Neuroeffector actions of prostaglandin D2 on isolated dog mesenteric arteries

Treatment with prostaglandin (PG) D₂ in concentrations (10⁻⁸ to 10⁻⁷ M) insufficient to alter the basal tone potentiated the contractile response of helical strips of dog mesenteric arteries to transmural electrical stimulation but did not influence the response to norepinephrine. The potentiating effect of PGD₂ was not prevented by treatment with diphloretin phosphate, a PG antagonist, whereas contractions of dog cerebral arteries induced by PGD₂ were suppressed. The ³H-overflow evoked by transmural stimulation in superfused mesenteric arterial strips previously soaked in ³H-norepinephrine containing media was significantly increased by PGD₂. It is concluded that PGD₂ increases the stimulation-evoked release of norepinephrine from adrenergic nerves innervating the arterial wall. PGD₂ appears to act differently on receptive sites responsible for increasing the release of norepinephrine and for producing arterial contraction.     

Specific antagonism by metoclopramide of dopamine-induced relaxation on isolated rabbit mesenteric arteries contracted with prostaglandin F2alpha.

On isolated rabbit mesenteric arteries pretreated with phenoxybenzamine (10-5M) and contracted with prostaglandin F_2α (PGF_2α) dopamine (10^?6M to 3×10^?4M) and isoprenaline (10-9M to 10-5M) caused a dose-related relaxation. Pindolol (10^?7M) significantly suppressed the effects evoked by isoprenaline, but did not affect those produced by dopamine. The dopamine receptor antagonist metoclopramide (5×10^?5 and 10^?4M), however, shifted the dose-response curve for dopamine-induced relaxation significantly to the right in a concentration dependent manner without affecting relaxations caused by isoprenaline or papaverine. These results demonstrate for the first time a specific antagonism to dopamine-induced relaxation on rabbit mesenteric arteries $\text{in vitro}$. They support the hypothesis of the existence of specific dopamine receptors in vascular smooth muscles.     

Different responsiveness of a variety of isolated dog arteries to prostaglandin D3

The addition of prostaglandin (PG) D₂ contracted helical strips of dog cerebral, coronary, renal and femoral arteries; the contraction was greatest in cerebral arteries. The contractile response of cerebral arteries was potentiated by aspirin and attenuated by polyphloretin phosphate. In the arterial strips contracted with PGF_2α, PGD₂ elicited a concetration-related relaxation; the relaxation was greatest in mesenteric arteries. In mesenteric arterial strips contracted with norepinephrine, a lesser degree of relaxation was induced, and in the K⁺-contracted arteries, only a contraction was induced. Treatment with PGD₂ attenuated the contractile responses of cerebral and mesentric arteries to PGF_2α or PGE₂; this inhibitory effect was approximately 10 times greater in mesenteric arteries. However, the response to serotonin (for cerebral arteries) or norepinephrine (for mesenteric) was unaffected. It may be concluded that the heterogeneity of response to PGD₂ of a variety of dog arteries is due to different contributions of vasoconstrictor and vasodilator mechanisms. PGD₂ appears top share the mechanism underlying arterial contraction with PGF_2α and PGE₂, and interferes with the effect of these PG's possibly on receptor sites.     

Effects of calcium antagonists and calmodulin inhibitors on angiotensin II-induced prostaglandin productions in the isolated dog renal arteries

Angiotensin II markedly potentiated both PGE2 and PGI2 productions in the isolated dog renal arteries. This angiotensin II-induced response was significantly reduced by the treatments of EGTA and calcium antagonists such as verapamil, nifedipine and 8-(N,N'-diethylamino)-octyl-3,4,5,-trimethoxybenzoate (TMB-8). Calmodulin inhibitors, trifluoperazine and W-7 also inhibited the angiotensin II-induced PG productions while an inactive analogue of W-7, W-5 did not have any effect. The results suggest that angiotensin II may enhance the intracellular Ca2+ level through the influx of extracellular Ca2+ and then, calmodulin activated with Ca2+ will stimulate both PGE2 and PGI2 productions via its activation of phospholipase A2 in the dog renal arteries.     

Nucleotide hydrolytic activity of isolated intact rat mesenteric small arteries.

