant activity on rat thoracic aorta.

Elastin peptides obtained in vivo from the enzymatic degradation of elastic fibers are present in the circulating human blood. In order to verify the role that these peptides may have in the regulation of the vascular tone, the activity of several peptides identified in the elastolytic digest of human elastin and some of their structural homologues has been tested. Three of these peptides show a vasorelaxant activity in isolated rat aorta precontracted by phenylephrine. The activity observed is higher in the absence of the endothelium; in these conditions the IC50 for the peptides Val-Gly-Val-Ala-Pro-Gly, Val-Gly-Val-Pro-Gly and Val-Gly-Val-Hyp-Gly was 40 +/- 2, 73 +/- 2 and 10 +/- 1 ng/ml, respectively. They are active in the range of the pathological circulating concentration and their role could be important in the regulation of vascular tone during several elastin degradative diseases.

Effects of phorbol ester on carbachol-induced contraction in bovine ciliary muscle: possible involvement of protein kinase C.

The aim of the research was to characterize muscarinic receptors of bovine ciliary muscle and to investigate the desensitization process. The role of protein kinase C was analyzed. The results show that muscarinic receptors of bovine ciliary muscle have the pharmacological characteristics of the M3 subtype. Acute exposure to phorbol esters (1 microM phorbol 12,13-dibutyrate, PDB, or 0.1 microM phorbol 12-myristate 13-acetate, PMA, for 15 and 5 min, respectively) resulted in antagonism of muscarinic receptor-mediated contraction. Long-term pretreatment (18 h) with PMA to down-regulate protein kinase C resulted in potentiation of carbachol-induced contraction, reduction of agonist-induced desensitization and loss of phorbol ester-induced desensitization. Staurosporine (3 microM) and H7 [1-(5-isoquinolinesulfonyl)-2-methyl-piperazine] (1 microM), protein kinase C inhibitors, produced a significant potentiation of the contractile effect of carbachol, reduced the desensitization produced by repeated addition of carbachol and suppressed that induced by phorbol esters. In vitro incubation with carbachol, PDB or PMA did not cause any modification of the binding of labeled [3H]quinuclidinyl benzilate. In vitro incubation with PDB and PMA produced, as expected, a significant translocation of protein kinase C from the cytosol to the membrane. The incubation of the ciliary muscle with carbachol, using the protocol of exposure that induced maximal desensitization of contractile responses, produced a significant redistribution of the enzyme from the cytosol to the membrane. These findings suggest that agonist-induced modulation of functional cholinergic sensitivity in ciliary muscle is correlated, at least partially, to the translocation of protein kinase C from the cytosol to the membrane. The desensitization by phorbol esters is completely due to protein kinase C activation; during the desensitization process, direct modification of the density and affinity of muscarinic receptors is not involved.

5-HT1A and D-2 receptor affinity of o-methoxyphenylpiperazine derivatives with terminal benzamide fragment on N-4 alkyl chain. 2.

The synthesis of some o-methoxyphenylpiperazines with a benzamide moiety on N-4 alkyl chain was accomplished and their affinity for dopamine and serotonin receptor subtypes was assayed by in vitro receptor binding. The results show that several derivatives had a good affinity both on D-2 and 5-HT1A receptors, the IC50s ranging from 10(-7) to 10(-8) M.

5-HT and DA receptor affinity of arylpiperazine derivatives with terminal benzamide fragment.

The synthesis of some arylpiperazines with a benzamide moiety on N-4 alkyl chain was accomplished and their dopaminergic and serotonergic affinity was assayed by in vitro receptor binding. The results of such investigation showed a moderate affinity on D-2 receptors and a lack of 5-HT receptor affinity.

Non-invasive haemodynamic study in hypertensive subjects after treatment with verapamil slow release.

