A. Filippelli

Nitric oxide participates in the hypotensive effect induced by adenosine A2 subtype receptor stimulation.

In a previous study, we demonstrated that adenosine plays an important role in the central control of the cardiovascular system with involvement of adenosine A2 rather than A1 subtype receptors. In the present study, we investigated the putative relationship between nitric oxide (NO) and adenosine in the central and peripheral control of the cardiovascular system. Adult male nor-motensive anesthetized rats were treated with N6-cyclohexyladenosine (CHA), an A1-punnoceptor agonist, and 5‘-N-cyclopropyl-carboxamidoadenosine (CPCA), an A2-purinoceptor agonist intracerebroventricularly (i.c.v. 3rd ventricle; 0.05–0.1–0.5 μg/rat) and by intravenous injection (0.5–1-5 μg kg−1 i.v.). CPCA and CHA induced a significant and dose-dependent decrease in arterial blood pressure (BP). CHA effects were less marked than CPA. Rats were pretreated with xanthine amine congener (XAC), and A1 adenosine antagonist, with 3,7-dimethyl-1-propargylxanthine (DMPX), an A2 adenosine antagonist (both administered at doses of 0.05 μg/rat i.c.v. or 0.5 μg kg−1 i.v.) and with N+-nitro-L-arginine methyl ester, an NO synthase inhibitor, (L-NAME, 90 μg/rat i.c.v. and 0.3 mg kg−1 i.v.). The intracerebroventricular and intravenous pretreatment with DMPX or L-NAME inhibited CPCA-induced hypotension; the effect of L-NAME was weaker than that of DMPX. The L-NAME inhibitory effect was reversed both in the central nervous system (CNS) and at the peripheral level by pretreatment with L-arginine (L-Arg; 90 mg kg−1 i.v.), a precursor of NO synthesis. Pretreatment with XAC, but not with L-NAME, reduced the hypotensive effect of CHA. Moreover, intracerebroventricular pretreatment with L-Arg (174 μg/rat) increased the hypotensive effect of CPCA. These results suggest that NO partially mediates the hypotensive effect induced by A2 subtype receptors stimulation in the CNS and at a peripheral level.

Effects of docosahexaenoic acid on calcium pathway in adult rat cardiomyocytes

In this study we examined the effect of polyunsaturated fatty acids (PUFAs), in particular of docosahexaenoic acid (DHA), on calcium homeostasis in isolated adult rat cardiomyocytes exposed to KCl, ET-1 and anoxia. Free [Ca(2+)](i) in rat cardiomyocytes was 135.7 +/- 0.5 nM. Exposure to 50 mM KCl or 100 nM ET-1 resulted in a rise in free [Ca(2+)](i) in freshly isolated cells (465.4 +/- 15.6 nM and 311.3 +/- 12.6 nM, respectively) and in cultured cells (450.8 +/- 14.8 nM and 323.5 +/- 14.8 nM respectively). An acute treatment (20 minutes) with 10 microM DHA significantly reduced the KCl- and ET-1-induced [Ca(2+)](i) increase (300.9 +/- 18.1 nM and 232.08 +/- 11.8 nM, respectively). This reduction was greater after chronic treatment with DHA (72 h; 257.7 +/- 13.08 nM and 192.18 +/- 9.8 nM, respectively). Rat cardiomyocytes exposed to a 20 minute superfusion with anoxic solution, obtained by replacing O(2) with N(2) in gas mixture, showed a massive increase in cytosolic calcium (1200.2 +/- 50.2 nM). Longer exposure to anoxia induced hypercontraction and later death of rat cardiomyocytes. Preincubation with DHA reduced the anoxic effect on [Ca(2+)](i) (498.4 +/- 7.3 nM in acute and 200.2 +/- 12.2 nM in chronic treatment). In anoxic conditions 50 mM KCl and 100 nM ET-1 produced extreme and unmeasurable increases of [Ca(2+)](i.) Preincubation for 20 minutes with DHA reduced this phenomenon (856.1 +/- 20.3 nM and 782.3 +/- 7.6 nM, respectively). This reduction is more evident after a chronic treatment with DHA (257.7 +/- 10.6 nM and 232.2 +/- 12.5 nM, respectively). We conclude that in rat cardiomyocytes KCl, ET-1 and anoxia interfered with intracellular calcium concentrations by either modifying calcium levels or impairing calcium homeostasis. Acute, and especially chronic, DHA administration markedly reduced the damage induced by calcium overload in those cells.

