Nicotine-induced stimulation of steroidogenesis in adrenocortical cells of the cat.

1. The effect of nicotine on steroid production and release from trypsin-dispersed cat adrenocortical cells was investigated. 2. Nicotine, like adrenocorticotrophin (ACTH), elicited a dose-dependent increase in steroidogenesis, which depended upon the presence of calcium in the medium. 3. Augmented steroid production evoked by submaximal concentrations of ACTH monobutyryl cyclic adenosine 3',5'-monophosphate (AMP), or prostaglandin E2 was further enhanced by steroidogenic concentrations of nicotine. 4. These results are discussed in relation to the possible mode of action of nicotine on cortical cells and to the potential consequences of smoking during stress.     

The release of catecholamines from the isolated cat heart by mephentermine

Summary In the Langendorff preparation of the cat heart superfusing an isolated segment of rabbit intestine, mephentermine released a substance into the perfusate which produced a relaxation of the intestine.The intestine-relaxing substance was not released by mephentermine from a heart taken from an animal pretreated with reserpine, but was again released following recovery from an infusion of levarterenol into the heart.The relaxing substance was released by mephentermine after treatment of the heart with dichloroisoproterenol, but the effects on the heart were blocked.Cocaine blocked both the release of this substance and the effects of mephentermine on the heart. Cocaine also blocked the action of nicotine.Nicotine released the relaxing substance under identical conditions with the exception of the reserpinized preparation where the action of nicotine was not readily restored by an infusion of levarterenol.Fluorometric analysis of the perfusate during the effect of a high concentration of mephentermine demonstrated a mean catecholamine concentration of 0.35 ng/ml.It is concluded that mephentermine releases a catecholamine, presumably norepinephrine, from the heart.With 6 Figures in the TextDedicated to Professor Otto Krayer on his 65th birthday.This investigation was supported in part by Public Health Service Research Grant HE-05291 and in part by grants from Wyeth Laboratories, Philadelphia.     

Compartmental prostaglandin release by angiotensin II and arginine-vasopressin in rabbit isolated perfused kidneys

The compartmental effects of angiotensin II (AII) and arginine-vasopressin (AVP) on renal prostaglandin (PG) formation were studied in the isolated perfused kidney of the rabbit by superfusion bioassay of venous and ureteral effluents (VE and UE) and radioimmunoassay (RIA). Comparable results were obtained with either bioassay or RIA when used to quantitate renal PG release. The effects on PG release into the VE were similar for AII and AVP, as were their pressor responses. However, their effects on PG release into the UE differed markedly. AII resulted in a 6-fold greater urinary efflux than venous of bioassayable PGs, whereas AVP-induced PG release into UE was slightly less than PG efflux into the VE at all doses of the peptide. The profile of released PGs varied according to the sampling source (VE or UE). Moreover, each peptide released a similar profile of PGs at all doses, i.e. UE PGE2 greater than PGF2 alpha greater than 6-keto PGF1 alpha; VE PGE2 greater than 6-keto PGF1 alpha greater than PGF2 alpha (TxB2 was not detected in either effluent). Thus, renal vascular PG release is similar for the vasoactive peptides, AII and AVP, whereas the urinary efflux of PGs is considerably greater in response to AII.     

Effects of heavy metals on corticosteroid production in cultured rat adrenocortical cells

Application of 10(-4) M Zn, 10(-5) M Ba, 10(-6) M Se, 10(-6) M Cd, 10(-6) M Hg, and 10(-6) M Mn did not affect the adrenocorticotrophic hormone (ACTH)-induced steroid production in cultured adrenocortical cells. The application of 10(-5) M Pb significantly reduced the ACTH-induced steroid production in cultured cells. However, Pb did not reduce the steroidogenesis induced by dibutyryl cyclic AMP (Dbc-AMP), suggesting that the plasma membrane is the site where Pb interferes with steroid production. The morphological changes induced by the addition of ACTH or Dbc-AMP plus the test metals were similar to those induced by ACTH or Dbc-AMP alone.     