Aim of this study is to investigate the haemodynamic effects, after the short and long-term antihypertensive treatment. After a wash-out period and a placebo treatment period, 30 hypertensive patients received verapamil SR (slow release, 240 mg o.d.) for 30 days. A significant decrease in systolic and diastolic blood pressure was obtained already 4 h after the first administration of verapamil; it was more evident and persistent throughout the study. No significant changes of heart rate or PR interval in ECG were observed. A significant decrease in total vascular resistances, both supine and upright, was evident already 4 h after the drug intake and observed throughout the study. The major effect was obtained after one month. No significant changes of cardiac output, cardiac index and stroke volume were recorded. Furthermore, plasma verapamil levels were measured to confirm that the haemodynamic effects are obtained by low drug concentrations. The present study provides evidence that the antihypertensive effect of verapamil, whose mechanism is the reduction of total vascular resistances, is progressive, long acting and achieved by low plasma levels, when slow release formulation is considered.

8-Methoxy- and p-dimethoxy-1-aminoethylhetero-tetralins: synthesis and DA, 5-HT receptors affinities.

Two series of compounds, 8-methoxy- and p-dimethoxy derivatives of 1-aminoethylhetero-tetralins, I, were evaluated for D-2, 5-HT1 and 5-HT2 receptors affinity. No significant serotoninergic affinity was observed, whereas p-dimethoxy-derivatives 7b e 11b showed a moderate D-2 affinity.

Oxygen isosteric derivatives of 3-(3-hydroxyphenyl)-N-n-propylpiperidine.

Some substituted 3-phenylmorpholines (10a-e,j,k) and 3-thienylmorpholines (10f,g), isosteres of 3-(3-hydroxy-phenyl)-N-n-propylpiperidine (3-PPP), were prepared and submitted to binding assays on D-2 dopaminergic and 5-HT1 and 5-HT2 serotonergic receptors, in comparison with 3-PPP and its analogue 3a,b. The results show the loss of D-2 affinity for all morpholines, while a certain activity was still observable for piperidine derivatives. Regarding the serotonergic affinity, only chloro and methoxy derivatives (10a-d) were moderately active on the 5-HT1A receptor, either when the substituent was in the C-2 or C-3 position, whereas no tested compounds showed affinity toward the 5-HT2 receptor.

Effects of prostaglandins and PAF on the contractility of the bovine ciliary muscle.

The present study has investigated the effect of prostaglandins and PAF on the contractility of the bovine ciliary muscle, a tissue involved in the control of aqueous outflow. The results show that the prostaglandins tested (PGI2, its stable analogue Iloprost, PGE2, PGE1, and PGF2 alpha) as well as PAF, were able to contract the ciliary muscle, although with different potencies and efficacies. PGI2 and Iloprost displayed parallel dose-effect curves with upper plateaus that did not differ significantly; however, PGI2 was slightly more potent than Iloprost. This is at variance with what is observed at the level of the platelet prostacyclin receptor. PGF2 alpha was equipotent with the PGEs tested, with a maximal effect not different from either PGI2 or PGEs. PAF was the most efficacious of the compounds tested.

Evidence for protein kinase C modulation of the ciliary muscle response to carbachol and desensitization.

The role of protein kinase C (PKC) in the desensitization of muscarinic receptor-mediated responses in bovine ciliary muscle was examined. Exposure of the bovine ciliary muscle to phorbol esters, used to activate PKC, resulted in antagonism of muscarinic receptor-mediated contraction. On the other hand, staurosporine, a known PKC inhibitor, caused a significant potentiation of the contractile effect induced by carbachol. Staurosporine reduced the desensitization induced by repeated additions of carbachol and completely suppressed that induced by phorbol esters. The results also indicate that desensitization mediated by phorbol esters as well as that mediated by muscarinic receptor agonists is heterologous.

Vascular eicosanoids and platelet-aortic wall interactions in spontaneously hypertensive rats.

