Interactions between apelin and angiotensin II on rat portal vein.

Apelin (AP), an endogenous ligand of the APJ receptor, is involved in the regulation of cardiovascular homeostasis. Regardless the multiple similarities between AP and angiotensin II (Ang II), their roles seem to be different. We studied both the interactions between Apelin 13 (AP13) and Ang II and to what extent, if any, nitric oxide (NO) is involved. The experiments were performed in endothelium-denuded or endothelium-intact rat portal vein in the presence of 10 microM N(G)-nitro L-arginine methyl ester or 10 microM aminoguanidine. AP13 did not modify the isolated rat portal vein tone by itself, but inhibited the Ang II-induced contractions acting mainly by a NO-dependent mechanism. Our results sustain the hypothesis that AP13 could increase the activity of both constitutive and inducible NO synthase on either endothelium intact or endothelium denuded rat portal vein rings.     

The actions of angiotensin II on the isolated portal vein of the rat

Isolated rat portal vein suspended in Krebs bicarbonate medium showed regular rhythmic activity and contracted in response to angiotensin II, noradrenaline, adrenaline, tyramine, acetylcholine, serotonin and histamine. Angiotensin II was 3–10 times as potent as noradrenaline on a weight basis. Responses to angiotensin II were unchanged in the presence of antagonists to these drugs, excepting high concentrations of phentolamine. Electrical stimulation or noradrenaline did not potentiate responses to angiotensin II; angiotensin II did not potentiate responses to noradrenaline. Angiotensin II acts directly on the smooth muscle of rat portal vein; its action is independent of noradrenaline. Spontaneous contractions and responses to angiotensin II and noradrenaline were transiently depressed and then potentiated in media made hyperosmolar by addition of NaCl, sucrose or urea. Responses were depressed in hypo-osmolar low NaCl solution but unaffected by isosmolar low NaCl. An increase of intracellular potassium concentration would not alone account for the entire effect of increased osmolarity.     

Positive inotropic action of angiotensin II in the pithed rat

Summary The cardiovascular effects of angiotensin II were examined in aortic blood pressure-controlled and-uncontrolled pithed rats. Angiotensin II induced a dose-dependent increase in diastolic blood pressure, left ventricular pressure (LVP), dP/dt (the first derivative of LVP) and heart rate in pithed rats. The maximal responses for these parameters were similar to those to noradrenaline, except for the rise in diastolic blood pressure, where noradrenaline caused a greater increase than angiotensin II. After treatment with propranolol, the positive chronotropic effect of angiotensin II was abolished. Angiotensin II produced a dose-dependent increase in diastolic blood pressure, which was similar to that of vasopressin, and an increase in dP/dt_max, which proved much greater than that of vasopressin. When aortic blood pressure was controlled and the -receptors were blocked by propranolol, angiotensin II caused a dose-dependent increase in dP/dt_max without affecting the left ventricular enddiastolic pressure. The same results were obtained after both - and -adrenoceptors were blocked by propranolol and phentolamine. Losartan but not PD 123177 caused parallel rightward shifts of the dose-response curve of angiotensin II for dP/dt_max in the aortic blood pressure controlled pithed rat without altering the maximal response. It is concluded that in the pithed rat angiotensin II produced an increase in myocardial contractile force which is not mediated by - or -adrenoceptors. The inotropic effect appears to be mediated by angiotensin receptors, of the AT₁-subtype.Correspondence to J. Zhang at the above address     