Neurotransmitted effect of prostaglandin F2 alpha in isolated cat jejunum

Prostaglandin F2 alpha (PGF2 alpha) (1 nM-0.1 microM) increased the amplitude of the spontaneous phasic contractions and produced tonic contraction of the isolated longitudinal muscle from the cat jejunum. The amplitude of the electrically-induced contractions increased by PGF2 alpha (1 nM-10 nM) in a dose-dependent degree. PGF2 alpha decreased the inhibitory effect of indomethacin (30 microM) on the contractile response to electrical stimulation. Hexamethonium did not influence the PGF2 alpha effect on the electrically-induced contractions but inhibited the PGF2 alpha-produced tonic contractions. The contractile effect of PGF2 alpha depended on the number of impregnated nerve cells and terminals preserved in the preparations long after dissection. The data suggest that PGF2 alpha increases the acetylcholine release and that there is a "stepwise" action of PGF2 alpha on the transmitter release from the postganglionic or preganglionic cholinergic terminals depending on the PGF2 alpha concentration and leading to phasic or tonic smooth-muscle contractions.     

Effects of adrenaline, noradrenaline, isoprenaline and salbutamol on the production and release of renin by isolated renal cortical cells of the cat.

1 Isolated renal cortical cells of the cat have been demonstrated to produce renin on incubation in vitro. After 2 h of incubation, without added agonist, the total amount of renin in the flask increased by a mean of 27.2 percent. The increase in renin content of the incubation flask was found to be present in the medium. 2 Noradrenaline (1.18 times 10-minus 4M) and adrenaline (1.09 times 10-minus 4M) added to the incubation medium stimulated renin production by 45 and 34% respectively, compared with the incubated controls. Most of the increase in renin production was present in the incubation medium. 3 Isoprenaline did not stimulate renin production. However, when added to the incubation medium at a concentration of 0.72 times 10-minus 4M there was a significant decrease in the cellular content and a significant increase in the medium content of renin. This increase was at least as great as that observed with adrenaline and noradrenaline. 4 Salbutamol had an effect similar to isoprenaline, i.e. it induced the release of renin into the medium without affecting production. In this respect it was about a third as potent as isoprenaline.     

Inhibition by ethanol and mepacrine of phospholipase-dependent prostaglandin release from the isolated perfused rat lung

To investigate the possibility that millimolar concentrations of ethanol have a membrane-directed inhibitory effect on phospholipase A2 and prostanoid generation (suggested from previous platelet experiments), we studied the release of prostacyclin, thromboxane A2 and prostaglandin E2 from isolated perfused rat lung. Prostanoid release was evoked by arachidonic acid, bradykinin and ionophore A23187 and was measured after extraction by radioimmunoassay. In these experiments, prostanoid release is dependent upon biosynthesis from fatty acid precursors as there is no endogenous prostanoid storage pool. Arachidonic acid and bradykinin caused enhanced release of more prostacyclin than thromboxane A2 with much less prostaglandin E2 and no detectable prostaglandin F2 alpha, whereas A23187 released equal proportions of prostacyclin and thromboxane A2 with less prostaglandin E2. Ethanol at 50 mM resembled mepacrine (46 microM) in that prostanoid release in response to bradykinin and A23187 was highly significantly reduced with little effect on release induced by arachidonic acid. We suggest that ethanol, like mepacrine, interferes with prostaglandin generation by an action at the phospholipase step. This may be secondary to a physical effect on membrane configuration.     

Mechanism of histamine release by alpha-chymotrypsin from isolated rat mast cells.

