Hormonal stimulation of arachidonic release from isolated perfused organs relationship to prostaglandin biosynthesis

The lipids of isolated Krebs perfused rabbit kidneys and hearts were labeled with [¹⁴C]arachidonic acid. Subsequent hormonal stimulation (e.g. bradykinin, ATP) of the pre-labelled tissue resulted in dose-dependent release of [¹⁴C]prostaglandins; little or no release of the precursor [¹⁴]arachidonic acid was observed. When fatty acid-free bovine serum albumin was added to the perfusion medium as a trap for fatty acids substantial release of [¹⁴C]arachidonic acid was detected following hormonal stimulation. The release of [¹⁴C]arachidonic acid was dose-dependent and >;3 fold that of [¹⁴C]prostaglandin release. Indomethacin by inhibiting the cyclo-oxygenase, completely inhibited release of [¹⁴C]prostaglandins and only slightly inhibited release of [¹⁴C]arachidonic acid. These results demonstrate that in both rabbit kidney and heart much more substrate is released by hormonal stimulation than is converted to prostaglandins. This suggests that either the deacylation reaction is not tightly coupled to the prostaglandin synthetase system or that there are two deacrylation mechanisms, one which is coupled to prostaglandin synthesis while the other is non-specific. It has previously been shown that prostaglandin release due to hormones such as bradykinin is transient despite continued presence of the hormone (tachyphylaxis). By utilizing albumin to trap released fatty acid, it was found that hormone-stimulated release of arachidonic acid is also transient. This directly demonstrates that tachyphylaxis occurs at a step prior to the cyclo-oxygenase.     

Changes induced by angiotensins and prostaglandin E2 on the release of transmitter from isolated perfused rabbit kidney during periarterial stimulation.

The influence of A II and PGE2 on the rise of perfusion pressure induced by periarterial stimulation and NA were studied in the rabbit isolated perfused kidney. Periarterial stimulation produced an increase in perfusion pressure and the venous outflow superfusing the rabbit aortic strip caused the muscle to contract. Both effects were found to be frequency dependent. NA induced similar effect when given into the renal artery. A II and its N-terminal analogs produced equal potentiation to periarterial stimulation without altering the effect of exogenous NA when added to the perfusion medium. DMGIA II which is a competitive inhibitor of A II inhibited the potentiating affect of A II. PGE2 also inhibited the effect of A II without altering the effect of exogenous NA. Addition of aspirin to the perfusion medium caused a potentiation to periarteral stimulation but did not change the effect of NA. A II added to the perfusion fluid containing aspirin still caused potentiation. From these results it was concluded that: (i) A II-induced potentiation to periarterial stimulation is mediated via specific receptors and probably due to facilitation of the release of transmitter from sympathetic nerve ending. (ii) PGE2 inhibited the release of transmitter. The effect of A II and PGE2 seemed to be mediated by independent mechanisms.     

Somatostatin release from isolated perfused rat stomach.

Gastric somatostatin release from the isolated rat stomach was studied using a perfusion technique. Somatostatin released from the isolated perfused rat stomach was found to be identical in molecular size and immunoreactively with synthetic somatostatin. Infusion of glucagon (10^?7 M) caused biphasic increase of gastric somatostatin release. Gastric somatostatin release was also stimulated by infusion of theophylline (10^?3 M) and dibutyryl cyclic AMP (10^?3 M). These results indicate the possible involvement of adenylate cyclase-cyclic AMP system in the regulatory mechanism of gastric somatostatin release.     

The prostaglandin system and insulin release. Studies with the isolated perfused rat pancreas

Using the isolated perfused rat pancreas PGE₂ (1 μM and 10 μM) had no effect on basal or glucose (10 and 20 mM)-induced insulin release (IR). PGF_2α stimulated basal IR at 1 μM and inhibited IR at 10 μM. The glucose-induced IR was unaffected by this PG. Furosemide (5 and 10 mM) led to a monophasic IR at low glucose (glu) and to a potentiation of IR at high glu. Only high indomethacin (Indo) (50 μg/ml) inhibited glu-induced IR. The stimulatory effect of furosemide on IR could not be inhibited by indomethacin. However mepacrine (0.1 mM) abolished the furosemide effect. Also glu-induced IR was inhibited by mepacrine. Acetylsalicylic acid (30 mg/100 ml) had no significant influence on glu-induced IR.These findings provide evidence that phospholipase activation rather than increased PG synthesis might primarily be involved in the secretory process of insulin.     

