Effects of crude and pure cholera toxin on prostaglandin release from the rabbit ileum

Investigations were made into the effects of crude and pure preparations of cholera toxin on the release of prostaglandin-like substances (PLS) from rabbit ileum. Perfusion of ileal loops with buffer containing crude toxin was followed by a release of PLS into the perfusate, in amounts up to 37.5 ng/30 min (PGE₂ equivalents). In contrast, no detectable PLS was released when ileal loops were perfused with pure toxin. Similarly, pieces of ileum opened longitudinally released PLS in amounts up to 107 ng PGE₂/g tissue when incubated with crude toxin for 1–4 hr, but no release of PLS was detected in the presence of pure toxin under comparable conditions.Treatment of rabbits with indomethacin, 1.6 mg/kg p.o., had no effect on the accumulation of fluid in ileal sacs injected with crude or pure cholera toxin. These results support the view that prostaglandins do not play an essential role in the action of cholera toxin.     

Effects of crude and pure cholera toxin on prostaglandin.

Investigations were made into the effects of crude and pure preparations of cholera toxin on the release of prostaglandin-like substances (PLS) from rabbit ileum. Perfusion of ileal loops in vivo with buffer containing crude toxin was followed by a release of PLS into the perfusate, in amounts up to 37.5 ng/30 min (PGE2 equivalents). In contrast, no detectable PLS was released when ileal loops were perfused with pure toxin. Similarly, pieces of ileum opened longitudinally released PLS in amounts up to 107 ng PGE2/g tissue when incubated with crude toxin for 1-4 hr, but no release of PLS was detected in the presence of pure toxin under comparable conditions. Treatment of rabbits with indomethacin, 1.6 mg/kg p.o., had no effect on the accumulation of fluid in ileal sacs injected with crude or pure cholera toxin. These results support the view that prostaglandins do not play an essential role in the action of cholera toxin.     

Evidence against the release of prostaglandin-like material from isolated intestinal tissue by pure cholera toxin

Prostaglandin-like material was released from finely cut guinea-pig ileum or human intestinal mucosa during incubation with Krebs solution. The tissue inactivated some of the released material and added PGE₂. There was no significant change in release of prostaglandin-like material when pure cholera toxin was incubated with guinea-pig ileum or human intestinal mucosa. The work is discussed in relation to the action of cholera toxin $\text{in vivo}$.     

Prostaglandin E2: A factor in the pathogenesis of cholera

Injection of cholera toxin $\text{in vivo}$ into loops of intestine in rats caused the production of an exudate. This was found to contain prostaglandin E₂ by assay on the rat stomach strip and by thin-layer chromatography. The amounts found ranged from 20 to 40 ng per loop of intestine. Introduction of 30 ng of prostaglandin E₂ into intestinal loops caused the production of an exudate similar in volume to that found after the introduction of cholera toxin. These results indicate that the exudate in cholera is caused by the action of prostaglandin liberated by the enterotoxin. It is suggested that an inhibitor of prostaglandin release could be added to the solutions used in treatment for the restoration of fluids and electrolytes, with the object of blocking the action of toxin still present in the intestinal lumen, thereby achieving a more rapid therapeutic result.     

Evidence against the release of prostaglandin-like material from isolated intestinal tissue by pure cholera toxin.

Prostaglandin-like material was released from finely cut guinea-pig ileum or human intestinal mucosa during incubation with Krebs solution. The tissue inactivated some significant change in release of prostaglandin-like material when pure cholera toxin was incubated with guinea-pig ileum or human intestinal mucosa. The work is discussed in relation to the action of cholera toxin in vivo.     

Production and release of heat-labile toxin by wild-type human-derived enterotoxigenic Escherichia coli

Production and release of heat-labile toxin (LT) by wild-type enterotoxigenic Escherichia coli (ETEC) strains, isolated from diarrheic and asymptomatic Brazilian children, was studied under in vitro and in vivo conditions. Based on a set of 26 genetically diverse LT(+) enterotoxigenic E. coli strains, cell-bound LT concentrations varied from 49.8 to 2415 ng mL(-1). The amounts of toxin released in culture supernatants ranged from 0% to 50% of the total synthesized toxin. The amount of LT associated with secreted membrane vesicles represented <5% of the total toxin detected in culture supernatants. ETEC strains secreting higher amounts of LT, but not those producing high intracellular levels of cell-bound toxin, elicited enhanced fluid accumulation in tied rabbit ileal loops, suggesting that the strain-specific differences in production and secretion of LT correlates with symptoms induced in vivo. However, no clear correlation was established between the ability to produce and secrete LT and the clinical symptoms of the infected individuals. The present results indicate that production and release of LT by wild-type human-derived ETEC strains are heterogeneous traits under both in vitro and in vivo growth conditions and may impact the clinical outcomes of infected individuals.     