Alpha-chymotrypsin (CT) was modified chemically and physically by the treatments with diisopropyl fluorophosphate, L-(1-tosylamide-2-phenyl) ethylchloromethylketone, hydrogen peroxide and heat. After these treatments, CT lost or decreased both the enzymic activity and ability of releasing histamine from rat mast cells. Ca++ was essential for histamine release by CT, while it enhanced only slightly the enzymic activity. Process of histamine release by CT could be separated into two stages: CT-dependent but not Ca++-dependent, and Ca++-dependent but not CT-dependent. The activated state of mast cells produced by CT decayed rapidly at 37 degrees C in the absence of Ca++, but these cells responded to Ca++ by adding CT once again, suggesting reconstitution of cell membrane structure affected by CT. Isoproterenol, epinephrine, prostaglandin E1, and dibutyryl-cyclic AMP (0.01-0.1 mM) did not inhibit release of histamine induced by CT. Neither theophylline (0.01-0.1 mM) alone nor the combinations of these cyclic AMP-active agents with theophylline inhibited the release of histamine. But, in the presence of papaverine (0.01-0.1 mM) a marked, dose-dependent inhibition was observed. These data suggest that 1) release of histamine by CT from rat mast cells is causally related to its hydrolytic activity, 2) this activity causes a reversible change on mast cell membrane which probably facilitates Ca++-influx through the cell membrane, and 3) there are subtle differences among CT, compound 48/80 and antigens concerning the effect of cyclic AMP-active agents in histamine-releasing mechanisms in mast cells.     

Reflex bradycardia and hypotension produced by prostaglandin F2alpha in the cat.

1 Intravenous administration of prostaglandin F2alpha results in a dose-dependent increase in pulmonary arterial pressure, decrease in systemic arterial pressure and a delayed bradycardia. Pulmonary vasoconstriction was observed at doses as low as 0.1 and 0.3 mug/kg. The systemic depressor and heart rate lowering effects were observed at 1 mug/kg doses and above. 2 A moderate bradycardia was still observed after atropine blockade but was abolished following bilateral vagotomy. Neither of these procedures affected the pulmonary vascular response. 3 Injections of submaximal doses of prostaglandin F2alpha (1--4 mug/kg) produced a greater and longer lasting bradycardia when injected into the left atrium than was observed following intravenous administration. In addition the latency of onset was much shorter following left atrial injection. These doses resulted in no change in heart rate and a minimal hypotension when injected into the brachiocephalic artery or into the aortic arch. 4 Small doses of prostaglandin F2alpha administered at the level of the origin of the coronary arteries produced marked decreases in heart rate and blood pressure whereas no change occurred following injection of the same amount into the ascending aorta at more distal sites. 5 These results suggest that prostaglandin F2alpha produces bradycardia and hypotension in the cat by activating 'receptors' located in the left heart or by acting on structures perfused by means of the coronary arteries.     

Procaine inhibits cyclic AMP-induced steroidogenesis in isolated bovine adrenocortical cells

Effects of procaine on dibutyryl adenosine 3',5'-cyclic monophosphate ((Bu)2cAMP)-, Ca2(+)- or forskolin-induced steroidogenesis were examined in isolated bovine adrenocortical cells. Procaine (less than 1.0 mM) caused a marked suppression of (Bu)2cAMP- or forskolin-induced steroidogenesis in the absence of extracellular Ca2+, but did not affect on Ca2(+)-induced steroidogenesis in the cells. (Bu)2cAMP decreased the cell associated 45Ca2+. However, procaine (300 microM) inhibited this effect of (Bu)2cAMP. These results suggest that procaine may abolish (Bu)2cAMP-induced Ca2+ release from intracellular calcium store(s) and inhibits steroidogenesis.     

