A comparison of vasopressin and noradrenaline on oxygen uptake by perfused rat hindlimb, kidney, intestine and mesenteric arcade suggests that it is in part due to contractile work by blood vessels

1. The rat hindlimb, kidney and intestine were each perfused in a nonrecirculating mode at 25 degrees C using an artificial perfusate (initial pressure 85 +/- 5 mmHg) and the effects of vasopressin and noradrenaline on oxygen uptake and perfusion pressure determined. 2. Both vasopressin (K0.5 = 0.1 nM) and noradrenaline (K0.5 = 2 nM) increased oxygen uptake as well as perfusion pressure by the perfused hindlimb; changes in oxygen uptake were closely matched by changes in pressure. The maximum increase in oxygen uptake was approx. 9 mumol/hr per g wet wt of hindlimb. 3. The perfused kidney also responded to vasopressin and noradrenaline with parallel increases in oxygen uptake and perfusion pressure for each agent. The largest increase in oxygen uptake was approx. 30 mumol/hr per g wet wt but this was not maximal. 4. Vasopressin increased oxygen uptake and pressure by the perfused intestine over the range 0.01-2 nM, but the changes in pressure only became significant at doses greater than 0.1 nM. 5. Noradrenaline inhibited oxygen uptake and increased perfusion pressure in a dose-dependent manner at pharmacological concentrations (greater than 30 nM) when shunting of perfusate may have contributed to unperfused regions. 6. A network of mesenteric blood vessels estimated to contain approx. 6% vascular tissue by weight, with the remainder white fat cells, lymphatics and connective tissue, was also perfused. 7. Vasopressin (K0.5 = 0.3 nM) and noradrenaline (K0.5 = 30 nM) each increased oxygen uptake and perfusion pressure in a dose-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prostaglandin action, release and inactivation by rat isolated perfused mesenteric blood vessels.

1 The following experiments were undertaken to confirm that prostaglandin is necessary for noradrenaline to exert its full vasoconstrictor effect in rat mesenteric blood vessels. Prostaglandin release and inactivation were also studied. 2 The cyclo-oxygenase inhibitor, 5, 8, 11, 14-eicosatetraynoic acid caused a significant depression of the concentration-effect curve to noradrenaline. As with indomethacin, responses were restored to control levels by prostaglandin E2 (PGE2) but PGE2 did not restore responses to noradrenaline depressed by papaverine. 3 PGE2-like activity was released from tissues at rest, equivalent to 50 +/- 20 pg PGE2/min. The substance was probably a stable prostaglandin since activity remained on acidifying and extracting into chloroform. The increase in release stimulated by noradrenaline was reduced below resting values by indomethacin. 4 There was a net loss of 7 +/- 1 and 1 +/- 0.2 ng PGE2/min from tissues perfused with 40 and 4 ng/min PGE2 respectively. No uptake occurred at lower PGE2 perfusion rates. 5 When indomethacin was used to depress responses to noradrenaline 15(S)-15-methyl PGE2 methyl ester was 12 times more potent than PGE2 in restoring responses to control values. The cyclic endoperoxide analogue U-46619 caused only partial restoration of indomethacin-depressed responses to noradrenaline but increased perfusion pressure at 2 ng/ml and above. 6 The results confirm that endogenous prostaglandin release, possible of PGE2, is obligatory to the full vasoconstrictor effect of noradrenaline. Noradrenaline increases the amount of prostaglandin released which may be taken up and inactivated by 15-hydroxy prostaglandin dehydrogenase or beta-oxidase. U-46619 may mimic both PGE2 and thromboxane A2.     

A close association between vasoconstrictor-mediated uracil and lactate release by the perfused rat hindlimb.