The role of A3 adenosine receptors in central regulation of arterial blood pressure

1 Pharmacological studies have suggested that A3 receptors are present on central neurons. Recently this adenosine receptor subtype has been identified in the rat and its presence in the central nervous system has been confirmed. 2 In this study we investigated the effects of acute intracerebroventricular (i.c.v.) injections of N6‐2‐(4‐aminophenyl)‐ethyladenosine (APNEA), a non‐selective A3 adenosine receptor agonist, on arterial blood pressure (ABP) and heart rate (HR), after treatment with 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX), a selective antagonist of A1 adenosine receptors. 3 Anaesthetized rats, after DPCPX (12 μg−1 kg i.c.v.), were treated with APNEA (0.4–4 μg kg−1 i.c.v.) resulting in a transitory and dose‐dependent decrease in arterial blood pressure without a change in heart rate. APNEA also induced hypotensive responses after i.c.v. pretreatment with aminophylline, at a dose of 20 μg kg−1. In contrast, pretreatment 48 h before, with 4 μg kg−1 i.c.v. of pertussis toxin reduced the hypotensive effect induced by APNEA. Administration of APNEA at a higher dose (20 μg kg−1 i.c.v.), after DPCPX, induced a decrease in ABP of −66±5.4 mmHg and after 3 min a decrease in heart rate of −62±6.0 beats min−1. Transection of the spinal cord abolished this significant fall in ABP, but not the decrease of HR. 4 These results suggest that a population of A3‐receptors is present in the CNS, whose activation induces a decrease in blood pressure with no change of heart rate.

Endothelin-1 in periaqueductal gray area of mice induces analgesia via glutamatergic receptors

&NA; The aim of the study was to examine whether endothelin‐1 (ET‐1) injected into dorsolateral periaqueductal gray (PAG) area of mice produces antinociception. ET‐1, from 1 to 4 pmol/mouse, induced antinociceptive effect in a dose‐dependent manner. This antinociceptive effect was prevented by NMDA receptor antagonists (2‐APV and MK‐801) injected in the same area (2‐APV) or by intraperitoneal route (MK‐801). CNQX, a non‐NMDA receptor antagonist, did not inhibit the ET‐1 effects. Prazosin, an &agr;1‐adrenergic blocking agent, also prevented the ET‐1 antinociceptive effect. We suggest that the activation of NMDA glutamatergic receptors in the PAG area may be a necessary step for ET‐1 induced antinociception.

Endothelin-1 in Rat Periaqueductal Gray Area Induces Hypertension Via Glutamatergic Receptors

We investigated the possible relationship between endothelin-1 injection into the dorsolateral periaqueductal gray area and the glutamatergic system in the control of cardiovascular function. Endothelin-1 was injected into the dorsolateral periaqueductal gray area of freely moving rats at doses ranging from 0.1 to 10 pmol. Endothelin-1 increased arterial blood pressure (from 7.0 +/- 1.6 to 55.0 +/- 4.1 mm Hg, mean +/- SEM) in a dose-dependent manner and induced characteristic behavioral changes such as longitudinal rolling of the body (barrel-rolling). DL-2-Amino-5-phosphonovaleric acid and (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[D-alpha] cyclohepten-5,10-imine hydrogen maleate, both selective N-methyl-D-aspartate excitatory amino acid receptor antagonists, but not 6-cyano-7-nitroquinoxaline-2,3-dione, a non-N-methyl-D-aspartate excitatory amino acid receptor antagonist, significantly decreased endothelin-1-induced cardiovascular and behavioral changes (P < .01). Prazosin and propranolol, adrenergic blocking agents, and reserpine, a depletor of catecholamine stores, also prevented these effects. We propose that the glutamatergic system may exert, via N-methyl-D-aspartate receptors, a significant influence on endothelin-1-induced cardiovascular and behavioral effects after its injection into the periaqueductal area.

