Annalisa Rubino

Contribution of P1-(A2b subtype) and P2-purinoceptors to the control of vascular tone in the rat isolated mesenteric arterial bed.

1 The direct vascular effects of adenosine and ATP were compared in the isolated and perfused mesenteric arterial bed of the rat. The actions of analogues of adenosine and ATP were also examined. 2 In preparations at basal tone, adenosine lacked vasoconstrictor actions, while ATP elicited dose‐dependent vasoconstrictor responses. When the tone of preparations was raised by adding methoxamine to the perfusate, adenosine and its stable analogue, 2‐chloroadenosine (2‐CADO) elicited dose‐dependent vasodilatation. The A2 adenosine receptor agonist, 5′‐N‐ethylcarboxamidoadenosine (NECA) was active at lower doses than adenosine, while the A2a‐selective agonist, CGS 21680 and the selective A1 agonist, N6‐cyclopentyladenosine (CPA) failed to induce vasodilatation. ATP and its analogue, 2‐methylthio ATP, elicited dose‐dependent vasodilatation at doses 400 fold lower than adenosine. 3 Vasodilator responses to adenosine and 2‐CADO were sensitive to antagonism by 1 μm 8‐sulphophenyltheophylline (8‐SPT) and were unaffected by inhibition of nitric oxide synthase by Nω‐nitro‐L‐arginine methyl ester (L‐NAME). In contrast, vasodilator responses to ATP were not sensitive to antagonism by 8‐SPT and were almost abolished by L‐NAME treatment. 4 These results indicate that in the rat mesenteric arterial bed, while both adenosine and ATP participate in the purinergic control of vascular tone, adenosine appears to be a weaker vasodilator than ATP and lacks vasoconstrictor action. A2b adenosine receptors account for the adenosine‐induced vasodilatation which is independent of the production of nitric oxide.

Evidence for a P2-purinoceptor mediating vasoconstriction by UTP, ATP and related nucleotides in the isolated pulmonary vascular bed of the rat

1 The vasoconstrictor effects of uridine 5′‐triphosphate (UTP), uridine 5′‐diphosphate (UDP), uridine 5′‐monophosphate (UMP) and uridine were tested in the isolated pulmonary vascular bed of the rat. Comparison was made with the effects of adenine nucleotides, adenosine 5′‐triphosphate (ATP), adenosine 5′‐diphosphate (ADP), adenosine 5′‐monophosphate (AMP) and with adenosine. The effect of P2x‐purinoceptor desensitization and blockade was compared on the vascular responses to uracil and adenine nucleotides. 2 At doses ranging from 10−8 to 10−5 mol, UTP elicited dose‐dependent vasoconstriction. UDP was equiactive to UTP, while UMP and uridine did not show vasomotor activity. Similarly, ATP showed dose‐related vasoconstrictor activity. ADP was less potent than ATP in eliciting vasoconstriction, while AMP was active only at the higher doses tested and adenosine was ineffective. 3 Vasoconstriction was produced by ATP analogues with the following order of potency: α,β‐methylene ATP > ATPγS > β,γ‐methylene ATP > 2‐methylthio ATP ≥ ATP. 4 Desensitization of P2x‐purinoceptors by the selective agonist α,β‐methylene ATP did not modify the vasoconstrictor activity of UTP and UDP and only partially reduced vasoconstrictor responses to ATP, while it abolished vascular responses to α,β‐methylene ATP itself. 5 The antagonists of P2‐purinoceptors, suramin and pyridoxalphosphate‐6‐azophenyl‐2′, 4′‐disulphonic acid (PPADS), did not affect vascular responses to UTP and UDP, but reduced vasoconstriction evoked by β,γ‐methylene ATP and ATP by about 70 and 30%, respectively. 6 This study demonstrates that uracil nucleotides, UTP and UDP, elicit vasoconstriction in the rat pulmonary vascular bed. In addition to confirming the presence of classical P2x‐purinoceptors, these results also suggest the presence of a distinct purinoceptor subtype which mediates UTP‐ and ATP‐evoked vasoconstriction in the rat pulmonary circulation.

