Effects of prostaglandins E2 and I2, and arachidonic acid on vascular reactivity to norepinephrine in isolated rat mesenteric artery,hind limb and splenic artery

The effects of prostaglandins E2(PGE2) and I2(PGI2), arachidonic acid, and indomethacin on the vascular reactivity to norepinephrine were tested in three different isolated rat vascular beds (mesenteric artery, hind limb and splenic artery) perfused with the Krebs bicarbonate solution. In these vascular beds PGE2 (0.25 – 16 ng/ml), PGI2 (0.1 – 100 ng/ml), arachidonic acid (0.1 – 10 μg/ml) or indomethacin (5 – 25 μg/ml) in the perfusate did not change the basal pressure. In the splenic artery, both PGE2 and PGI2 attenuated the vascular response to norepinephrine in a dose-related manner. In the mesenteric vascular bed and the hind limb, however, PGE2 potentiated the vascular response to norepinephrine, while PGI2 attenuated this response. Arachidonic acid, a prostaglandin precursor, potentiated the vasoconstrictor response to norepinephrine in the mesenteric artery and the hind limb, whereas in the splenic artery, attenuation of the response to norepinephrine occurred. In these three vascular beds, indomethacin, a prostaglandin synthetase inhibitor, attenuated the vascular response to norepinephrine. In the mesenteric artery and the hind limb, PGE2 and not PGI2 reversed the effect of indomethacin, while in the splenic artery, neither PGE2 nor PGI2 reversed the inhibitory effect of indomethacin. These results suggest that, at least in the rat mesenteric artery and the hind limb where the modulating effect of arachidonic acid is similar to that of PGE2, PGE2 and not PGI2 is a primary endogenous prostaglandin in determining the vascular reactivity to norepinephrine.     

Effects of prostaglandin E1 and indomethacin on fetal and neonatal lamb mesenteric artery responses to norepinephrine.

The effects of prostaglandin E1 (PGE1) and indomethacin on isolated fetal and neonatal lamb mesenteric artery responses to norepinephrine were investigated. PGE1 (1.5 micrometer) significantly reduced vasoconstriction responses to 0.5 to 5 micrometer norepinephrine. Indomethacin (1 micrometer) markedly potentiated the constrictor effects of 0.5 to 10 micrometer norepinephrine. PGE1 prevented the potentiating effect of indomethacin. Neither PGE1 nor indomethacin altered basal muscle tension. These results suggest that endogenous PGs modify adrenergic responses in the isolated mesenteric arteries of preterm and newborn lambs.     

Bile acids are able to reduce blood pressure by attenuating the vascular reactivity in spontaneously hypertensive rats

Effects of the synthetic bile acids on blood pressure were examined in spontaneously hypertensive rats. Continuous intravenous administration of the bile acids at the rate of 1 mg/min for 20 min significantly lowered the blood pressure by 12 mmHg. In order to examine its blood pressure lowering mechanism, the isolated mesenteric arterial perfusion system was employed. Bile acids in the perfusate inhibited vascular reactivity to norepinephrine and KCl in a dose-dependent manner. This inhibitory action diminished as the concentration of potassium in the perfusate decreased. When the perfusate was free from potassium, its inhibitory action completely disappeared. These results in vivo and in vitro studies strongly suggest that bile acids act directly on the vascular beds and attenuate vascular response to norepinephrine.     

Prostaglandins--modulation of adrenergic nervous system.

Prostaglandins (PGs) affect vascular tone by a direct action on the vascular smooth muscle and by influencing vascular reactivity to adrenergic simuli and several vasoactive substances. Thus, in the isolated Tyrode's perfused rabbit renal, mesenteric and splenic vasculature PGE2 inhibited adrenergically induced vasoconstriction. Since the vasoconstrictor responses to renal nerve stimulation were enhanced by the blockade of PG synthesis and were reduced by stimulation of PG synthesis with arachidonic acid, this suggests that PGE2 functions as an inhibitory modulator of the adrenergic nervous system. However, our demonstration that PGE2 enhanced adrenergically induced vasoconstriction in the renal and mesenteric vasculature of the rat, but had opposite effects in the rat splenic vasculature indicates that the modulatory-effect of PGE-compounds on the adrenergic neuromuscular junction is species dependent and varies in different vascular beds within the same species. Prostaglandins, the release of which is evoked by several vasoactive substances including angiotensins, kinins, and adenine nucleotides, may also contribute to the regulation of vascular tone by either opposing or amplifying the vascular actions of vasoactive substances.     