Cardiotoxicity of doxorubicin: Effects of 21-aminosteroids

The purposes of this study were to investigate in vivo the effects of two lazaroids,U-74389G (21-[4-(2,6-di-1-pyrrolidinyl-4-pyrimidinyl)-1-piperazinyl]-pregna-1,4,9 (11)-triene-3,20-dione (2)-2-butenenedionate) and U-83836E (-)-2-[[4-(2,6-di-1-pyrrlidinyl-4-pyrimidinyl)-1-piperazinyl]methy l]-3,4-dihydro-2,5,7,8-tetramethyl-2H-1-benzopyran-6-ol, dihydrochloride against the cardiotoxicity induced by doxorubicin in rat and the mechanisms underlying such a toxicity. Doxorubicin (DXR) administered intraperitoneally (5 mg/kg 4 times per week for 1 week) induced significant decrease of body weight, ECG alterations and 100% mortality. The lazaroids used in this study did not protect from DXR-induced cardiotoxicity. Our results showed that the compound U-74389G delayed, but did not reduce DXR-induced mortality, and did not prevent body weight loss and ECG changes. The compound U-83836E was unable to modify any toxic effects induced by DXR. These data indicate that oxygen free radicals and the subsequent increase in intracellular calcium are only steps of DXR progressive general toxicity that leads to cardiac injury. In conclusion, we propose that the 21-aminosteroids, potent inhibitors of membrane lipid peroxidation, alone are not enough to protect from DXR toxic effects.

Effects of docosahexaenoic acid on [Ca2+]i increase induced by doxorubicin in ventricular rat cardiomyocytes

The clinical use of doxorubicin (DXR) is limited by cardiotoxicity partially due to interference with intracellular Ca(2+) homeostasis and involving the activation of the sarcoplasmic reticulum (SR) Ca(2+) release channels. It is known that docosahexaenoic acid (DHA) is able to potentiate the sensitivity of cancer cells to DXR. The aim of our study was to further evaluate the effects of DHA on [Ca(2+)](i) overload induced by DXR in adult rat ventricular cardiomyocytes in order to verify if DHA interferes with DXR-induced cardiotoxicity too. [Ca(2+)](i) was measured by microfluorimetry. Our data demonstrated that 100 microM DXR induced a statistically significant [Ca(2+)](i)-increase in cardiomyocytes perfused with CaCl(2) Krebs solution (from 135.7 +/- 15 nM to 560.2 +/- 49 nM, n = 9, p < 0.01) and with Ca(2+)-free Krebs solution (from 89.3 +/- 15 nM to 551.1 +/- 35 nM, n = 9, p < 0.01). Treatment with 10 microM DHA for 20 min significantly suppressed DXR [Ca(2+)](i)- increase in cells perfused with CaCl(2) Krebs solution (142.3 +/- 12 nM, n = 9, p < 0.01) and in Ca(2+)-free procedures (100.4 +/- 12 nM, n = 9, p < 0.01). Caffeine 10 mM significantly increased [Ca(2+)](i) in cardiomyocytes perfused with CaCl(2) Krebs solution (from 135.7 +/- 15 nM to 979.2 +/- 17.8 nM, n = 9, p < 0.01) and with Ca(2+)-free Krebs solution (from 89.3 +/- 15 nM to 891.1 +/- 30 nM, n = 9, p < 0.01). Treatment with 10 microM DHA for 20 min suppressed caffeine [Ca(2+)](i)-increase in cardiomyocytes perfused with CaCl(2) Krebs solution (174.2 +/- 28 nM, n = 9, p < 0.01) and in Ca(2+)-free procedures (161.9 +/- 34 nM, n = 9, p < 0.01). In conclusion, our results suggest that DHA is able to prevent acute modifications of calcium homeostasis induced by DXR probably interfering with SR Ca(2+) release channels.

Involvement of periaqueductal gray area NMDA receptors in endothelin-induced behavioural effects.

We investigated the behavioural effects induced by endothelin-1 injected into the lateral-caudal periaqueductal gray matter of freely moving rats. Endothelin-1 induced a dose-dependent longitudinal rolling of the body (barrel rolling) which was prevented by D,L-2-amino-5-phosphonovalerate (2-APV), an N-methyl-D-aspartate (NMDA) receptor antagonist, but not by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA receptor antagonist. This effect, not reproducible with NMDA alone, indicates that the activation of the NMDA receptors in the periaqueductal gray area may be a necessary, but not sufficient, step for the triggering of endothelin-1-induced barrel-rolling behaviour.