1. Angiotensin II (5 nM) increased perfusion pressure, O2 uptake and the release of lactate, uracil and uric acid from the perfused rat hindlimb. The release of all three substances was greatest 5 min after commencement of angiotensin II infusion and then decreased over the next 20 min to reach a plateau value that was approx. 2.5-fold basal values. Following removal of angiotensin, pressure, O2 uptake as well as lactate, uracil and uric acid release each returned to pre-infusion (basal) values. 2. Cyanide (1 mM) when added during angiotensin II (5 nM) infusion blocked the pressor effect and completely inhibited all O2 uptake. Cyanide (1 mM) also inhibited the angiotensin-induced increase in uric acid, uracil and lactate release, but the effects differed. Whereas uric acid release remained inhibited throughout the cyanide infusion, uracil and lactate release were only temporarily interrupted and a secondary release of both ensued. 3. Nitroprusside (0.5 mM) when added during angiotensin II (5 nM) infusion blocked pressure and O2 uptake. Lactate and uracil release were partly blocked and returned to pre-infusion (basal) values. However uric acid release was totally blocked and no release occurred when nitroprusside was present with angiotensin II. 4. Combined data showed a significant correlation (r = 0.831; P less than 0.001) between effluent lactate and effluent uracil. 5. It is concluded that lactate and uracil release which increase markedly during vasoconstriction of the hindlimb reflect an association between glycolysis and uracil nucleotide turnover within the same tissue, possibly vascular smooth muscle.     

Arecoline inhibits catecholamine release from perfused rat adrenal gland

AIM: To study the effect of arecoline, an alkaloid isolated from Areca catechu, on the secretion of catecholamines (CA) evoked by cholinergic agonists and the membrane depolarizer from isolated perfused rat adrenal gland. METHODS: Adrenal glands were isolated from male Sprague-Dawley rats. The adrenal glands were perfused with Krebs bicarbonate solution by means of a peristaltic pump. The CA content of the perfusate was measured directly using the fluorometric method. RESULTS: Arecoline (0.1-1.0 mmol/L) perfused into an adrenal vein for 60 min produced dose- and time-dependent inhibition in CA secretory responses evoked by acetylcholine (ACh) (5.32 mmol/L), 1.1-dimethyl-4-phenyl piperazinium iodide (DMPP) (100 micromol/L for 2 min) and 3-(m-choloro-phenyl-carbamoyl-oxy)-2-butynyl trimethyl ammonium chloride (McN-A-343) (100 micromol/L for 2 min). However, lower doses of arecoline did not affect CA secretion of high K(+) (56 mmol/L); higher doses greatly reduced CA secretion of high K(+). Arecoline also failed to affect basal catecholamine output. Furthermore, in adrenal glands loaded with arecoline (0.3 mmol/L), CA secretory response evoked by Bay-K-8644 (10 micromol/L), an activator of L-type Ca(2+) channels, was markedly inhibited, whereas CA secretion by cyclopiazonic acid (10 micromol/L), an inhibitor of cytoplasmic Ca(2+)-ATPase, was not affected. Nicotine (30 micromol/L), which was perfused into the adrenal gland for 60 min, however, initially enhanced ACh-evoked CA secretory responses. As time elapsed, these responses became more inhibited, whereas the initially enhanced high K(+)-evoked CA release diminished. CA secretion evoked by DMPP and McN-A-343 was significantly depressed in the presence of nicotine. CONCLUSION: Arecoline dose-dependently inhibits CA secretion from isolated perfused rat adrenal gland evoked by activation of cholinergic receptors. At lower doses arecoline does not inhibit CA secretion through membrane depolarization, but at larger doses it does. This inhibitory effect of arecoline may be mediated by blocking the calcium influx into the rat adrenal medullary chromaffin cells without the inhibition of Ca(2+) release from the cytoplasmic calcium store. There seems to be a difference in the mode of action of nicotine and arecoline in rat adrenomedullary CA secretion.     

Control of vascular tone by purines and pyrimidines

In this Commentary, the roles of uridine adenosine tetraphosphate as an endothelium-derived contracting or relaxing factor described in the paper by Tölle et al. are considered and put into the wider context of the mechanisms of control of vascular tone by purinergic signalling via receptors located on both smooth muscle and endothelial cells.     