Segments of isolated intact rat mesenteric small arteries were incubated in physiological bicarbonate buffer in the presence of nano- to millimolar concentrations of ATP. ATP was hydrolysed, and when the vessel was transferred from one incubation to another, the enzyme activity was transferred with the vessel, consistent with the presence of an ecto-ATPase. The substrate, ATP, was shown to induce a modification of the hydrolytic activity which occurred the more rapidly the higher the concentration of ATP. The modified system hydrolysed ATP with a decreased substrate affinity. As the substrate induced a modification of the hydrolytic activity, steady-state velocity measurements for determination of kinetic parameters could not be obtained. Nevertheless, it was possible to compare the modification caused by ATP and UTP, and to compare the hydrolysis rates measured with [32P]ATP, [32P]UTP and [32P]GTP. It was concluded that the hydrolytic activity of the vessels did not distinguish between the nucleoside triphosphates (NTPs). In a histidine buffer, the activity was shown to be activated by micromolar concentrations of either Ca2+ or Mg2+, and not to be influenced by inhibitors of P-type, F-type and V-type ATPases. Functional removal of the endothelium before assay did not reduce the measured NTP hydrolysis. At millimolar concentrations of trinucleotide the hydrolysis rate was 10-15 mumol per min per gram of tissue or 0.11-0.17 mumol per min per 10(6) vascular smooth muscle cells. This value is equivalent to the maximal velocity obtained for the Ca2+ or Mg(2+)-dependent NTPase released to the medium upon 2 s of sonication of the vessels (Plesner, L., Juul, B., Skriver, E. and Aalkjaer, C. (1991) Biochim. Biophys. Acta 1067, 191-200). Comparing the characteristics of the released NTPase to the characteristics of the activity of the intact vessel, they showed a strong resemblance, but the substrate-induced modification of the enzyme was seen only in the intact preparation.     

Differential effects of tetrodotoxin on the sialogenous and vasodilator actions of prostaglandin E2 in the dog salivary gland

Prostaglandin E₂ (PGE₂) (0.1–300 nmol) and acetylcholine (ACh) (0.3–300 nmol) were injected into the glandular artery through which the mandibular gland of the dog was perfused with blood at a constant pressure of about 100 mm Hg. Either substance produced salivary secretion and an increase in blood flow rate in a dose-related manner. The effects of PGE₂ were far more powerful and long-lasting than those of ACh. The potency of PGE₂ was about 1/300 that of prostaglandin F_2α on a molar basis in producing the two effects. During complete nerve-block attained by intra-arterial infusion of tetrodotoxin (0.3 nmol/min) the sialogenous action of PGE₂ was abolished, although not completely, whereas the vasodilator effect remained in larger part. Both sialogenous and vasodilator effects of ACh were not affected by tetrodotoxin. These results indicate that the sialogenous effect of PGE₂ was due in most part to a neural, probably parasympathetic, excitant action, whereas the vasodilator action was due in larger part to a direct one on the vasculature.     

Somatostatin,prostaglandin E2 and atropine inhibition of the gastric actions of bombesin in the dog

Bombesin, acetylcholine, prostaglandins and somatostatin are all thought to be involved in the regulation of gastrin release and gastric secretion. We have studied the effects of low doses of atropine, 16-16(Me)₂-prostaglandin E₂ (PGE₂) and somatostatin-14 on bombesin-stimulated gastrin release and gastric acid and pepsin secretion in conscious fistula dogs. For reference, synthetic gastrin G-17 was studied with and without somatostatin. Bombesin, in a dose-related manner, increased serum gastrin, which in turn stimulated gastric acid and pepsin secretion in a serum gastrin, concentration-dependent manner. Somatostatin inhibited gastrin release by bombesin as well as the secretory stimulation by G-17; the combination of sequential effects resulted in a marked inhibition of bombesin-stimulated gastric acid and pepsin secretion. PGE₂ also strongly inhibited gastrin release and acid and pepsin secretion. Atropine had no significant effect on gastrin release, but greatly inhibited gastric secretion. Thus somatostatin and PGE₂ inhibited at two sites, gastrin release and gastrin effects, while atropine affected only the latter.     

Distinctive effect of angiotensin II on prostaglandin production in dog renal and femoral arteries

The effect of angiotensin II (Ang II) on prostaglandin (PG) production in dog renal and femoral vasculature was examined in vivo and in vitro. In pentobarbital anesthetized dogs, the reduction of blood flow induced by intra-arterial infusion of Ang II was potentiated by pre-treatment with indomethacin (5 mg/kg) in the renal but not the femoral vasculature. Isolated renal and femoral arterial strips were incubated and the release of PGE2 and PGI2 (as 6-keto-PGF1 alpha) into the medium was measured by radioimmunoassay. Basal PGE2 and PGI2 production by renal and femoral arterial strips was approximately the same. PGI2 production was predominant for both strips. Ang II stimulated PG production in renal but not femoral arteries. In the renal artery, Ang II-induced PG production was inhibited by indomethacin (10(-6) M), mepacrine (10(-4) M) and saralasin (10(-6) M). These results suggest that Ang II stimulates PG production by the renal artery per se and the Ang II receptor is linked to phospholipase A2 in the renal but not the femoral artery.     