Regulation of vascular tone by UTP and UDP in isolated rat intrapulmonary arteries

Vasoconstrictor and vasodilator responses of isolated rat intrapulmonary arteries to the pyrimidine nucleotides UTP and UDP were evaluated and compared with vascular responses to ATP and its analogues. UTP and UDP (1-500 microM) were equipotent in inducing concentration-dependent vasoconstriction, unaffected by the P2 receptor antagonists suramin (100 microM) and Reactive blue 2 (50 microM); ATP (10-500 microM) produced weaker vasoconstriction. UTP and UDP lacked vasodilator activity, while ATP and its analogue 2-methylthio ATP evoked endothelium-dependent vasodilatation. These results indicate that UTP and UDP evoke vasoconstriction of rat intrapulmonary arteries whereas ATP is predominantly a vasodilator at the same arteries. Furthermore, the pharmacological profile of the native UTP/UDP receptor differs from that of the known P2Y2, P2Y4 and P2Y6 recombinant receptors for pyrimidine nucleotides.

Nitric Oxide and Endothelin-1 in Coronary and Pulmonary Circulation

Since the discovery of the vasorelaxant properties of nitric oxide and the vasoconstrictor effect of endothelin-1, there have been many studies of the distribution and functional significance of these agents in various vascular beds. In the coronary and pulmonary circulation nitric oxide and endothelin-1 actions have been largely investigated in terms of an imbalance between the opposing effects of these vasoactive agents leading to pathophysiological conditions. This article review functional and immunocytochemical studies with emphasis on the ultrastructural localization of nitric oxide synthase and endothelin-1 in the coronary and pulmonary vascular beds. Localization of nitric oxide synthase (type III or I or II) has been shown in endothelial cells, smooth muscle, and perivascular nerves of the coronary and pulmonary vascular beds and in the neurons, nerve fibers, and the small granule-containing cells within cardiac ganglia. Endothelin-1 was mainly localized in subpopulations of coronary and pulmonary endothelial cells. These immunocytochemical studies provide information about the sources of nitric oxide and endothelin-1 that contribute to the vasomotor control of cardiac and pulmonary circulation under normal and pathophysiological conditions.

Possible role of diadenosine polyphosphates as modulators of cardiac sensory‐motor neurotransmission in guinea‐pigs.

1. Isolated guinea‐pig atria were used to study the neuromodulatory effect of diadenosine polyphosphates (APnA) on cardiac capsaicin‐sensitive sensory‐motor neurotransmission. 2. In the presence of atropine, guanethidine and propranolol, electrical field stimulation (EFS) of the atrial preparations evoked a positive inotropic response which is known to be mediated by release of calcitonin gene‐related peptide (CGRP) from sensory‐motor nerves. P1,P2‐diadenosine pyrophosphate (AP2A), P1,P3‐diadenosine triphosphate (AP3A), P1,P4‐diadenosine tetraphosphate (AP4A), P1,P5‐diadenosine pentaphosphate (AP5A) and P1,P6‐diadenosine hexaphosphate (AP6A) inhibited in a concentration‐dependent way (0.1‐30 microM) cardiac responses to EFS. The inhibitory effect of APnA was mimicked by adenosine. 3. All the APnA tested had a direct negative inotropic effect, by reducing in a concentration‐dependent manner the basal contractile tension. The inotropism of APnA was comparable to that of adenosine. 4. Both inhibition of cardiac responses to EFS and negative inotropism of AP2A, AP3A and AP4A were sensitive to the antagonism by the A1 adenosine receptor antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX; 0.1‐1 nM). The extent of antagonism of DPCPX for the APnA tested was comparable to that for adenosine. 5. Despite the direct negative inotropism, AP4A tested at the highest concentration used did not affect the cardiac responses to the neurotransmitter CGRP, applied exogenously. 6. These results have demonstrated that in isolated guinea‐pig atria APnA inhibited sensory‐motor neurotransmission, without affecting cardiac responses to exogenous CGRP. The effect of APnA was sensitive to antagonism by DPCPX, which suggests it operates via the activation of prejunctional A1 adenosine receptors. A postjunctional negative inotropism was also shown, mediated by myocardial A1 adenosine receptors.

Recovery after dietary vitamin E supplementation of impaired endothelial function in vitamin E-deficient rats.

1 Thoracic aortae, isolated from rats supplemented with dietary vitamin E after vitamin E deficiency, were analysed for changes in vascular reactivity. 2 Following 4 or 12 months of dietary vitamin E deficiency, endothelium‐dependent vasodilator responses to acetylcholine were significantly impaired. However, when animals were fed after the first 4 months of vitamin E deprivation with a vitamin E‐supplemented diet for 8 months, endothelium‐mediated responses were completely restored. 3 In contrast, the endothelium‐independent vasodilator or vasoconstrictor responses to sodium nitroprusside and noradrenaline, respectively were not altered either by vitamin E deficiency or supplementation. 4 These data indicate that vitamin E supplementation reversed the impairment of endothelial cell function which occurs during vitamin E deficiency.