Histamine stimulates renin release from the isolated perfused rat kidney

Summary The renal effects of histamine, histamine receptor agonists and antagonists were studied in the isolated rat kidney, which was perfused with a synthetic medium at constant perfusion pressure in a single pass system.Histamine induced a concentration-dependent increase of renin release ranging from a two-fold increase at 0.5 M to a four-fold increase at 10 M. No change in renal vascular resistance, glomerular filtration rate and sodium excretion occurred. Histamine-H₂-antagonists (ranitidine and cimetidine) were more effective to block the response to histamine than was the histamine H₁-antagonist diphenhydramine.Histamine-H₂-agonists (impromidine and dimaprit, 2.5 M each) were potent stimulators of renin release, their effect was blunted by H₂-antagonists.The histamine-H₁-agonist pyridyl-2-ethylamine had a low stimulatory activity at 10 M final concentration, which may reflect partial H₂-agonistic effects.It is concluded that histamine stimulates renin release via H₂-receptor activation.Part of this work was presented at the 8th Internat. Congress of Pharmacology, Tokyo 1981     

嘌呤与嘧啶受体的生理学意义

在脊椎类和无脊椎类动物的多种组织中 ,嘧啶与嘌呤类物质具有广泛的特异性作用。这些化合物的重要生理学作用可归纳为短时程作用与长时程作用两种类型。其中短时程作用涉及神经信号传递、外分泌和内分泌以及免疫细胞功能的调节作用 ;长时程作用涉及个体发育和再生过程中嘧啶与嘌呤类物质对细胞生长、细胞分化、以及细胞增生过程的影响。     

嘌呤与嘧啶受体的生理学意义

在脊椎类和无脊椎类动物的多种组织中,嘧啶与嘌呤类物质具有广泛的特异性作用.这些化合物的重要生理学作用可归纳为短时程作用与长时程作用两种类型.其中短时程作用涉及神经信号传递、外分泌和内分泌以及免疫细胞功能的调节作用;长时程作用涉及个体发育和再生过程中嘧啶与嘌呤类物质对细胞生长、细胞分化、以及细胞增生过程的影响.     

Atrial natriuretic factor inhibits norepinephrine release evoked by sympathetic nerve stimulation in isolated perfused rat mesenteric arteries

The effect of atrial natriuretic factor (ANF) on [3H]norepinephrine release evoked by sympathetic nerve stimulation was examined in the isolated perfused rat mesenteric arteries. ANF (1 nM to 0.1 microM) caused a dose-dependent inhibition of [3H]norepinephrine release during nerve stimulation. The present result indicates that ANF inhibits noradrenergic neurotransmission in the rat mesenteric arteries through a prejunctional mechanism. This prejunctional effect of ANF may in part contribute to its vasodilation action.     

Cyanide release from nitroprusside in the isolated, perfused, bloodless liver and hindlimbs of the rat

When sodium nitroprusside in artificial medium was perfused through the isolated liver and hindlimbs of a rat at the near physiological flow rate of 8.5 ml min-1, free cyanide was found in the perfusate. The liver reached a steady-state ratio of cyanide released/nitroprusside perfused of about 1.5 (or approximately 30% of the total nitroprusside cyanide) within 15 min, and maintained that rate for about 1.5 hr. In the hindlimbs cyanide was released at a much slower rate (7.5 to 18.8% of the total), and the release did not achieve a steady state even after 1.5 hr. Even after small corrections for cyanide extraction by both tissues, the rate of cyanide release by either tissue was probably more rapid than that resulting from static incubations in blood.     