Adenosine as a natural prostaglandin antagonist in vascular smooth muscle

Adenosine has actions on smooth muscle similar to those of prostaglandin (PG) antagonists. Like some PG antagonists it is a phosphodiesterase inhibitor and seems to interfere with calcium effects. It has agonist/antagonist interactions with theophylline, a PG antagonist. In rat mesenteric vascular smooth muscle adenosine blocked responses to noradrenaline which depend on release of intracellular calcium but not those to potassium ions which depend on calcium entry from extracellular fluid. Partial inhibition of endogenous PG synthesis by indomethacin enhanced the adenosine effect. In preparations in which vascular reactivity had been abolished by indomethacin and then partly restored by 1 or 5 ng/ml PGE2, adenosine also inhibited responses to noradrenaline: the curve for the 5 ng/ml PGE2 concentration was to the right of and parallel to the 1 ng/ml curve consistent with a competitive interaction between adenosine and PGE2. Similar interactions between adenosine and PGE2 were shown in human lymphocytes in which activation also depends on calcium release. These findings suggest how calcium-dependent metabolic responses may be controlled and indicate further reasons for caution in the interpretation of cyclic AMP experiments.     

Adrenomedullin release in the rat mesenteric resistance artery

Adrenomedullin (AM) is a potent vasodilator peptide whose major source is the vascular wall. In the present study, the mechanism of release of AM was investigated in the rat mesenteric resistance artery. The isolated mesenteric vascular bed was perfused with Krebs solution at a constant flow rate (5 ml/min) and AM in the perfusate was measured by a highly sensitive enzyme immunoassay (Immunoenzymometric assay; IEMA) method. In preparations without endothelium, spontaneous release of AM was detected in the perfusate (68.7+/-5.8 fmol/ml, n=45). Periarterial nerve stimulation (PNS, 4 and 8 Hz) caused 11.4+/-3.9% (4 Hz) and 9.1+/-3.5% (8 Hz) decreases in the spontaneous release of AM. Removal of Ca2+ from the medium did not affect the spontaneous AM release, but abolished the PNS-induced inhibition of spontaneous AM release. Perfusion of 10nM calcitonin gene-related peptide (CGRP) or 0.1 microM capsaicin (inducer of CGRP release) inhibited significantly the spontaneous AM release. PNS (8 Hz)-induced inhibition of spontaneous AM release was antagonized by CGRP(8-37) (CGRP receptor antagonist). These results suggest that AM is mainly released from vascular smooth muscle cells of the rat mesenteric artery and endogenous or exogenous CGRP inhibits AM release.     

Methyl xanthine phosphodiesterase inhibitors behave as prostaglandin antagonists in a perfused rat mesenteric artery preparation.

The methyl xanthines, theophylline, caffeine and 3-isobutyl-1 methyl xanthine (MIX) inhibited the pressure responses to noradrnealine, angiotensin II and potassium ions in the isolated perfused mesenteric vascular bed of the male rat. The ID50s for inhibition of responses to noradrenaline were 1.85 mug/ml (0.83 x 10(-5) M) for MIX, 18 mug/ml (1 x 10(-4)M) for theophylline and 133 mug/ml (6.8 x 10(-4) M) for caffeine. Similar ID50 concentrations were found for responses to angiotensin II and potassium. We have previously found that substances which inhibit the three pressor agents equally may be prostaglandin (PG) synthesis inhibitors or PG antagonists. Xanthine itself, cyclic AMP and dibutyrl cyclic AMP had no inhibitory effects on the preparation up to concentrations of 10-2 M. Partial inhibition of PG synthesis by indomethacin shifted the % inhibition/log concentration curve to the left, while addition of exogenous PGE2 shifted it to the right. In preparations completely inhibited by sufficient indomethacin added to the perfusate to block PG synthesis, and then restored by adding 1 or 5 ng/ml PGE2 in addition to the indomethacin, the methyl xanthines again inhibited responses suggesting that they were PG antagonists rather than inhibitors of synthesis or release. In preliminary experiments MIX also inhibited effects of PGF2alpha on rat uterus and PGE1 on guinea pig ileum. Effective concentrations of theophylline were similar to the therapeutic levels in human plasma. PG antagonists may be a major action of methyl xanthines requiring reinterpretation of many experiments which have attributed their effects to PDE inhibition. PGs may also be involved in regulating PDE action.     