Effects of disulfiram on excitation-contraction coupling in rat soleus muscle

The aim of this study was to analyze whether disulfiram could affect excitation-contraction coupling in rat slow-twitch (soleus) muscle.In small bundles of intact fibers, the amplitude and the time constant of relaxation of twitch and potassium contractures were dose-dependently and reversibly reduced by disulfiram at concentrations up to 27 M. At larger concentrations (up to 67.5 M) these effects were still present but less pronounced. In the presence of disulfiram (27 M), the relationship between the amplitude of potassium contractures and membrane potential was shifted to more positive potentials whereas, the steady state inactivation curve was unchanged. These observations suggest that disulfiram has no effect on voltage sensors. In saponin-skinned fibers, the amount of Ca²⁺ taken up, estimated by using the amplitude of 10 mM of caffeine contracture, was increased by disulfiram (27 M). By contrast no significant modification was observed in the sensitivity of the ryanodine receptors to caffeine (contractures generated at 5 mM of caffeine) and in the myofibrillar Ca²⁺ sensitivity (Triton X-100 skinned fibers).These results indicate that disulfiram induces a dose-dependent reversible effect on the contractile responses of soleus mammalian skeletal muscle by acting mainly on the sarcoplasmic reticulum Ca²⁺-ATPase activity.     

Characterization of the angiotensin II-receptor subtype in the longitudinal smooth muscle of the rat portal vein

Summary The purpose of the present study was to identify the angiotensin II-receptor subtype involved in the enhancement of the amplitude of the phasic contractions by angiotensin II in the isolated rat portal vein preparation.At an extracellular Ca²⁺ concentration of 0.9 mmol/l and a K⁺ concentration of 4 mmol/l, angiotensin II induced concentration-dependent increases in the amplitude of the phasic contractions. The enhancement of phasic contraction amplitude caused by angiotensin II was not significantly altered by pretreatment of the rat portal vein with indomethacin 10^–5 mol/l or nitro-L-arginine 10^–4 mol/l, indicating that neither prostaglandins nor the endothelium derived-relaxing factor (NO) are involved. Losartan (DuP 753), a nonpeptide selective AT₁-receptor antagonist, concentration-dependently shifted the concentration-response curve for the effect of angiotensin II on the amplitude of the contractions to the right, without reducing the maximal response (pA₂ = 8.6, slope = 0.98), thus suggesting competitive antagonism at the level of AT₁-receptors. By contrast, PD 123177, a nonpeptide selective AT₂-receptor antagonist, even at 10^–5 mol/l, caused no significant change of the phasic myogenic response to angiotensin II, indicating the absence of AT₂-receptor involvement. Dithiothreitol, a disulfide-reducing agent which is known to inactivate AT₁-receptors in various tissues, markedly inhibited (3 mmol/l) or even abolished (5 mmol/l) the contractile response of the rat portal vein to angiotensin II, supporting the conclusion that these receptors can be classified as AT₁-receptors.In conclusion, the receptor subtype mediating the angiotensin II-induced potentiation of the spontaneous phasic contractions in the rat portal vein appears to belong to the AT₁-receptor subtype.Preliminary data were presented at the Spring Meeting of the German Pharmacological Society, Mainz, 1992Correspondence to J. S. Zhang at the above address     

Antagonism of the dipsogenic action of intraseptal angiotensin II in the rat

The injection of 0.01 to 2.0 μg of antiotensin II (A II) into the medial septal area of unanesthetized rats in normal water balance caused dose-dependent drinking, during the 60 min period following drug administration. A hyperbolic dose-response curve, rectified by a log dose scale was obtained. Pretreatment with 5 and 10 μg of locally injected haloperidol, 15 min prior to A II (0.3 μg), partially antagonized the dipsogenic effects of A II and a dose of 25 μg of haloperidol completely blocked this effect. A cataleptic-like state followed haloperidol administration. The injection of doses as high as 25 μg of dopamine in the same brain site caused no drinking. Pretreatment with 3 μg of intraseptal Sar¹, Ala³, Ile⁸ — angiotensin I, a competitive antagonist of A II at peripheral receptors, completely antagonized the dipsogenic effect of A II. The same dose (3 μg) of the A II analog alone caused only a mild but significant drinking response. These results suggest that A II acts of specific receptors in the CNS that may be similar to peripheral angiotensin receptors. On the other hand, the role of brain catecholamines in the mediation of A II-induced drinking remains uncertain.     

An electrophysiological study of alpha-adrenoceptor mediated excitation-contraction coupling in the smooth muscle cells of the rat saphenous vein.