Stimulatory effects of prostaglandin E1 on rat anterior pituitary cyclic amp and luteinizing hormone release

The effect of prostaglandin E₁ (PGE₁) on rat anterior pituitary cyclic AMP accumulation and luteinizing hormone (LH) release was studied both in vivo and in vitro. Addition of PGE₁ to incubation medium over a concentration range of 10^-6 to 10^-4 M produced a graded increase in pituitary cyclic AMP. At the lowest concentration (10^-6 M) there was no significant increase in LH release, but proportional increments in LH release were seen with increasing concentrations of PGE₁.Ten minutes after intravenous administration of 5 μg of PGE₁ to adult male rats, pituitary cyclic AMP was substantially increased while serum LH levels were not changed. Administration of a higher dose of PGE₁ (20 μg) produced a greater increase in pituitary cyclic AMP; and, at this dose serum LH was significantly increased. These results suggest that the PGE₁ effect on LH release is mediated by the adenyl cyclase — cyclic AMP system.     

α-Adrenergic stimulants, prostaglandins and catecholamine release from the adrenal gland in vitro

In rat adrenal glands incubated in Locke's solution in vitro norepinephrine and phenylephrine inhibited the release of epinephrine. PGE₂ and PGE₁ also inhibited the release of catecholamines but PGF_α1 had no effect on the adrenal. Thus, catecholamine release from adrenal cells may be regulated by the same mechanisms as in adrenergic nerve endings.     

Somatocrinin (growth hormone releasing factor) in vitro bioactivity; Ca++ involvement, cAMP mediated action and additivity of effect with PGE2

The release of GH induced by purified hypothalamic GRF or native or synthetic tumor-derived GRF is antagonized by the presence of CoCl²; it is simulated by 8Br .cAMP, IBMX, cholera toxin, forskolin, with identical maximal effects (E_max). Somatocrinin (GRF) stimulates the efflux of cAMP by the pituitary cells in parallel to the release of GH. Addition of either 8Br .cAMP, IBMX, cholera toxin or forskolin to a maximally stimulating dose of GRF does not increase the response which remains GRF-E_max. In contradistinction with these results PGE₂ releases GH with a dose-response curve different from that of GRF, and the combination of PGE₂ + GRF produces an E_max far greater than that due to either agonist alone; showing a true additivity. The name $\text{somatocrinin}$ is proposed to replace the acronym GRF.     

The mechanism of action of soluble lymphocyte mediators. IV. Effect of migratio inhibitory factor (MIF) on macrophage cyclic AMP and on responsiveness to adenylate cyclase stimulators.

Intracellular levels of cyclic 3′,5′-adenosine monophosphate (cAMP) in purified guinea pig peritoneal macrophages were elevated following incubation with the adenylate cyclase stimulators prostaglandins E₁ and E₂ (PGE₁, PGE₂), isoproterenol, and cholera toxin. Exposure of macrophages to antigen-stimulated lymphocyte culture supernatants, containing migration inhibitory factor (MIF), resulted in a moderate but consistent decrease in the cAMP level, which was best expressed after 1–2 hr of incubation. Incubation of macrophages with MIF-containing supernatants or partially purified MIF for 1–2 hr resulted in reduced cAMP accumulation in response to PGE₁, PGE₂, isoproterenol, and cholera toxin (nonspecific refractoriness). These findings indicate that MIF-induced inhibition of macrophage migration is not due to an increase in the cellular level of cAMP and that the reduction in cAMP concentration, caused by MIF, is probably a secondary phenomenon unrelated to the inhibition of cellular motility.     

Lincomycin increases synthetic rate and periplasmic pool size for cholera toxin.

Increased enterotoxigenicity of Vibrio cholerae 569B grown with low concentrations of lincomycin, previously described in terms of increased extracellular biological activity (capillary permeability factor and fluid accumulation in ligated rabbit ileal loops), was further characterized. Polyacrylamide gel electrophoresis and single radial immunodiffusion showed that lincomycin-stimulated cells produced increased molar quantities of cholera toxin (CT) both extra- and intracellularly. The intracellular CT was released in comparable amounts by sonication, deoxycholate extraction, and polymyxin B treatment. Polymyxin B release of CT was nearly complete under conditions wherein only 6% of total cellular beta-galactosidase was released, implying a periplasmic pool of CT in stimulated cells. No intracellular choleragenoid (CT subunit B) was found in stimulated cells by polymyxin B release. No proteolysis of 14C-labeled CT was detected after prolonged incubation with sonicated nonstimulated cultures or sonicated concentrated cells. These data support the conclusion that the stimulatory effect of lincomycin involves an increase in the rate of synthesis of the CT molecule, and argue against alternative models involving inhibition of putative normal degradation of CT, increased release of otherwise cell-bound CT, or activation of inactive, or less active, forms of CT.     