Effect of angiotensin ii and norepinephrine on release of prostaglandins E2 and I2 by the perfused rat mesenteric artery

Prostaglandin E2 (PGE2) and 6 keto-PGF1 alpha, the stable metabolite of prostacyclin (PGI2), have been measured in the effluent of perfused rat mesenteric arteries by the use of a sensitive and specific radioimmunoassay (RIA) method. The PGE2 and 6 keto-PGF1 alpha were continuously released by the unstimulated mesenteric artery over a period of 145 min. After 100 min of perfusion the release of PGE2 and 6 keto-PGF1 alpha was 45.1 +/- 8.4 pg/min and 254 +/- 75 pg/min respectively, which is in accord with the general belief that PGI2 is the major PG synthesized by arterial tissue. Angiotensin II (AII) (5 ng/ml) induced an increase of PGE2 and 6 keto-PGF1 alpha release without changing the perfusion pressure. The effect of norepinephrine (NE) injections on release of PGs depended on the duration of the stabilization period. The changes of perfusion pressure induced by NE were not related to changes in release of PGs. Thus, it seems that the increase of PG release induced by AII and NE was due to a direct effect of the drugs on the vascular wall. This may represent an important modulating mechanism in the regulation of vascular tone.     

Endothelin inhibits presynaptic adrenergic neurotransmission in rat mesenteric artery

The effect of endothelin(ET) on adrenergic neurotransmission was examined in isolated perfused rat mesenteric arteries. Porcine ET(10(-12) to 10(-10)M) attenuated the pressor response to sympathetic nerve stimulation (NS). It also stimulated the release of prostaglandin E2 (PGE2), but its inhibition of the pressor response to NS was not affected by indomethacin treatment. ET also caused dose-dependent inhibition of [3H]norepinephrine release during NS. Higher doses of ET rather enhanced the pressor response to NS. These results suggest that ET inhibits presynaptic adrenergic neurotransmission without mediation of PGE2, while it potentiates the responsiveness of the postsynaptic alpha-adrenergic receptor. Thus ET appears to act directly on the neuroeffector junction as well as on the peripheral vasculature.     

The neurogenic constrictor response of isolated rat saphenous artery is reduced after 2-week tail suspension

Under real or simulated microgravity conditions the control of arterial vascular tone is greatly disturbed. The low arterial vessel reactivity to sympathetic influences may be the cause of an increase in flow in hind limb skeletal muscles in tail-suspended (TS) rats. Our previous experiments with constant pressure perfusion of rat hind limb demonstrated the reduced vasoconstrictor responses to sympathetic nerve stimulation in TS rats. Responses to exogenous noradrenaline depended on the perfusion conditions. It is known that the vessels of various branching orders noticeably differ in nerve density and in sensitivity to vasoconstrictor agonists. So under neurogenic or exogenous noradrenaline influences the vascular resistance may be increased at different levels of vascular bed, thus making the data analysis seriously complicated. This uncertainty may be overcome by investigation of a single vessel isolated from hind limb vascular bed. The saphenous artery, a resistance artery with dense innervation, is a very convenient object for this purpose. Thus, this study was aimed at comparing the effects of 2-week tail suspension upon the constrictor responses of isolated saphenous artery to neurogenic and exogenous noradrenaline stimuli in rats.     