The effects of perivascular nerve stimulation and application of exogenous alpha-adrenoceptor agonists on the rat saphenous vein were studied by simultaneous recordings of electrical and mechanical activities. The resting membrane potential of the saphenous vein averaged -65 mV. Perivascular nerve stimulation elicited excitatory junction potentials (e.j.ps) and slow depolarizations. Contraction was observed when either the e.j.p. or the slow depolarization reached a critical threshold of about -55 mV. Exogenously applied noradrenaline, B-HT 920 and clonidine induced depolarization and contraction similar to the slow depolarization. The responses to these agonists and the slow depolarizations were antagonized by yohimbine, but not by prazosin. The selective alpha 1-adrenoceptor agonists phenylephrine and methoxamine had very little effect on the electrical and mechanical activities of the saphenous vein. It was concluded that in the rat saphenous vein, only alpha 2-adrenoceptors are present and that these receptors mediate the slow depolarization and contraction induced by nerve stimulation.     

Specific antagonists for the myotropic action of angiotensin II and angiotensin I on the isolated rat colon

Substitutions of 8 phenylalanine with L-alanine and D-phenylalanine abolish the myotropic action of the angiotensin II (AT(II)) analogues and confer inhibitory properties on the molecule. [8-L-Ala]-AT(II) and [8-D-Phe]-AT(II) antagonize specifically the myotropic action of AT(II) and angiotensin I (AT(I)) on the rat colon, while the action of other myotropic agents (acetylcholine, 5 hydroxytryptamine) is not modified.     

Are multiple angiotensin receptor types involved in angiotensin (1-7) actions on isolated rat portal vein.

Angiotensin (1-7) [Ang (1-7)] is a bioactive component of the renin angiotensin system. Ang (1-7) may interact with angiotensin type 1 (AT1) or type 2 (AT2) receptors and with Ang (1-7) - specific receptors. We examined the interactions between different doses of Ang (1-7) (1 nM-1 microM) and angiotensin II (Ang II) (10 and 100 nM) on isolated rat portal vein. In endothelium-denuded portal vein rings, Ang (1-7) inhibited contractile effects induced by Ang II. The effects of Ang (1-7) were modified by indomethacin, N(G)-nitro-L-arginine methyl ester (L-NAME), (D-Ala7)-Angiotensin (1-7) (H-2888) and losartan. Our results suggest that on rat isolated portal vein rings without endothelium, Ang (1-7) reduces Ang II-induced contractions by acting mostly on Ang (1-7) specific receptors, and this effect is mediated by vasodilatatory prostaglandins. At high concentrations, Ang (1-7) effects are mediated by AT1-receptors, though to a lesser extent than by Ang (1-7) specific receptors.     

Effects of buphenin on the rat portal vein

The vasodilator and tocolytic substance buphenin (10 mumol/l) stimulated the spontaneous phasic activity of some (8 out of 18) isolated rat portal vein preparations; 0,1-1 mmol/l buphenin diminished or abolished the activity in all preparations. The isotonic and isometric tonic contractions of portal vein in response to adrenaline, noradrenaline and phenylephrine (0,1-1 mumol/l) disappeared almost completely after addition of buphenin in equimolar concentrations, whereas acetylcholine contractions persisted. The beta-adrenergic blocking agents propranolol and dichloisoprenaline (10 mumol/l) only slightly antagonized the inhibitory effect of buphenin on the contractile responses to catecholamines. It is concluded that buphenin exerts dual action upon rat portal vein: the drug partially stimulates the beta-receptors and partially blocks the alpha-adrenergic receptors.     

The involvement of potassium channels in the action of ciclazindol in rat portal vein.