Nitric oxide synthase-independent release of nitric oxide induced by KCl in the perfused mesenteric bed of the rat

The aim of the present study was to test whether the contractile responses elicited by KCl in the rat mesenteric bed are coupled to the release of nitric oxide (NO). Contractions induced by 70 mM KCl were coincident with the release of NO to the perfusate. The in vitro exposure to the nitric oxide synthase (NOS) inhibitor L-N(omega)-nitro-L-arginine methyl ester, L-NAME (1-100 microM) potentiated the vascular responses to 70 mM KCl and, unexpectedly, increased the KCl-stimulated release of NO. Moreover, even after the chronic treatment with L-NAME (70 mg/kg/day during 4 weeks), the KCl-induced release of NO was not reduced, whereas the potentiation of contractile responses was indeed achieved. The possibility that NOS had not been completely inhibited under our experimental conditions can be precluded because NOS activity was significantly inhibited after both L-NAME treatments. After the in vitro treatment with 1 to 100 microM L-NAME, the inhibition of NOS was concentration-dependent (from 50% to 90%). With regard to the basal release of NO, the inhibition caused by L-NAME was not concentration-dependent and reached a maximum of 40%, suggesting that basal NO outflow is only partially dependent on NOS activity. An eventual enhancement of NOS activity caused by KCl was disregarded because the activity of this enzyme measured in homogenates from mesenteric beds perfused with 70 mM KCl was significantly reduced. On the other hand, endothelium removal, employed as a negative control, almost abolished NOS activity, whereas the incubation with the Ca(2+) ionophore A23187, employed as a positive control, induced an increase in NOS activity. It is concluded that in the mesenteric arterial bed of the rat, the contractile responses elicited by depolarization through KCl are coincident with a NOS-independent release of NO. This observation, which differs from the results obtained with noradrenaline, do not support the use of KCl as an alternative contractile agent whenever the participation of NO is under study.     

Regulation of bone resorption and formation by purines and pyrimidines

Growing evidence suggests that extracellular nucleotides, signalling through P2 receptors, might play important roles in the regulation of bone and cartilage metabolism. ATP and other nucleotides can exert impressive stimulatory effects on the formation and activity of osteoclasts (bone-resorbing cells) in addition to inhibiting bone formation by osteoblasts. In this review, the current understanding of the actions of nucleotides on skeletal cells and the probable receptor subtypes involved are discussed.     

Storage,release, uptake,and inactivation of purines

Adenine nucleotides are released into the interstitial space during platelet thrombus formation and neurotransmission. ATP has also been reported to be released from the heart and endothelial cells in some studies. Ecto ATPase, ADPase, and 5′-nucleotidase activities capable of hydrolyzing ATP sequentially to adenosine are present in many cell types and may serve to terminate the actions of the nucleotides. The opposing effects of adenosine and ATP on the same cell types have suggested a modulatory role for adenosine of the actions of extracellular ATP and that the rates of hydrolysis of nucleotides might be regulated. Consistent with this it has been found that the balance between feedforward inhibition of 5′-nucleotidase by ADP and/or ATP and preferential delivery of AMP from ADPase to 5′-nucleotidase determines the rate of adenosine production and that this differs in different cell types. Alternatively, adenosine may be produced intracellularly as a result of an imbalance between energy demand and supply. There are at least two different cytosolic forms of 5′-nucleotidase. Degradation of ATP during increased metabolic activity results in an increase in intracellular AMP concentration. Either cytosolic enzyme has a high KM (2–5 mM) and would thus respond to this increase with a proportional rise in the rate of adenosine production. The nucleoside transporter is essential to allow the diffusion of adenosine to extracellular receptor sites. In general, adenosine must be taken up via the nucleoside transporter before it is inactivated either by phosphorylation by adenosine kinase in the micromolar range or by deamination by adenosine deaminase at higher concentrations. © 1993 Wiley-Liss, Inc.     