We studied the aggregation of collagen and ADP-stimulated platelet-rich plasma (PRP) and the formation of thromboxane B2 (TxB2) by collagen-stimulated PRP in spontaneously hypertensive rats (SHR) and in Wistar-Kyoto control rats (WKY). In addition, we evaluated the inhibition of the aggregation of PRP following homologous or heterologous perfusions through isolated aortas, the release of 6-keto-prostaglandin (PG)F1 alpha from these arteries perfused with PRP, and the sensitivity of PRP to the antiaggregatory activity of the stable PGI2 analogue, iloprost, in both SHR and WKY. The lower activities (aggregation induced by ADP and collagen, collagen-stimulated TxB2 production) of SHR platelets, were not accompanied by morphological differences from WKY platelets. These changes were associated with a greater release of arterial 6-keto-PGF1 alpha, with greater platelet antiaggregatory activity of the arterial wall and with higher sensitivity of platelets to iloprost. The lower reactivity of platelets to aggregating agents, and the greater sensitivity to prostacyclin, associated with a greater production of arterial prostacyclin were the major changes observed in SHR animals. These alterations in the SHR vs. normotensive WKY may lead to an enhanced risk of hemorrhage in the hypertensive state.

Biochemical and functional evidence for the presence of dopamine D1 receptors in the bovine ciliary body.

The present paper reports both functional and biochemical evidence for the presence of dopamine D1 receptors in the bovine ciliary body. Dopamine (DA) and dopamine D1 agonists (such as SKF 38,393) but not D2 agonists (such as LY 141,865) produced a concentration-related decrease in the tone induced by a maximally active concentration of carbachol (1 x 10(-4)-5 x 10(-4) M). The maximal relaxation obtained was 100% of the carbachol response using 10(-5) M dopamine or 5 x 10(-6) M SKF 38,393. SCH 23,390, a D1 antagonist, but not (-)-sulpiride, antagonized the effect of DA and SKF 38,393. In accordance with the functional data, radioreceptor binding experiments revealed the existence of a high affinity saturable [3H]SCH 23,390 binding to membranes prepared from ciliary body (Bmax: 344 fmol mg protein-1; Kd: 0.87 nM). The binding was specifically displaced by SCH 23,390, dopamine and dopamine D1 agonists, but not by norepinephrine, D2 agonists, or antagonists such as LY 141,865 and sulpiride. No specific binding was found when using dopamine D2 ligands, such as tritiated spiroperidol.

[Reactivity of the aorta from spontaneously hypertensive rats before and after endothelial removal]. / Reattività dell'aorta di ratti spontaneamente ipertesi prima e dopo rimozione del'endotelio.

In the present study the mechanic activity of SHR and Wistar rat's aorta was evaluated, in vitro, after stimulation by chloride of potassium, phenylephrine, norepinephrine, histamine, serotonin and acetylcholine, before and after the removal of the endothelium. The aorta rings of the rats were taken before the development of the hypertensive state (7th week) and at 18th week (when the SHR rats already showed established hypertension starting since IXth week of life), and successively suspended in a bath for isolated organ. The mechanic activity was measured by isometric transductor. The obtained findings show an increased sensitivity, in SHRs, to (K+), NA and FeE, if these are compared with the control group, since the prehypertensive stage (7th week). The removal of the endothelium didn't modify the response amplitude to K in both the breeds, while the maximum response amplitude, provoked by NA and FeE, significantly increased in SHRs compared to controls. The relaxation induced by the vasodilator agents (Ach-H-5HT) was completely absent in the SHR rats' aorta with established hypertension. In conclusion, these results suggest a functional deterioration of the endothelial cells, in the hypertensive animals, that could contribute to increase the peripheric vascular resistances observed during hypertension.

Changes of vascular smooth muscle reactivity in hypertensive rats.

Mechanical responses produced by high potassium solution (high-K), norepinephrine (noradrenaline; NA) and phenylephrine (Phe) were examined in the thoracic aorta isolated from control (WKY) and spontaneously hypertensive rats (SHR). In the SHRs the tissues showed increased sensitivity to high potassium compared with those from the control rats. Mechanical removal of endothelium in tissues from the controls did not change the response. The effects produced by NA and Phe were also increased in tissues from SHRs. The amplitudes of contractions were enhanced after removal of the endothelium in tissues from the controls. The relaxation in response to endothelium-dependent vasodilators (acetylcholine and histamine) was significantly depressed in aortic rings from SHRs. Experiments using modified sandwich preparations suggest that the defect in the relaxant ability of vascular smooth muscle is coupled to a reduced functionality of the endothelium.