1. In whole portal veins, ciclazindol (0.3-10 microM) increased the amplitude and duration, but decreased the frequency of spontaneous contractions. Glibenclamide (0.3-10 microM) produced a small increase in contraction amplitude and duration with a small reduction in contraction frequency. 2. In whole portal veins, ciclazindol (1-10 microM) antagonized the relaxant effects of BRL 38227 in a non-competitive manner. Under identical conditions, the effects of glibenclamide (0.3-10 microM) appeared to be competitive. 3. In whole portal veins loaded with 42K, ciclazindol itself (up to 3 microM) had no detectable effect on basal 42K exchange. However, the increase in 42K efflux produced by BRL 38227 (5 microM) was antagonized by ciclazindol (3 microM). Similar effects were produced by glibenclamide (up to 3 microM). 4. In freshly-isolated portal vein cells examined by the whole-cell voltage-clamp technique, ciclazindol (1-100 microM) inhibited the slowly-activating and inactivating transient outward current (ITO) which could be generated at potentials more positive than -30 mV. In addition ciclazindol (1-10 microM) inhibited the non-inactivating K-current (IKCO) induced by BRL 38227 (10 microM). 5. In freshly-isolated portal vein cells under current-clamp conditions, the hyperpolarization produced by BRL 38227 (10 microM) was reversed by ciclazindol (1-10 microM). 6. In porcine brain membrane fragments, glibenclamide (0.65 nM) displaced 50% of the binding of [3H]-glibenclamide whereas ciclazindol (up to 10 microM) had no effect. 7. It is concluded that ciclazindol is a K-channel blocker. Its action is not selective for the channel(s) which carry IKCO but also extends to those which carry ITO.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of action of angiotensin II in human isolated subcutaneous resistance arteries

1. Human isolated subcutaneous arteries were mounted in a myograph and isometric tension measured. In some experiments, intracellular calcium [Ca(2+)]i was also measured using fura-2. 2. Angiotensin II (100 pM - 1 microM) increased [Ca(2+)]i and tone in a concentration-dependent manner. The effects of angiotensin II (100 nM) were inhibited by an AT1-receptor antagonist, candesartan (100 pM). 3. Ryanodine (10 microM), had no effect on angiotensin II-induced responses, but removal of extracellular Ca(2+) abolished angiotensin II-induced rise in [Ca(2+)]i and tone. Inhibition of Ca(2+) entry by Ni(2+) (2 mM), also inhibited angiotensin II responses. The dihydropyridine, L-type calcium channel antagonist, amlodipine (10 microM), only partially attenuated angiotensin II responses. 4. Inhibition of protein kinase C (PKC) by chelerythrine (1 microM), or by overnight exposure to a phorbol ester (PDBu; 500 nM) had no effect on angiotensin II-induced contraction. 5. Genistein (10 microM), a tyrosine kinase inhibitor, inhibited angiotensin II-induced contraction, but did not inhibit the rise in [Ca(2+)]i, suggesting that at this concentration it affected the calcium sensitivity of the contractile apparatus. Genistein did not affect responses to norepinephrine (NE) or high potassium (KPSS). 6. A selective MEK inhibitor, PD98059 (30 microM), inhibited both the angiotensin II-induced contraction and rise in [Ca(2+)]i, but had no effect on responses to NE or KPSS. 7. AT1 activation causes Ca(2+) influx via L-type calcium channels and a dihydropyridine-insensitive route, but does not release Ca(2+) from intracellular sites. Activation of tyrosine kinase(s) and the ERK 1/2 pathway, but not classical or novel PKC, also play a role in angiotensin II-induced contraction in human subcutaneous resistance arteries.     

Selective local action of angiotensin II on dopaminergic neurons in the rat hypothalamus in vivo

Summary Microdialysis was used to investigate whether angiotensin II modulates the basal and K⁺-induced release of endogenous noradrenaline, dopamine and their metabolites 3,4-dihydroxyphenylglycol (DOPEG) and 3,4-dihydroxyphenylacetic acid (DOPAC) from the anterior hypothalamus of the anaesthetized rat. The release of the amines was stimulated twice (ST1 and ST2) with either 50 mmol/l or 100 mmol/l K⁺. The release of each amine induced by K⁺ was reproducible and concentration-dependent. Angiotensin II when present in the perfusion fluid after ST1 at a concentration of 0.1 and 10 mol/l (chosen after in vitro experiments had shown that the recovery of the peptide across the dialysis membrane was only 3.6%), had no significant effect on amine release. However, 10 mol/l angiotensin II induced an immediate, significant increase in basal DOPAC outflow which reached a maximum of 89% in the 100 mmol/l K⁺ and 53% in the 50 mmol/l K⁺ experiments. No such effect was observed with DOPEG outflow. In a separate experimental series, addition of angiotensin II without a preceding K⁺ stimulation period did not significantly affect the outflow of the amines and metabolites.The results suggest that angiotensin II can selectively influence dopamine metabolism in the anterior hypothalamus in vivo but does not act locally to acutely facilitate the release of endogenous catecholamines from this brain area.     