Indomethacin inhibits responses to all vasoconstrictors in the rat mesenteric vascular bed: Restoration of responses by prostaglandin E2

Indomethacin added to the perfusing buffer inhibited pressor responses to noradrenaline, angiotensin II, arginine vasopressin, histamine, serotonin, calcium ions and potassium ions in the male rat mesenteric vascular bed. For every pressor agent the indomethacin concentration which inhibited response amplitude by 50% was about 7 microg/ml (2.1 × 10^?5 M). With every pressor agent, prostaglandin (PG) E2 could restore normal responsiveness in indomethacin-blocked preparations even while the indomethacin was still present in the buffer. The concentration of PGE2 required was proportional to the concentration of indomethacin. Preparations completely inhibited by indomethacin needed about 5ng/ml PGE2 for complete restoration of normal responses. Aspirin and mefenamic acid could also inhibit responses to all pressor agents tested but with these drugs only a partial restoration could be achieved by PGE2.     

Methyl xanthine phosphodiesterase inhibitors behave as prostaglandin antagonists in a perfused rat mesenteric artery preparation

The methyl xanthines, theophylline, caffeine and 3-isobutyl-1 methyl xanthine (MIX) inhibited the pressure responses to noradrenaline, angiotensin II and potassium ions in the isolated perfused mesenteric vascular bed of the male rat. The ID50s for inhibition of responses to noradrenaline were 1.85 μg/ml (0.83 × 10⁻⁵M) for MIX, 18 μg/ml (1 × 10⁻⁴M) for theophylline and 133 μg/ml (6.8 × 10⁻⁴M) for caffeine. Similar ID50 concentrations were found for responses to angiotensin II and potassium. We have previously found that substances which inhibit the three pressor agents equally may be prostaglandin (PG) synthesis inhibitors or PG antagonists. Xanthine itself, cyclic AMP and dibutyryl cyclic AMP had no inhibitory effects on the preparation up to concentrations of 10⁻²M. Partial inhibition of PG synthesis by indomethacin shifted the % inhibition/log concentration curve to the left, while addition of exogeneous PGE2 shifted it to the right. In preparations completely inhibited by sufficient indomethacin added to the perfusate to block PG synthesis, and then restored by adding 1 or 5 ng/ml PGE2 in addition to the indomethacin, the methyl xanthines again inhibited responses suggesting that they were PG antagonists rather than inhibitors of synthesis or release. In preliminary experiments MIX also inhibited effects of PGF2α on rat uterus and PGE1 on guinea pig ileum. Effective concentrations of theophylline were similar to the therapeutic levels in human plasma. PG antagonism may be a major action of methyl xanthines requiring reinterpretation of many experiments which have attributed their effects to PDE inhibition. PGs may also be involved in regulating PDE action.     

Effect of somatostatin on organ blood flow in anaesthetized cats

Effect of somatostatin infusion (100 ng/min-61 pmol/min) on organ blood flow was studied in anaesthetized cats. Total blood flow in the superior mesenteric, left renal, lienal, inferior caval veins and the sagittal sinus was measured by the H2-clearance method. Vascular resistance decreased in the small intestine, in the hind limb and in the kidney due to somatostatin infusion. Somatostatin seems to have direct vasodilatory effect in different vascular beds.     

Copper inhibits pressor responses to noradrenaline but not potassium. Interactions with prostaglandins E1, E2, and I2 and penicillamine.

Low concentrations of copper inhibited responses to norepinephrine and angiotensin (IC50 3 X 10(-6) M) but not to potassium in rat mesenteric vascular preparations perfused either with buffer or indomethacin and prostaglandin (PGE2). The dose-response curve was not shifted by indomethacin, imidazole, or PGE2 but was moved to the right by 2.8 X 10(-11) M PGE1 and to the left by 2.8 X 10(-7) M PGE1. These effects of copper are similar to the effects of PGI2 in the preparation. Copper moved the PGI2 dose-response curve against noradrenaline in parallel to the left, suggesting that the two were interacting at some point. Penicillamine, which may stimulate PGE1 synthesis, had PGE1-like interactions with the copper effect, suggesting that its value in Wilson's disease may be partly due to antagonism of the biological action of copper as well as to its copper-chelating properties.     