Effect of polyphloretin phosphate and 7-oxa-13-prostynoic acid on the vasoactive actions of prostaglandin E2 and 5-hydroxytryptamine on isolated human umbilical arteries

Two prostaglandin antagonists, polyphloretin phosphate (PPP) and 7-oxa-13-prostynoic acid (EC-I-148) were examined for their ability and selectivity to block the vasoactive actions of prostaglandin E₂ and 5-hydroxytryptamine on isolated human umbilical arteries. Polyphloretin phosphate was found to be a weak antagonist of prostaglandin E₂ and exhibited a low degree of selectivity since responses to 5-hydroxytryptamine were also blocked. EC-I-148 (3.25 × 10^?5M) demonstrated a strong and selective antagonism to the contractions produced by prostaglandin E₂ because contractions to 5-hydroxytryptamine were not altered. EC-I-148 contracted umbilical artery strips in concentrations which antagonized responses to PGE₂.     

Metabolism of prostaglandin D2 in isolated rat lung: the stereospecific formation of 9 alpha,11 beta-prostaglandin F2 from prostaglandin D2

The metabolic transformation of exogenous prostaglandin D2 was investigated in isolated perfused rat lung. Dose-dependent formation (2-150 ng) of 9 alpha,11 beta-prostaglandin F2, corresponding to about 0.1% of the perfused dose of prostaglandin D2, was observed by specific radioimmunoassay both in the perfusate and in lung tissue after a 5-min perfusion. To investigate the reason for this low conversion ratio, we analyzed the metabolites of tritium-labeled 9 alpha,11 beta-prostaglandin F2 and prostaglandin D2 by boric acid-impregnated TLC and HPLC. By 5 min after the start of perfusion, 9 alpha,11 beta-prostaglandin F2 disappeared completely from the perfusate and the major product formed remained unchanged during the remainder of the 30-min perfusion. The major product was separated by TLC and identified as 13,14-dihydro-15-keto-9 alpha,11 beta-prostaglandin F2 by GC/MS. In contrast, pulmonary breakdown of prostaglandin D2 was slow and two major metabolites in the perfusate increased with time, each representing 56% and 11% of the total radioactivity at the end of the perfusion. The major product (56%) was identified as 13,14-dihydro-15-ketoprostaglandin D2 and the minor one (11%) was tentatively identified as 13,14-dihydro-15-keto-9 alpha,11 beta-prostaglandin F2 based on the results from radioimmunoassays, TLC, HPLC, and the time course of pulmonary breakdown. These results demonstrate that the metabolism of prostaglandin D2 in rat lung involves at least two pathways, one by 15-hydroxyprostaglandin dehydrogenase and the other by 11-ketoreductase, and that the 9 alpha,11 beta-prostaglandin F2 formed is rapidly metabolized to 13,14-dihydro-15-keto-9 alpha,11 beta-prostaglandin F2.     

Allicin relaxes isolated mesenteric arteries through activation of PKA-KATP channel in rat

Allicin is a natural effective organosulfur compound isolated from garlic, which possesses many beneficial properties, such as antibacterial, anti-inflammatory, antimicrobial, hypotensive and hypolipidemic. In the present study, we investigated the effects and the underlying mechanisms of allicin on isolated mesenteric arteries (MAs). We examined MAs relaxation induced by allicin on rat-isolated mesenteric artery (MA) rings, the K_ATP channels with patch, and the expression of Kir6.1 and SUR2B with western blotting and NO production with Diaminofluorescein-FM diacetate (DAF-FMDA) in rat mesenteric artery smooth muscle cells (MASMCs). The results showed that allicin elicited the dose-dependent vasorelaxation effect with phenylephrine (PE) precontracted rat MA rings. The vasorelaxation effect was endothelium and NO independent but could be diminished by inhibition of PKA and K_ATP channels in the vascular smooth muscle. Allicin activated K_ATP channels in rat MASMCs, and the activation of K_ATP channels was inhibited by the inhibitors of PKA and K_ATP channels. But allicin had no effect on the expression of K_ATP subtypes Kir6.1 and SUR2B. These observations suggest that allicin exerts vasorelaxation effect through activation of PKA-K_ATP^-signaling pathway.     