Enhanced sympathetic neurotransmission in the tail artery of 1,3‐dipropyl‐8‐sulphophenylxanthine (DPSPX)‐treated rats

1 Sympathetic neurotransmission and noradrenaline content of the tail artery of Wistar rats treated for 7 days with the adenosine antagonist, 1,3‐dipropyl‐8‐sulphophenylxanthine (DPSPX), were examined 1 Systolic blood pressure of the DPSPX‐treated rats (164.0 ± 2.9 mmHg; n = 6) was significantly greater than saline‐treated controls (140.0 ±2.8 mmHg; n = 5) after 7 days treatment 3 The pressor responses of the arterial rings to transmural nerve stimulation (65 V, 0.1 ms, 4–64 Hz, for 1 s) were markedly enhanced in the DPSPX‐treated compared with the saline‐treated animals. Both noradrenergic and purinergic components of perivascular sympathetic neurotransmission were enhanced during DPSPX‐induced hypertension 4 Vasoconstrictor responses to exogenous noradrenaline (0.1–300 μm) and adenosine 5′‐triphosphate (0.01–3 mM) were unaffected after DPSPX treatment, indicating prejunctional alteration of sympathetic cotransmission during DPSPX‐induced hypertension 5 Acute exposure to DPSPX (10 μm) did not modify vasoconstrictor responses to transmural nerve stimulation, thus supporting the claim that the enhancement of sympathetic neurotransmission only results from long‐term DPSPX treatment 6 The noradrenaline content of the tail arteries of DPSPX‐treated (4.498 ±0.26 ng cm−1; n = 4) was significantly greater than saline‐treated (3.440 ± 0.30 ng cm−1; n = 5) animals 7 These findings show that chronic inhibition of the actions of endogenous adenosine by DPSPX results in an elevation of systolic blood pressure accompanied by enhanced sympathetic cotransmission and enhanced noradrenaline content of the rat tail artery.

Age‐related changes in purinergic and adrenergic components of sympathetic neurotransmission in guinea‐pig seminal vesicles

1 Purinergic and adrenergic components of sympathetic neurotransmission and contractile responses to exogenous α,β‐methylene ATP and noradrenaline have been investigated in the seminal vesicles of 1‐day (new‐born), 2‐weeks (young), 12‐weeks (adult) and 2‐years old (aged) guinea‐pigs. 2 In seminal vesicles of new‐born guinea‐pigs electrical field stimulation (EFS; 80 V, 0.5 ms for 30 s, 2–32 Hz) evoked tonic frequency‐related contractions. In 2‐weeks old guinea‐pigs the tonic contraction masked an initial phasic component of the neurogenic responses, whereas in 12‐weeks and 2‐years old guinea‐pigs, neurogenic responses were biphasic, a phasic response being followed by a tonic contraction. In all experimental groups, prazosin (10−6 M) blocked the tonic contraction while desensitization of P2X receptors by α,β‐methylene ATP (10−4 M) abolished the phasic responses. 3 The phasic purinergic component of the neurogenic response was significantly higher in 12‐weeks and 2‐years old animals, compared with 2‐weeks old guinea‐pigs. At 32 Hz phasic contractions were (mN mg−1 tissue): 0.047±0.012, 0.018±0.040 and 0.147±0.026 in 2‐weeks, 12‐weeks and 2‐years old guinea‐pigs, respectively. In contrast, the tonic adrenergic component of the neurogenic contraction significantly declined at 12‐weeks and 2‐years compared with 2‐weeks old guinea‐pigs. 4 Contractile responses (mN mg−1 tissue) to the highest concentration of α,β‐methylene ATP tested were significantly higher in 2‐weeks (0.248±0.022) than in 1‐day old animals (0.113±0.012) and decreased in 12‐weeks (0.163±0.016) and 2‐years old guinea‐pigs (0.200±0.008). The pD2 values for the purinoceptor agonist were also significantly lower in adult (4.74±0.20) and aged guinea‐pigs (5.22±0.08) compared with 2‐weeks old animals (5.91±0.27). Conversely, responses to the highest concentration of noradrenaline gradually decreased with age, without significant changes in the pD2 values. Contractile responses to KCl (240 mM) did not differ significantly between the experimental groups. 5 These results demonstrate age‐related changes in purinergic and adrenergic components of sympathetic neurotransmission in the guinea‐pig seminal vesicles. The purinergic component is absent in new‐born animals and it appears fully developed in adult and old guinea‐pigs, while the adrenergic component decreases with age. Pre‐ and postjunctional mechanisms contributing to the age‐related changes of sympathetic neurotransmission are discussed.