Blunted responses to vasoconstrictors in mesenteric vasculature but not in portal vein of spontaneously hypertensive rats treated with relaxin

Relaxin (RLX), an ovarian polypeptide hormone that is particularly associated with gestation in viviparous species, has recently been shown to decrease blood pressure in virgin spontaneously hypertensive rats (SHR) upon chronic infusion. In this investigation, vascular reactivity to angiotensin II, arginine-vasopressin, and norepinephrine was studied in the perfused mesenteric artery and isolated portal vein of control and RLX-treated virgin spontaneously hypertensive rats. The latter received an intravenous infusion of 75 ng/hr purified rat RLX for 2 days, whereas the controls were given an equal infusion of saline. All of the animals were then killed and their tissues processed for in vitro study. In the perfused mesenteric artery, the concentration-response curves for arginine-vasopressin and norepinephrine were shifted to the right by a factor of about 2 (P less than 0.05 and P less than 0.005, respectively) after RLX treatment. In the isolated portal vein, the response to angiotensin II was not affected; the effect of norepinephrine was slightly displaced to the right (increase in EC50) and the maximum response remained unchanged. These results demonstrate that RLX treatment for 42 hr blunted the vascular response to vasoconstrictor agents in the mesenteric vasculature and are consistent with similar observations reported previously in the same tissue of 20-day-old pregnant rats. It is concluded that RLX may be involved in the blunted response to vasoconstrictor agents during gestation in the rat.     

Phosphoinositide 3-kinase contributes to diabetes-induced abnormal vascular reactivity in rat perfused mesenteric bed

Phosphatidylinositol 3-kinase (PI3K) is a signaling enzyme that plays key roles in vascular growth, proliferation, and cellular apoptosis and is implicated in modulating vascular smooth muscle contractility. The aim of this study was to determine whether PI3K contributes to development of diabetes-induced abnormal vascular reactivity to selected vasoactive agonists. The effect of 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), a selective PI3K inhibitor, on isolated perfused mesenteric vascular bed from streptozotocin (STZ)-diabetic rats was investigated. Changes in perfusion pressure, which reflected peripheral resistance, were measured using isolated perfused mesenteric vascular beds. Our results showed that STZ treatment produced an increase in the vasoconstrictor response to norepinephrine (NE), angiotensin II (Ang II) and endothelin-1 (ET-1), and an attenuated vasodilator response to carbachol and histamine in the isolated perfused mesenteric vascular bed from STZ-diabetic animals. Chronic inhibition of PI3K with LY294002 resulted in prevention of diabetes-induced abnormal vascular reactivity to the vasoactive agonists. However, the high blood glucose levels were not normalized. Results of this study indicate that selective inhibition of PI3K can attenuate the development of diabetes-induced abnormal vascular responsiveness in the isolated perfused mesenteric vascular bed.     

Hydrogen sulfide-induced relaxation of resistance mesenteric artery beds of rats

Hydrogen sulfide (H2S) has been shown recently to function as an important gasotransmitter. The present study investigated the vascular effects of H2S, both exogenously applied and endogenously generated, on resistance mesenteric arteries of rats and the underlying mechanisms. Both H2S and NaHS evoked concentration-dependent relaxation of in vitro perfused rat mesenteric artery beds (MAB). The sensitivity of MAB to H2S (EC50, 25.2 +/- 3.6 microM) was about fivefold higher than that of rat aortic tissues. Removal of endothelium or coapplication of charybdotoxin and apamin to endothelium-intact MAB significantly reduced the vasorelaxation effects of H2S. The H2S-induced relaxation of MAB was partially mediated by ATP-sensitive K+ (KATP) channel activity in vascular smooth muscle cells. Pinacidil (EC50, 1.7 +/- 0.1 microM, n=6) mimicked, but glibenclamide (10 microM, n=6) suppressed, the vasorelaxant effect of H2S. KATP channel currents in isolated mesenteric artery smooth muscle cells were significantly augmented by H2S. L-cysteine, a substrate of cystathionine-gamma-lyase (CSE), at 1 mM increased endogenous H2S production by sixfold in rat mesenteric artery tissues and decreased contractility of MAB. DL-propargylglycine (a blocker of CSE) at 10 microM abolished L-cysteine-dependent increase in H2S production and relaxation of MAB. Our results demonstrated a tissue-specific relaxant response of resistance arteries to H2S. The stimulation of KATP channels in vascular smooth muscle cells and charybdotoxin/apamin-sensitive K+ channels in vascular endothelium by H2S represents important cellular mechanisms for H2S effect on MAB. Our study also demonstrated that endogenous CSE can generate sufficient H2S from exogenous L-cysteine to cause vasodilation. Future studies are merited to investigate direct contribution of endogenous H2S to regulation of vascular tone.     