Regional differences in the prostanoid receptors mediating prostaglandin F2 alpha-induced contractions of cat isolated arteries

U46619, a stable thromboxane A2 (TXA2) mimetic, and prostaglandin F2 alpha (PGF2 alpha) contracted helical strips of cat coronary, renal and mesenteric arteries in a concentration-dependent manner. The EC50 values for U46619 did not differ significantly in these arteries, but those for PGF2 alpha were in the order of coronary less than renal less than mesenteric arteries. Contractions induced by U46619 were antagonized by S-145, a selective TXA2 receptor antagonist, with similar activity in these arteries. On the other hand, contractions induced by low concentrations of PGF2 alpha (10(-9) to 10(-7) M) were not influenced by treatment with S-145 in coronary arteries, although those induced by high concentrations (5 x 10(-7) to 10(-5) M) were partially attenuated. These contractions resistant to the TXA2 antagonist were antagonized by diphloretin phosphate (DPP), a non-selective PG antagonist. Contractions induced by PGF2 alpha (5 x 10(-7) to 5 x 10(-5) M) in mesenteric arteries were inhibited by S-145 in a concentration-dependent manner. Contractions induced by PGF2 alpha in renal arteries were partially inhibited by S-145. The inhibitory activity of S-145 to PGF2 alpha-induced contractions at EC50 was in the order of coronary less than renal less than mesenteric arteries. Treatment with indomethacin slightly potentiated the contractions induced by PGF2 alpha in mesenteric arteries. Removal of the endothelium did not affect the contractile responses induced by PGF2 alpha and the inhibitory activity of S-145 in the arteries. These results suggest that the contractile responses induced by low concentrations of PGF2 alpha (up to 10(-7) M) are associated with their action via PG receptor(s), which is different from TXA2 receptor, and those induced by high concentrations of PGF2 alpha (5 x 10(-7) M or higher) interact with TXA2 receptors in cat vascular smooth muscles. It appears that the functional expression of this PG receptor(s) is greater in coronary arteries than in renal arteries, and that it is not found in mesenteric arteries.     

Influence of extracellular calcium and calcium antagonists on contractions induced by potassium and prostaglandin F2 alpha in isolated cerebral and mesenteric arteries of the cat.

1 The effects of a number of calcium antagonists (diltiazem, nifedipine, nimodipine and verapamil) have been studied on feline isolated pial arteries contracted by potassium (127 mM) or prostaglandin F2 alpha (PGF2 alpha, 2.5 microM) and mesenteric arteries contracted by potassium (127 mM). 2 Withdrawal of Ca2+ from the extracellular medium for 30 min reduced the contractile response to potassium in cerebral vessels by 92% and in mesenteric vessels by 96%. Subsequent addition of Ca2+ caused reproducible contractions which were inhibited by both nifedipine and nimodipine. 3 The four calcium antagonists relaxed the isolated middle cerebral artery contracted either by potassium or PGF2 alpha, and mesenteric arteries contracted by potassium, in the following order of potency: nimodipine greater than nifedipine greater than verapamil greater than diltiazem. 4 Nimodipine was more potent than nifedipine in cerebral arteries, and more potent in cerebral than in mesenteric arteries. Otherwise, the potassium-contracted cerebral and mesenteric vessels showed no major differences in sensitivity to calcium antagonists.     

Inhibition by glucocorticoids of prostaglandin release from adipose tissue in vitro.

1 When rabbit chopped adipose tissue was incubated with a lipolytic agent (adrenocorticotrophic hormone, ACTH1-24, 0.1 microng/ml) in Krebs solution, prostaglandin E2 was formed in the tissue and about the same amount was found in the medium. 2 In the presence of indomethacin (1 microng/ml) the appearance of prostaglandin E2 was almost abolished both in the tissue and in the medium. 3 When the incubation was carried out in the presence of hydrocortisone or betamethasone (1-10 microng)ml) the concentration of prostaglandin E2 leaking or carried into the medium was significantly reduced, whereas that remaining in the tissue was significantly increased. This action of the steroids was not reversed by increasing substrate (arachidonic acid) concentration in the medium. 4 The steroids did not affect lipolysis, nor did they influence prostaglandin metabolism since such activity was not detectable in the adipose tissue. 5 Anti-inflammatory steroids therefore did not reduce prostaglandin formation but increased the tissue/medium ratio, which supports the view that they inhibit the release of prostaglandins after these have been synthesized.     