Augmented sensory‐motor vasodilatation of the rat mesenteric arterial bed after chronic infusion of the P1‐purinoceptor antagonist, DPSPX

1 . The effect of long‐term antagonism of P1‐purinoceptors on vascular function was examined in the perfused mesenteric arterial bed isolated from rats which had received constant infusion of either the non‐selective P1‐purinoceptor antagonist, 1–3‐dipropyl‐8‐sulphophenylxanthine (DPSPX, 30 μg kg−1 h−1, i.p.) or saline for seven days. Sympathetic and sensory‐motor neurotransmission, smooth muscle and endothelial function were assessed. 2 . Basal tone was similar in mesenteric arterial preparations from control and DPSPX‐treated rats. Continuous perfusion with methoxamine (7–70 μm) induced similar increases in tone in control and DPSPX‐treated preparations. In the presence of guanethidine (5 μm), electrical field stimulation (EFS; 1–12 Hz, 60V, 0.1 ms, 30 s) elicited frequency‐dependent vasodilatation due to activation of sensory‐motor nerves. In tissues from DPSPX‐treated rats the nerve‐mediated vasodilator responses were markedly augmented at all frequencies. Maximal relaxation at 8 Hz was 38.34 ± 4.76% (n = 5) in controls and 65.92 ± 3.68% (n = 5) after DPSPX‐treatment (P < 0.01). Adenosine (3 μm) inhibited the frequency‐dependent sensory‐motor neurotransmission similarly in preparations from controls and DPSPX‐treated rats. 3 . In raised‐tone preparations calcitonin gene‐related peptide (CGRP; 5, 15 and 50 pmol), the principal vasodilator transmitter of sensory‐motor nerves in rat mesenteric arteries, produced similar relaxations in control and DPSPX‐treated preparations. Vasodilator responses to the sensory neurotoxin capsaicin (50 and 500 pmol) were also similar between the groups. 4 . Assay of tissue CGRP levels of the superior mesenteric artery by enzyme‐linked immunosorbent assay showed no significant difference in tissue levels of CGRP in controls, 120.25 ± 26.34 pmol g−1 tissue (n = 6) and with DPSPX‐treatment, 82.12 ± 24.42 pmol g−1 tissue (n = 6). 5 . In raised‐tone preparations dose‐dependent endothelium‐dependent vasodilatation to acetylcholine and ATP, and endothelium‐independent vasodilatation to sodium nitroprusside were similar in control and DPSPX‐treated preparations. 6 . EFS (4–32 Hz, 90V, 1 ms, 30 s) elicited frequency‐dependent vasoconstriction due to activation of sympathetic nerves which was similar in controls and in DPSPX‐treated preparations. Adenosine (10 and 30 μm) inhibited sympathetic neurotransmission similarly in control and DPSPX‐treated preparations. Dose‐dependent vasoconstriction to noradrenaline (NA) and ATP, and to KC1 (0.15 mmol) was similar between the groups. 7 . High performance liquid chromatographic analysis of tissue NA showed no significant difference in NA content of the superior mesenteric artery from DPSPX‐treated (1.38 ± 0.09 ng mg−1, n = 6) and control rats (1.46 ± 0.17 ng mg−1, n = 6). 8 . In conclusion, in rats with hypertension due to 7 days treatment with the P1‐purinoceptor antagonist, DPSPX, there is an increase in sensory‐motor vasodilatation of the mesenteric arterial bed. There is no change in sympathetic nerve, endothelial or smooth muscle function. Augmented sensory‐motor neurotransmission, which does not involve a change in postjunctional responsiveness to CGRP or in the CGRP content of sensory‐motor nerves, could be a compensatory change in response to the DPSPX‐induced hypertension.

Non-adrenergic non-cholinergic (NANC) neural control of the atrial myocardium

1. Current concepts in the regulation of atrial contractility by non-adrenergic non-cholinergic (NANC) sensory nerves are reviewed. 2. There is now evidence that in addition to sympathetic and parasympathetic innervation capsaicin-sensitive sensory nerves contribute to the local control of atrial contractility by releasing NANC transmitters, such as calcitonin gene-related peptide (CGRP). 3. Certain chemical and physical stimuli affect atrial contractility by inducing the release of CGRP from sensory nerves. In addition, as widely recognized for the sympathetic and vagal atrial innervation, NANC neurotransmission is under the inhibitory control of several endogenous modulators. 4. Cardioexcitatory actions of NANC neurotransmission on the atrial myocardium are considered. 5. Pharmacological modulation of NANC neurotransmission and functional evidence for cross-talk between NANC and sympathetic neurones are also discussed.