SQ 14,225 attenuates the vascular response to norepinephrine in the rat mesenteric arteries.

SQ 14, 225, a new angiotensin-converting enzyme inhibitor, attenuated the vascular contraction induced by norepinephrine in the perfused rat mesenteric vascular bed, while SQ 20, 881, an another converting enzyme inhibitor, did not have any effect on the vascular reactivity. Furthermore, this effect of SQ 14, 225 was not altered in the presence of bradykinin or angiotensin II in the perfusate. These results suggest that SQ 14, 225 may have a direct antihypertensive effect which is not mediated by the inhibition of angiotensin-converting enzyme.     

Failure of prostaglandins E1 and E2 to alter canine gastric mucosal cyclic nucleotides.

The action of prostaglandins and indomethacin on gastric mucosal cyclic nucleotide concentrations was evaluated in 18 anesthetized mongrel dogs. Prostaglandins E1 (PGE1) and E2 (PGE2) (25 microgram/kg bolus, then 2 micrograms/kg/min) were administered both intravenously (4 experiments; femoral vein) and directly into the gastric mucosal circulation (10 experiments; superior mesenteric artery). The possible synergistic effect of pre-treatment and continuous arterial infusion of indomethacin (5 mg/kg bolus for 5 min, then 5 mg/min), a prostaglandin synthetase inhibitor, with PGE2 was studied in 4 experiments. Antral and fundic mucosa were biopsied and measured by radioimmunoassay for cyclic nucleotides. Doses of PGE1 and PGE2 which inhibited histamine-stimulated canine gastric acid secretion did not significantly alter antral or fundic mucosal cyclic nucleotide concentrations. Concomitant infusion of PGE2 with indomethacin did not potentiate the mucosal nucleotide response compared to PGE2 alone. These studies fail to implicate cyclic nucleotides as mediators of the inhibitory acid response response induced by PGE1 or PGE2 in intact dog stomach.     

Modification of vasodilator response in streptozotocin-induced diabetic rat

Functional dilatory response in streptozotocin-induced diabetic rats was investigated using thoracic aortas, isolated hearts, and mesenteric beds. Dose-response curves to the PGI2 analogue iloprost on phenylephrine-preconstricted rings of diabetic rats and controls were comparable. In contrast, decreased vasodilation in diabetic rats was observed when dose-response curves to iloprost were performed in hearts and on phenylephrine-preconstricted mesenteric beds. Dose-response curves to forskolin, an adenylyl cyclase activator, performed with hearts and phenylephrine-preconstricted aortic rings and isolated mesenteric beds of diabetic rats and controls were comparable. However, a decreased vasodilation to the ATP-sensitive potassium channel (K(ATP)) activator lemakalim was observed in diabetic hearts, but not in aortic rings and mesenteric beds. In conclusion, under our experimental conditions, diabetes mellitus affects the vasodilation to iloprost in both coronary and mesenteric beds, but not in the aorta. In the heart, this modification of vascular reactivity may be due to a decrease in K(ATP) channel mediated response and not to a decreased activity of adenylyl cyclase. At this time, in the isolated mesenteric bed, the mechanism of this modification in vascular reactivity remains unknown.