Analysis of the vasorelaxant action of angiotensin II in the isolated rat renal artery

Taking into consideration that mechanisms involved in the vasodilatator actions of angiotensin II have not yet been completely elucidated, the present study was undertaken in order to examine the mechanisms underlying the angiotensin II-induced relaxation of rat renal artery (RRA). Angiotensin II produced concentration-dependent and endothelium-independent relaxation of isolated RRA. Angiotensin II-induced relaxation was partially reduced by inhibitors of nitric oxide synthase and guanylyl cyclase. The remaining dilatation was inhibited by a potassium channel blocker, charybdotoxin. Precontraction of RRA with high concentration of K(+) partially reduced angiotensin II-evoked relaxation, while indomethacin, glibenclamide, apamin and barium did not alter the angiotensin II concentration-response curve. Losartan had no effect on angiotensin II effect. Oppositely, HOE 140 and PD123319, separately or in combination, partially antagonized vasorelaxation induced by angiotensin II. Complete blockade of RRA response was obtained after simultaneous incubation of all three receptor antagonists HOE-140, PD123319, and losartan; L-NOARG plus HOE-140; or PD123319 plus charybdotoxin. These results indicate that angiotensin II produces endothelium-independent relaxation of RRA, which is most probably mediated by the interaction of the NO-cGMP pathway and K(+) channels. Moreover, we can assume that AT(1), AT(2), and B(2) receptors are involved in the vasorelaxant effect of angiotensin II.     

Peripheral action of angiotensin II

The effects of angiotensin II were studied on isolated atrial preparations of nonreserpinised and reserpinised rabbits, before and after treating the preparations by propranolol. Peripheral action of angiotensin was cardioaccelerator via direct stimulation of beta-receptors of the atria in isolated atrial preparations.     

Effects of angiotensin II and 1-sar., 8-isoleu. angiotensin II on electrical and mechanical properties of the portal vein from rats of different ages

The effects of angiotensin II (Ag-11) and 1-sar., 8-isoleu. angiotensin 11 (anti-Ag-11) on the membrane and mechanical properties of smooth muscle cells of the rat portal vein were investigated in three different age groups (6--8 weeks, immature rat; 3--5 months old, young rat and 13--15 months old, adult rat). Application of Ag-11 (10(-10) g/ml) did not depolarize the membrane, but did increase the spike frequency and potentiated the frequency and amplitude of twitch contraction. In a concentration of more than 10(-9) g/ml Ag-II, the membrane was depolarized and a phasic contracture was developed in three age groups. Depolarization of the membrane produced by Ag-II could be classified into two components, i.e. phasic and tonic depolarizations. In adult rats, Ag-II produced the highest amplitude of the depolarization in three age groups but tonic depolarization showed nearly the same amplitude of smooth muscle cell membrane to Ag-II increases with age up to 3--5 months and that an increased sensitivity is accompanied by a generation of desensitization. Tachyphylaxis to Ag-II was also observed by repetitive applications, but the appearance depended on the stimulus conditions. Anti-Ag-II, itself slightly increased the spike frequency and the amplitude of twitch contraction. However, under pretreatment with anti-Ag-II (10(-9) or 10(-7) g/ml), the actions of Ag-II on the electrical and mechanical activities were markedly suppressed in the three age groups. When the dose-response curve was obtained from the mechanical response produced by Ag-II, the relation shifted to the right in the presence of anti-Ag-II in all age groups. In the presence of 10(-7) g/ml anti-Ag-II, no contracture was evoked by application of 10(-6) g/ml Ag-II in three age groups. Anti-Ag-II seems to possess a higher affinity to the angiotensin receptor than does Ag-II.     