Nitrendipine-induced stimulation of renin release by the isolated perfused rat kidney

The direct effects of the organic calcium antagonist nitrendipine upon renin release were assessed using the isolated rat kidney perfused at constant pressure. This model circumvents the indirect actions of vasodilating agents by artificially maintaining perfusion pressure constant, thereby avoiding the hypotensive effects associated with the systemic administration of such agents. Renin release as assessed by radioimmunoassay was stimulated 2.6-fold upon the administration of 10(-6) M nitrendipine. Since this stimulation of renin release occurred in the absence of any alteration in perfusion pressure, we conclude that it represents a direct action of nitrendipine. This finding is in support of the current hypothesis concerning the inverse relationship between cytosolic Ca2+ and renin secretory rate, and suggests that Ca entry into the juxtaglomerular cells of the juxtaglomerular apparatus is sensitive to blockade by organic calcium antagonists such as nitrendipine.     

Volume-regulatory potassium release from isolated perfused rat kidney

The present study has been performed to test for cell volume regulatory potassium release from the isolated perfused rat kidney exposed to hypotonic perfusate and for its sensitivity to potassium channel blocker barium and calcium channel blocker verapamil. Replacement of 25 mmol/l NaCl with 50 mmol/l mannitol has little effect on effluent potassium activity, whereas subsequent omission of mannitol from the perfusate leads to a transient increase of effluent potassium activity, reflecting volume regulatory potassium release. Barium (1 mmol/l) leads to a marked transient decrease of effluent potassium activity, pointing to net cellular uptake of potassium. Verapamil (1 mumol/l) leads to a slight decrease of effluent potassium activity. Both barium and verapamil virtually abolish the rapid, transient increase of effluent potassium activity upon exposure to hypotonic perfusates. Thus, the substances either block or markedly retard volume regulatory potassium release. The apparent renal vascular resistance is transiently increased by exposure to hypotonic perfusates and by barium, but is reduced by verapamil. Cell volume regulation of isolated perfused mouse straight proximal tubules is retarded but not abolished by verapamil (0.1 mmol/l). In conclusion, cellular potassium release from rat kidney can be determined by continuous measurement of effluent potassium activity. The volume regulatory potassium release and cell volume regulation are impaired by both barium and verapamil. The persisting cell volume regulation could be due either to slow potassium release and/or some mechanism independent of potassium.     

Atriopeptin release from the isolated perfused rabbit heart

Mammalian atrial extracts have been shown to contain bioactive peptides which exert natruiretic, diuretic, and smooth muscle relaxant effects. These extracts include several low molecular weight (< 5,000 Mr) atrial peptides (atriopeptins) which exhibit identical sequences over a central core region which are derived from the high molecular weight peptide (atriopeptigen) precursor which has been purified and sequenced. In the current study we found that extracts of rabbit atria possess both high and low molecular weight bioactive atrial peptides, however, the coronary venous effluent obtained from the isolated perfused rabbit heart only contained the low molecular weight peptide. This trypsin labile activity causes a dose-dependent relaxation of rabbit aorta and chicken rectum assay strips. Separation of the bioactivity with gel filtration chromatography and reversed phase HPLC indicates the heart releases a single substance similar to atriopeptin III. There was no evidence that atriopeptigen was released from the isolated perfused rabbit heart. We suggest that atriopeptigen is proteolytically processed in the atria to an atriopeptin which is subsequently the released form of the atrial peptide.     

Metabolism of deoxycytidine in isolated perfused organs

Summary The metabolism of deoxycytidine-U-¹⁴C was studied in isolated perfused normal and regenerating rat liver, in perfused rat intestine and in isolated perfused mouse liver. A technique to perfuse mouse liver is described. Catabolism of dCyd is very slow in organs of the rat whereas it is rapid in those of the mouse. Nevertheless, incorporation into liver DNA does not differ markedly between the species. The quantitative aspects of dCyd metabolism in the isolated organs are discussed.

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Zusammenfassung Der Stoffwechsel von Deoxycytidin-U-¹⁴C wurde in isoliert perfundierter normaler und regenerierter Rattenleber, in Rattendarm und in normaler Mäuseleber untersucht. Eine Methode zur Perfusion der Mäuseleber wird beschrieben. Der Abbau von Deoxycytidin ist langsam in Rattenorganen, jedoch rasch in Mäuseleber. Dabei sind zwischen den beiden Tierarten keine großen Unterschiede beim Einbau in Leber-DNA zu verzeichnen. Die quantitativen Aspekte des Deoxycytidinstoffwechsels in isolierten Organen werden diskutiert.

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Abbreviations used in text and figures dCyd deoxyoytidine - dThd deoxythymidine - dUrd deoxyuridine - dCMP deoxyoytidine monophosphate - BAIBA ß-aminoisobutyrio acid This is publication No. 754 of the Euratom Biology Division, Contract 078-69 BIAC.     