Endothelin-1 increases cholinergic nerve-mediated contraction of human bronchi via tachykinin synthesis induction

In some asthmatics, muscarinic receptor antagonists are effective in limiting bronchoconstrictor response, suggesting an abnormal cholinergic drive in these subjects. There is a growing body of evidences indicating that cholinergic neurotransmission is also enhanced by endothelin‐1 (ET‐1) in rabbit bronchi, mouse trachea and in human isolated airway preparations. We investigated the role of secondary mediators in ET‐1 induced potentiation of cholinergic nerve‐mediated contraction in human bronchi, in particular the possible role of neuropeptides in this phenomenon. Bronchial tissues after endothelin treatment were exposed to a standard electrical field stimulation (EFS) (30% of EFS 30Hz)‐induced contraction. In addition, in some experiments, preparations were treated with a tachykinin NK2 receptor antagonist and subsequently exposed to the same protocol. HPLC and RIA were performed on organ bath fluid samples. Moreover, the human bronchi were used for the β‐PPT (preprotachykinin) mRNA extraction and semiquantitative reverse transcription polymerase chain reaction (RT – PCR), prior to and 30 – 40 min following ET‐1 challenge. The selective tachykinin NK2 receptor antagonist, SR48968, was effective to reduce ET‐1 potentiation of EFS mediated contraction. HPLC or RIA showed significant increased quantities of NKA in organ bath effluents after EFS stimulation in bronchi pretreated with ET‐1. Finally, β‐PPT mRNA level after stimulation of bronchi with ET‐1 was increased about 2 fold respect to control untreated bronchi. In conclusion, this study demonstrated that, at least in part, the ET‐1 potentiation of cholinergic nerve‐mediated contraction is mediated by tachykinin release, suggesting that in addition to nerves, several type of cells, such as airway smooth muscle cell, may participate to neuropeptide production.

Distribution of endothelin-1-receptor subtypes in rat portal vein.

Endothelin-1 (ET-1), a potent vasoactive peptide, was first isolated from cultured porcine endothelial cells. Subsequent studies revealed the existence of two additional related peptides, ET-2 and ET-3, and at least two distinct ET-receptor subtypes, ETA (selective for ET-1) and ETB (nonselective for ET isopeptides). These isopeptides and receptors are widely distributed in many tissues and are involved in numerous biological responses. The aim of this study was to identify the eventual distribution of the two distinct endothelin-receptor subtypes in isolated endothelium-denuded rat portal vein rings (PVRs) and strips (PVSs). BQ-123 (0.6, 1, and 6 microM) and PD-145065 (0.06, 0.1, 0.6, and 6 microM) were used to differentiate the subtypes because they are selective antagonists for ETA and nonselective antagonists for ETA-ETB receptors, respectively. To characterize the ET receptors further, sarafotoxin-S6c (a selective ETB-receptor agonist) and IRL-1038 (a selective ETB-receptor antagonist) were used. In PVRs, cumulative additions of ET-1 (0.1-100 nM) caused graded and slow contractions and potentiated spontaneous rhythmic contractions. The EC50 values and maximal response to 100 nM of ET-1 were 2.72 nM and 0.75 g, respectively (n = 7). PVSs showed ET-1 EC50 values very similar to those of PVRs, but Emax values to 100 nM of ET-1 were significantly lower (Emax = 0.33 g; n = 7). Moreover, ET-1 clearly increased the amplitude and frequency of spontaneous contractions in both types of specimens, although these were greater in the PVSs. Thirty-minute incubation with the selective ETA-receptor antagonist BQ-123 blunted ET-1-induced effects in PVS specimens but only weakly antagonized ET-1-induced contractions in PVRs. In contrast, the nonselective ETA-ETB-receptor antagonist PD-145065 significantly shifted the ET-1 concentration-response curve to the right in PVRs and partially inhibited ET-1 effects in PVSs. Moreover, sarafotoxin-S6c (0.1-100 nM) contracted PVRs and PVSs in a similar manner to ET-1; its effects were antagonized by IRL-1038 only at the PVR level. The differences observed in PVR and PVS specimens in response to agonists and antagonists of ET confirmed the great heterogeneity of endothelin-sarafotoxin receptors. In our experimental models, functionally ETB-like (or non-ETA) receptors seem mostly to mediate vasoconstriction.