Effect of volatile anesthetics on steroidogenesis in isolated bovine adrenocortical fasciculata cells

To examine effects of volatile anesthetics (VAs) on steroidogenesis, cell suspensions of isolated bovine adrenocortical cells were incubated with several steroidogenic agents in the presence or absence of halothane and sevoflurane. The adrenocortical cells were dispersed by trypsin digestion of bovine adrenal cortex. The cortisol level was measured fluorometrically. VAs inhibited adrenocorticotropic hormone-, acetylcholine-, angiotensin-II-, and KCl-stimulated steroidogenesis in a concentration-dependent manner with extracellular Ca(2+). However, dibutyryl cyclic adenosine monophosphate-stimulated steroidogenesis was not inhibited by VAs. These results suggest that VAs inhibit steroidogenesis by blocking Ca(2+)-influx from the extracellular space without influencing the action of intracellular cyclic nucleotides.     

Antagonism of saikosaponin-induced prostaglandin E2 release by baicalein in C6 rat glioma cells

There are several Kampo medicines (Chinese herbal medicines) containing both Bupleuri Radix and Scutellariae Radix, which are used for the treatment of inflammation. Saikosaponins are derived from Bupleuri Radix, and baicalein is from Scutellariae Radix. The present study was undertaken to investigate the pharmacological interaction of saikosaponin b1 and baicalein in prostaglandin E2 (PGE2) release from C6 rat glioma cells in vitro. Saikosaponin a, b1 and d potently stimulated PGE2 release, while saikosaponin b2 and c moderately stimulated PGE2 release. Saikosaponin b1 caused an irreversible elevation of intracellular Ca2+ concentration, which was eliminated by removing extracellular Ca2+. On the other hand, baicalein inhibited saikosaponin b1-induced PGE2 release in a concentration-dependent manner. These results suggest that saikosaponins are activators of PGE2 release, and baicalein is one of the functional inhibitors of PGE2 release by saikosaponins.     

Intracellular coupling of prostaglandin inhibition of acid secretion in isolated rabbit gastric parietal cells.

Acid secretion from isolated rabbit gastric parietal cells can be stimulated by gastric secretagogues, histamine (cyclic-AMP pathway) and carbachol (inositol phosphate pathway). Prostaglandins (PG) from E series are potent inhibitors of acid secretion. The intracellular mechanism of this inhibition was examined by using a stable PGE1-analogue, misoprostol. Aminopyrine (AP) accumulations due to histamine, IBMX and forskolin were dose-dependently inhibited by misoprostol, whereas a weak but significant biphasic effect on carbachol-induced AP accumulation was observed. The cyclic-AMP formation induced by histamine and IBMX were also inhibited by misoprostol in a non-competitive way. The potent effect of forskolin on cyclic-AMP levels was not modified by misoprostol in parietal cells, whereas it was potentiated in non-parietal cells. The inhibitory effect of misoprostol on AP accumulation was reduced by incubation of parietal cells with Bordetella pertussis toxin (IAP) but not with Cholera toxin (CT). Pretreatment of the cells with IAP did not alter cyclic-AMP levels of resting and histamine-stimulated parietal cells but abolished the inhibitory effect of misoprostol. Treatment with CT increased basal and histamine-stimulated cyclic-AMP levels and masked the inhibitory effect of misoprostol. The biphasic effect of misoprostol on carbachol-stimulated AP accumulation in parietal cells was confirmed on carbachol-stimulated phospholipase C activity and on [Ca2+]i stimulated by carbachol. These data confirm a direct and specific effect of the prostanoid on the Gi-subunit of the adenylate cyclase coupled to the histamine H2-receptor, and a biphasic effect on the phospholipase C pathway of the parietal cells.