Corticosteroids inhibit prostaglandin release from perfused mesenteric blood vessels of rabbit and from perfused lungs of sensitized guinea pig.

Infusion of norephinephrine (NE) (1 - 3 mug/ml/min) into the isolated mesenteric vascular preparation of rabbit resulted in a rise in perfusion pressure, which was associated with the release of prostaglandin E-like substance (PGE) at a concentration of 2.81 +/- 0.65 ng/ml in terms of PGE2. Indomethacin (3 mug/ml) abolished the NE-induced release of PGE. Arachidonic acid (0.2 mug/ml) in the presence of indomethacin did not restore the NE-induced release of PGE. Hydrocortisone (10 - 30 mug/ml) and dexamethasone (2 - 5 mug/ml) also inhibited the NE-induced release of PGE. The inhibitory action of both corticosteroids was abolished by arachidonic acid (0.2 mug/ml). Antigen-induced release of a prostaglandin-like substance (PGs) (43.1 +/- 3.8 ng/ml in terms of PGE2 and a rabbit aorta contracting substance (RCS) from perfused lungs of sensitized guinea pigs was completely abolished by indomethacin (5 mug/ml) or by hydrocortisone (100 mug/ml). Indomethacin, however, increased histamine release up to 280% of the control level, which was 470 +/- 54 ng/ml, while hydrocortisone diminished histamine release down to 30% of the control level. A superimposed infusion of arachidonic acid (1 mug/ml) into the pulmonary artery reversed the hydrocortisone-induced blockade of the release of RCS and PGs. It may be concluded that corticosteroids neither inhibit prostaglandin synthetase nor influence prostaglandin transport through the membranes but they do impair the availability of the substrate for the enzyme.     

Effect of prostaglandin E2 on the pressor response to periarterial stimulation and norepinephrine of the isolated perfused rabbit kidney.

The interaction of prostaglandin E2 (PGE2) and aspirin with the responses to peri-arterial stimulation (PS) and norepinephrine (NE) was studied in the isolated kidney of rabbit perfused through the renal artery at constant flow with Krebs' solution. NE and PS increased vascular perfusion pressure of kidney and caused a contraction on the isolated rabbit aortic strip superfused with the effluent from kidney. Addition of PGE2 to the perfusion medium decreased the PS-induced rise in perfusion pressure without changing the effect of exogenous NE. In contrast, addition of aspirin to the perfusion medium induced a potentiation of the response to PS but not to NE. These results suggest that PGE2 modulates the effect of PS probably by inhibiting the releases of NE from sympathetic nerve endings.     

Cholinergic stimulation of neuropeptide Y secretion from the isolated perfused rat stomach.

Neuropeptide Y (NPY) is present in both extrinsic sympathetic adrenergic nerve terminals and intrinsic nerves of the gastrointestinal (GI) tract. Based on this localization a number of functions have been attributed to GI NPY including regulation of blood flow, intestinal fluid and electrolyte transport, and motility. There is nothing currently known, however, about the regulation of its secretion from GI nerves. The effect of cholinergic agonists and antagonists on secretion of NPY immunoreactivity (NPY-IR) from the isolated perfused rat stomach was investigated in the present study. Perfusate samples were extracted and concentrated on SepPak cartridges. Basal levels of NPY-IR varied between 98 and 147 fmol/min. Release was stimulated by high potassium concentrations (50 mM) and acetylcholine (ACh; 1 microM). ACh-induced secretion was unaffected by atropine, but inhibited by hexamethonium. Further evidence for a nicotinic component in the regulation of NPY-IR secretion was obtained by the finding of hexamethonium-induced reduction in basal secretion and stimulation of secretion by 1,1-dimethyl-4-phenyl-piperazinium (DMPP). In conclusion, cholinergic agonists and antagonists can modulate gastric NPY-IR secretion, and the cholinergic stimulatory effects are probably mediated via nicotinic receptor stimulation at the level of the intrinsic ganglia.     

Perfusion rate dependent prostaglandin release from isolated rabbit kidneys

Isolated rabbit kidneys were perfused with 37 degrees C Krebs-Henseleit solution aerated with 95% O2 + 5% CO2. Perfusion rate was varied from 1 to 10 ml/min. This was accompanied by parallel changes of perfusion pressure, prostaglandin excretion and release of radioactivity from kidneys with 14C-arachidonic acid incorporated into the tissue lipid pool. It is suggested that enhancement of perfusion rate raises the intrarenal pressure which increases renal prostaglandin release due to increased substrate availability.     