Vascular effects of capsaicin in isolated perfused rat mesenteric bed

The effects of intra- and extraluminal capsaicin administration were evaluated in isolated perfused rat mesenteric bed. Capsaicin (10 nM-1 microM) produced a potent concentration-dependent relaxation of the tonic vasoconstriction induced by norepinephrine (1 microM) but not by high-K+ (60 mM). The capsaicin-induced relaxation was nearly abolished in preparations pretreated in vitro with a high concentration of capsaicin (1 microM, for 10 min, 1 h before). Capsaicin-induced relaxation was reduced but not abolished in preparations obtained from rats pretreated neonatally with capsaicin. The capsaicin effects were unaffected by atropine, guanethidine, propranolol, hexamethonium or tetrodotoxin. The observation that capsaicin (0.1 microM)-induced relaxation was virtually abolished in presence of the proteolytic enzyme alpha-chymotrypsin (1 U/ml) supports the involvement of neuropeptide(s) in this response. Bolus injections of calcitonin gene-related peptide (CGRP) elicited a potent and rapidly ensuing relaxation which underwent tachyphylaxis. However, no cross-desensitization with capsaicin was observed. It is concluded that activation of capsaicin-sensitive sensory fibers could release neuropeptides locally with a potent effect on intestinal blood flow.     

Kinetics of propranolol uptake in muscle, skin, and fat of the isolated perfused rat hindlimb.

The tissue distribution kinetics of a highly bound solute, propranolol, was investigated in a heterogeneous organ, the isolated perfused limb, using the impulse-response technique and destructive sampling. The propranolol concentration in muscle, skin, and fat as well as in outflow perfusate was measured up to 30 min after injection. The resulting data were analysed assuming (1) vascular, muscle, skin and fat compartments as well mixed (compartmental model) and (2) using a distributed-in-space model which accounts for the noninstantaneous intravascular mixing and tissue distribution processes but consists only of a vascular and extravascular phase (two-phase model). The compartmental model adequately described propranolol concentration-time data in the three tissue compartments and the outflow concentration-time curve (except of the early mixing phase). In contrast, the two-phase model better described the outflow concentration-time curve but is limited in accounting only for the distribution kinetics in the dominant tissue, the muscle. The two-phase model well described the time course of propranolol concentration in muscle tissue, with parameter estimates similar to those obtained with the compartmental model. The results suggest, first that the uptake kinetics of propranolol into skin and fat cannot be analysed on the basis of outflow data alone and, second that the assumption of well-mixed compartments is a valid approximation from a practical point of view (as, e.g., in physiological based pharmacokinetic modelling). The steady-state distribution volumes of skin and fat were only 16 and 4%, respectively, of that of muscle tissue (16.7 ml), with higher partition coefficient in fat (6.36) than in skin (2.64) and muscle (2.79).     

Phospholipase inhibition and the mechanism of angiotensin-induced prostacyclin release from rat mesenteric vasculature

Generation of prostaglandins by arterial vasculature of rats was measured by perfusing the isolated mesenteric arterial vascular bed with Krebs-Henseleit solution. The effluent directly superfused bioassay tissues in cascade, and aliquots were collected for subsequent chromatography and radioimmunoassay. Injection of arachidonate (1–10 μg) or angiotensin II (0.1–0.5 μg) through the mesentery caused release of a PGI₂-like substance. After extraction and Chromatographic separation of the mesenteric effluent, it was confirmed by radioimmunoassay that 6-oxo-PGF_1α (the hydration production of PGI₂) is the predominant prostanoid generated from exogenous arachidonate. Release of 6-oxo PGF_1α and PGE₂ from mesentery was also stimulated by injection of angiotensin II (0.05–0.5 μg). Treatment of the mesentery with indomethacin (1 μg/ml) abolished all effects of angiotensin II and arachidonate. Perfusion of the mesentery with dexamethasone (3 μg/ml) or mepacrine (33 μg/ml) both of which have been reported to inhibit phospholipase A₂ activity, reduced PGI₂ release induced by angiotensin II, but did not affect conversion of exogenous arachidonate. It is concluded that PGI₂ is the major prostanoid generated in perfused mesenteric arterial vasculature of rats, and angiotensin II releases PGI₂ by activation of a phospholipase.     