Effect of gallopamil on excitation-contraction coupling

• 1.1. Investigations performed in skeletal muscle have suggested that phenylalkylamine calcium antagonists, particularly gallopamil, affect excitation-contraction coupling independently of their effect on the sarcolemmal calcium current.
• 2.2. Sarcoplasmic reticulum and single channel studies have provided evidence that phenylalkylamine calcium antagonists inhibit calcium release through the sarcoplasmic reticulum calcium channel/ryanodine receptor. This action has not been observed with dihydropyridine calcium antagonists.
• 3.3. Binding experiments have confirmed the existence of intracellular binding sites for phenylalkyl-amines, and have shown that gallopamil interferes with the binding of ryanodine to its low affinity sites.
• 4.4. The dose-response relationship for the effect of gallopamil on excitation-contraction coupling has not been definitely established. However, there is evidence that gallopamil may be effective at concentrations that are close to the therapeutic range.

     

The receptor subtype mediating the action of angiotensin II on intracellular sodium in rat proximal tubules

1. An investigation was undertaken to explore the subtype of receptor involved in mediating the actions of angiotensin II on intracellular sodium content in suspensions of isolated proximal tubules of the rat.
2. Intracellular sodium content of the proximal tubules was measured with ²³Na n.m.r. spectroscopy and under these conditions basal sodium content of the tubular cells was 69.04±1.73 nmol mg⁻¹ dry weight and the ATP levels, at 8.3±0.9 nmol ATP mg⁻¹ protein, were consistent with active respiration by the tissue.
3. In the presence of 10⁻⁴ M PD123319, a selective non-peptide AT₂ receptor antagonist, intracellular sodium levels rose from steady state by 30% (P<0.01; n=7) within 10 min of exposure to angiotensin II 10⁻¹¹ M. Over the subsequent 30 min steady state levels were re-established. Administration of angiotensin II 10⁻¹¹ M, in the presence of the selective AT₁ receptor antagonist, losartan at either 10⁻⁶ M (n=5) or 10⁻⁴ M (n=6), was without effect on intracellular sodium levels, which were significantly different (P<0.001) from those observed when PD 123319 was present.
4. Angiotensin II 10⁻⁵ M, administered to the tubular suspension in the presence of 10⁻⁴ M PD123319, decreased (P<0.01, n=6) intracellular sodium content by 16% in the first 5 min, but in the following 25 min returned to steady state levels. However, in the presence of losartan 10⁻⁴ M, angiotensin II 10⁻⁵ M had no effect on intracellular sodium content which was markedly different (P<0.001) from that obtained in the presence of PD123319.
5. These findings show that at both the high and low concentrations of angiotensin II, its modulation of intracellular sodium levels within the proximal tubule cells is mediated via the activation of AT₁ receptors. The intracellular mechanism underlying this effect remain to be investigated.

     

Mechanism of action of alpha-adrenoceptor activation in single cells freshly dissociated from the rabbit portal vein.

1. The action of noradrenaline was studied in freshly dispersed cells of the rabbit portal vein using microelectrode techniques. 2. In normal physiological salt solution, the ionophoretic application of noradrenaline evoked an alpha-adrenoceptor-mediated depolarization and sometimes a beta-adrenoceptor-mediated hyperpolarization. Experiments were carried out in the presence of propranolol to study the membrane mechanism associated with alpha-adrenoceptor activation. 3. In the current clamp mode of recording, the equilibrium potential of the noradrenaline-evoked depolarization was -1.9 mV. The depolarization was brought about by an increase in membrane conductance. 4. Under voltage clamp conditions, noradrenaline produced an inward current with a reversal potential of -7 +/- 3 mV (mean +/- s.e. mean). 5. The relationship between the noradrenaline-induced inward current and clamp potential was non-linear. Depolarization enhanced the conductance elicited by noradrenaline. 6. Evidence is presented which suggests that an additional conductance mechanism (probably an increase in potassium conductance) is also evoked by alpha-adrenoceptor stimulation in dispersed cells of rabbit portal vein.