Perfusion rate dependent prostaglandin release from isolated rabbit kidneys

Isolated rabbit kidneys were perfused with 37°C Krebs-Henseleit solution aerated with 95% O₂ + 5% CO₂. Perfusion rate was varied from 1 to 10 ml/min. This was accompanied by parallel changes of perfusion pressure, prostaglandin excretion and release of radioactivity from kidneys with ¹⁴C-arachidonic acid incorporated into the tissue lipid pool. It is suggested that enhancement of perfusion rate raises the intrarenal pressure which increases renal prostaglandin release due to increased substrate availability.     

Carbamylcholine, but not somatostatin or neurotensin, stimulates prostaglandin E2 release from the isolated perfused rat stomach

Prostaglandin E2 release by carbamylcholine (10(-6) M), somatostatin (10(-10)-10(-8) M) and neurotensin (10(-10) - 10(-8) M) has been evaluated in the isolated perfused rat stomach. Carbamylcholine significantly stimulated gastric PGE2 release and increased the perfusion pressure, whereas somatostatin and neurotensin had no effect. Combination of carbamylcholine with somatostatin or neurotensin produced no increase over that found with carbamylcholine alone. The relationship between perfusion-pressure and PGE2 release was not causal. The present findings do not support a role for prostaglandins in the mechanism of somatostatin or neurotensin action in the stomach.     

Corticosteroids inhibit prostaglandin release from perfused mesenteric blood vessels of rabbit and from perfused lungs of sensitized guinea pig

Infusion of norephinephrine (NE) (1 – 3 μg/ml/min) into the isolated mesenteric vascular preparation of rabbit resulted in a rise in perfusion pressure, which was associated with the release of a prostaglandin E-like substance (PGE) at a concentration of 2.81 ± 0.65 ng/ml in terms of PGE₂. Indomethacin (3 μg/ml) abolished the NE-induced release of PGE. Arachidonic acid (0.2 μg/ml) in the presence of indomethacin did not restore the NE-induced release of PGE. Hydrocortisone (10 – 30 μg/ml) and dexamethasone (2 – 5 μg/ml) also inhibited the NE-induced release of PGE. The inhibitory action of both corticosteroids was abolished by arachidonic acid (0.2 μg/ml). Antigen-induced release of a prostaglandin-like substance(PGs) (43.1 ± 3.8 ng/ml in terms of PGE₂ and a rabbit aorta contracting substance (RCS) from perfused lungs of sensitized guinea pigs was completely abolished by indomethacin (5 μg/ml) or by hydrocortisone (100 μg/ml). Indomethacin, however, increased histamine release up to 280% of the control level, which was 470 ± 54 ng/ml, while hydrocortisone diminished histamine release down to 30% of the control level. A superimposed infusion of arachidonic acid (1 μg/ml) into the pulmonary artery reversed the hydrocortisone-induced blockade of the release of RCS and PGs. It may be concluded that corticosteroids neither inhibit prostaglandin synthetase nor influence prostaglandin transport through the membranes but they do impair the availability of the substrate for the enzyme.     

Corticosteroids inhibit prostaglandin release from perfused mesenteric blood vessels of rabbit and from perfused lungs of sensitized guinea pig

Infusion of norephinephrine (NE) (1 – 3 μg/ml/min) into the isolated mesenteric vascular preparation of rabbit resulted in a rise in perfusion pressure, which was associated with the release of a prostaglandin E-like substance (PGE) at a concentration of 2.81 ± 0.65 ng/ml in terms of PGE₂. Indomethacin (3 μg/ml) abolished the NE-induced release of PGE. Arachidonic acid (0.2 μg/ml) in the presence of indomethacin did not restore the NE-induced release of PGE. Hydrocortisone (10 – 30 μg/ml) and dexamethasone (2 – 5 μg/ml) also inhibited the NE-induced release of PGE. The inhibitory action of both corticosteroids was abolished by arachidonic acid (0.2 μg/ml). Antigen-induced release of a prostaglandin-like substance (PGs) (43.1 ± 3.8 ng/ml in terms of PGE₂ and a rabbit aorta contracting substance (RCS) from perfused lungs of sensitized guinea pigs was completely abolished by indomethacin (5 μg/ml) or by hydrocortisone (100 μg/ml). Indomethacin, however, increased histamine release up to 280% of the control level, which was 470 ± 54 ng/ml, while hydrocortisone diminished histamine release down to 30% of the control level. A superimposed infusion of arachidonic acid (1 μg/ml) into the pulmonary artery reversed the hydrocortisone-induced blockade of the release of RCS and PGs. It may be concluded that corticosteroids neither inhibit prostaglandin synthetase nor influence prostaglandin transport through the membranes but they do impair the availability of the substrate for the enzyme.