Transmitter release modulated by isoprenaline in the dog isolated mesenteric vein

1. The effects of isoprenaline on the release of transmitter substances from perivascular adrenergic nerves were estimated from the excitatory junction potential (ejp) and outflow of noradrenaline in the dog mesenteric vein. 2. Isoprenaline increased the ejp amplitude and decreased the evoked release of noradrenaline. Yohimbine potentiated the former and converted the latter to an increased outflow. 3. Therefore, stimulation of prejunctional beta-adrenoceptor by isoprenaline increases the release of noradrenaline from perivascular adrenergic nerves. 4. Possible involvement of prejunctional alpha-adrenoceptors in the isoprenaline-induced modulation of transmitter release was also suggested.     

Effect of prostaglandin E2 on vascular reactivity to norepinephrine in isolated rat mesenteric artery,hind limb and splenic artery

The effects of prostaglandin E2 (PGE2) 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 (0.1–64 ng/ml) or indomethacin (0.1–96 μg/ml) in the perfusate did not change the basal pressure. In the mesenteric vascular bed and the hind limb, PGE2 dose-dependently potentiated the vascular response to norepinephrine, whereas PGE2 dose-dependently inhibited the vascular response to noreinephrine in the splenic artery. In these three vascular beds indomethacin in the perfusate dose-dependently attenuated the vascular response to norepinephrine. In the mesenteric artery and the hind limb PGE2 restored the effect of indomethacin, but in the splenic artery PGE2 did not restore the inhibitory effect of indomethacin. These results indicate that the modulating effect of exogenously administrated PGE2 on the vascular action to norepinephrine varies in different vascular beds. It is also suggested that the contribution of endogenous PGE2 synthesized in the vascular wall to the vascular reactivity to norepinephrine is, as well as the effect of exogenous PGE2, different in different vascular beds.     

Methyl ester of prostaglandin D2 as a delivery system of prostaglandin D2 into brain

We examined the permeability of the blood-brain barrier to a methyl ester of prostaglandin D2 and the brain uptake was assessed by radioactivity measurements and radioimmunoassay. When the methyl ester (1 mg/kg) was administered intravenously into mice, it was rapidly taken up by the brain (189 ng/g brain at 30 s) and disappeared from the brain with a half-life of 9 s, whereas it was hardly detectable in the blood. The methyl ester transported into the brain was hydrolyzed to prostaglandin D2 and the time course of prostaglandin D2 levels showed an accumulation phase with a peak at 30 s. The total amount of prostaglandin D2 and its methyl ester was 279 ng/g brain at 30 s after injection, corresponding to 0.5% of the administered dose and being 6-times higher than that after prostaglandin D2 injection. The advantage of the methyl ester over prostaglandin D2 for brain uptake was observed at doses higher than 0.2 mg/kg where the methyl ester which escaped from hydrolysis in the blood was taken up more effectively than prostaglandin D2. In in vitro experiments, the esterase activity on the methyl ester was shown to be 20-times greater in the plasma than in the brain homogenate. These results indicate that the esterification of prostaglandin D2 may serve as a good system for the delivery of prostaglandin D2 into the brain.     

Age-related changes in adenosine 5'-triphosphate-induced constriction of isolated, perfused mesenteric arteries of rats

In isolated mesenteric arteries of rats, dose-dependent increase in perfusion pressure by adenosine 5'-triphosphate (ATP, 0.1 approximately 3000 nmole) diminished with age. ATP responses of both 4- and 32-week-old rats were enhanced by indomethacin (5 microM), and further by the combination of indomethacin and N(G)-nitro-L-arginine methyl ester (L-NAME, 5 microM). The enhancement with each of the treatments was less in 32-week-old rats than that in 4-week-old rats, and there was no enhancement in 75-week-old rats. The ATP response was enhanced by removing the endothelium only in 4-week-old rats. The constrictions in response to ATP (1000 nmole) in both 4- and 32-week-old rats were equally enhanced by reactive blue 2 (30 micromole) and were inhibited by pyridoxal-phosphate-6-azophenyl-2',4-disulphonic acid (PPADS, 30 microM) and alpha, beta-methylene ATP (alpha, beta-mATP, 100 nmole) to a similar extent. The increased tone which was produced by the perfusion with physiological solution containing 100 mM potassium chloride was greater in older animals. This age-related change in the vascular tone disappeared when the responses were potentiated by L-NAME. These results demonstrate that in rat mesenteric arteries, ATP-induced constriction decreases with age. The age-related decline of vasoconstriction is not likely to arise from the changes in the contractility of smooth muscle, from the counterbalancing regulation by the endothelium, or from the cooperation of P2 purinoceptor subtypes. The density of purinoceptors and some post-receptor signal transduction mechanisms in the vascular smooth muscle cells may change with age. The enhanced ATP response might have special physiological significance in rats during development.