The interaction between noradrenaline and 5-hydroxytryptamine in the isolated perfused rat hindlimb

The vascular response to bolus injections of 5-hydroxytryptamine (5HT) over the range 0.01-10.0 microgram was measured in isolated perfused rat hindlimbs. The hindlimb vasculature displayed a vasoconstrictor response to 5HT, which was potentiated by the addition of 0.01 microgram/ml noradrenaline (NA) to the perfusate at all doses tested. The response to 5HT was also enhanced by the monoamine oxidase (MAO) inhibitor, tranylcypromine (0.05 mg/ml), suggesting that MAO located in the vascular wall is involved in the termination of the response to 5HT in the peripheral circulation. MAO inhibition abolishes the potentiation of 5HT by NA, indicating that this potentiation results from competition for MAO by both amines, leaving more amine intact to activate membrane receptors. Corticosterone (0.01 mg/ml), catecholamine extraneuronal uptake inhibitor, did not alter the response to 5HT, nor the increase of this response by NA, it thus appears that 5HT and NA have different membrane transport systems in the peripheral vasculature.     

Prostanoid-induced modulation of neuropeptide Y and noradrenaline release from the rat mesenteric bed

1. A variety of prostanoids were examined for their ability to alter the periarterial nerve stimulation-induced release of noradrenaline (NA) and neuropeptide Y immunoreactive compounds (NPY-ir) from the perfused mesenteric arterial bed of the rat. 2. Periarterial nerve stimulation (16 Hz) increased the overflow of NA, NPY-ir and perfusion pressure. 3. The prostacyclin (PGI2) analogues, carbaPGI2 and cicaprost both produced a concentration-dependent attenuation of the nerve stimulation-induced increase in NA, NPY-ir overflow and perfusion pressure. 4. The prostaglandin (PG) analogue PGE2 attenuated the evoked increase in NPY-ir overflow as well as a modest decrease in NA. 5. PGE1, sulprostone and iloprost attenuated the nerve stimulation-induced increase in NA overflow but not NPY-ir. 6. Neither PGF2alpha nor the thromboxane A2 analogue U46619 altered the evoked increase in NA or NPY-ir overflow. 7. The results support the view that sympathetic co-transmitter release can be differentially modulated by paracrine/autocrine mediators at sympathetic neuroeffector junctions.     

Vascular actions of 17beta-oestradiol in rat aorta and mesenteric artery

1. It has been proposed that the cardiovascular protective actions of 17beta-oestradiol may involve calcium antagonistic actions. We have examined the effects of 17beta-oestradiol on contractions to noradrenaline and KCl in male rat small mesenteric artery and aorta. 2. In rat mesenteric artery, 17beta-oestradiol (10 microM) significantly reduced the maximum contraction to noradrenaline (67.7 +/- 5.8% of control) and KCl (38.8 +/- 3.1% of control) without affecting potency. 3. In rat aorta, 17beta-oestradiol (10 microM) also significantly reduced contractions to noradrenaline (77.5 +/- 4.8% of control), and the effects were mimicked by droloxifene (10 microM). The effect of oestrogen was not prevented by the protein synthesis inhibitor cycloheximide (10 microM). In experiments carried out in calcium-free solution in which calcium stores were depleted, 17beta-oestradiol (10 microM) significantly reduced the contraction to calcium restoration in rat aorta. 4. In aorta from female rats, 17beta-oestradiol (10 microM) significantly reduced contractions to noradrenaline (73.6 +/- 10.8% of control), but this effect of oestrogen was not prevented by cycloheximide (10 microM). 5. In summary, 17beta-oestradiol diminishes the maximum contractile response to noradrenaline in both rat small mesenteric artery and aorta, an effect which at least in the aorta is mimicked by the oestrogen receptor antagonist/partial agonist droloxifene, and may be due to restriction of calcium entry by a nongenomic action.