Cholera Toxin Induces Cyclic AMP-Independent Down-Regulation of Gsα and Sensitization of α2-Autoreceptors in Chick Sympathetic Neurons

Abstract: The role of the stimulatory GTP-binding protein (G_S) in the α₂-autoinhibitory modulation of noradrenaline release was investigated in cultured chick sympathetic neurons. The α₂-adrenoceptor agonist UK 14,304 caused a concentration-dependent reduction of electrically evoked [³H]noradrenaline release with half-maximal effects at 14.0 ± 5.5 nM. In neurons treated with 100 ng/ml cholera toxin for 24 h, the half-maximal concentration was lowered to 3.2 ± 1.4 nM without changes in the maximal effect of UK 14,304. The pretreatment with cholera toxin also increased the inhibitory action of 10 nM UK 14,304 when compared with the inhibition of noradrenaline release in untreated cultures derived from the same cell population. In cultures treated with either 10 µM forskolin or 100 µM 8-bromo-cyclic AMP, neither the half-maximal concentration nor the maximal effect of UK 14,304 was altered. Cholera toxin, forskolin, and 8-bromo-cyclic AMP all induced an increase in spontaneous outflow and a reduction in electrically evoked overflow, effects not observed after a pretreatment with dideoxyforskolin. Exposure of neurons to cholera toxin, but not to forskolin or 8-bromo-cyclic AMP, induced a translocation of α-subunits of G_s (G_sα) from particulate to soluble fractions and led ultimately to a complete loss of G_sα from the neurons. In contrast, no effect was seen on the distribution of either α-subunits of G_i- or G_o-type G proteins or of β-subunits. These results indicate that cholera toxin causes a selective, cyclic AMP-independent down-regulation of G_sα. This down-regulation of G_sα is associated with the sensitization of α₂-autoreceptors.     

Impaired prostaglandin release from the kidneys of salt-loaded and hypertensive rats

Prostaglandin (PG) release by the isolated perfused kidney of the rat has been stimulated by nor-adrenaline infusion and measured by bioassay. There was no basal output of PGE₂-like activity, but stimulated release reached mean concentrations of 9.1 ng/g kidney/ml perfusate in kidneys from female albino rats drinking water and 2.9 ng/g/ml in those from animals given 1.5% NaCl to drink. Kidneys from uninephrectomised animals with mock-clipped renal arteries released 7.3 ng PG/g/ml and those from rats with uninephrectomy and constricted renal arteries 3.3 ng/g/ml.     

Metabolism of [14C] arachidonic acid in the isolated rabbit spleen

Infusion of [¹⁴C] arachidonic acid (AA) into the isolated, Tyrode perfused rabbit spleen resulted in the release of a substance into the venous effluent with the musculotropic activity and chromatographic properties of prostaglandin (PG)E₂. Smaller amounts of radioactive materials with the chromatographic properties of PGF_2α, 6-keto-PGF_1α, and PGD₂ were also released. The radiolabeled material released in largest amounts from the spleen was identified as PGE₂ on the basis of: 1) Co-chromatography with PGE₂ in three solvent systems, 2) Conversion of the radioactive material and of authentic [³H] PGE₂ to similar products by treatment with sodium borohydride and with potassium hydroxide, and 3) Stability of the musculotropic activity in Tyrode solution at 37°C. Release of the major and minor radioactive products was inhibited by pretreatment of the spleen with either indomethacin or 5,8,11,14-eicosatetraynoic acid.     

Cyclic AMP formation and release by cultured bone cells stimulated with prostaglandin E2

PGE₂ produced a marked and dose-related increase in cAMP content of cultured bone cells and in the release of cAMP into the incubation medium. The amount of cAMP released from the cells by PGE₂ was proportional to the cellular concentration, and was dependent upon the time of incubation with PGE₂. The cAMP levels released into the media increased slowly at a linear rate during a 60 min treatment with PGE₂. This release was blocked by theophylline, probenecid, ouabain and dinitrophenol, suggesting that the release of cAMP was not a simple diffusive process and required energy. SC-19220 reduced the formation of cAMP more than the release, suggesting that the formation and the release may arise from separate events. Inability of D600 to inhibit PGE₂-induced release of cAMP indicates that the release does not require calcium.     

Interactions of prostaglandin E1 (PGE1) and LRH on anterior pituitary function

Numerous biochemical pathways influence the synthesis and release of anterior pituitary hormones. Releasing factors extracted from the hypothalamus and prostaglandins (PGs) appear to alter a common biochemical activity, adenyl cyclase, in pituitary cells. Luteinizing hormone releasing hormone (LRH), prostaglandin (PGE₁), 7 oxa-13-prostynoic acid and cycloheximide were tested for individual and interacting effects on the $\text{in vitro}$ release of FSH, LH and prolactin from hemipituitaries of 15 day old female rats. LRH (10 ng/ml) consistently released both LH and FSH in all $\text{in vitro}$ experiments and inhibited prolactin release in 1 of 2 experiments. Lower concentrations (5 and 1 ng/ml) also stimulated LH and FSH release but did not influence prolactin release. Concurrent depletion of stored LH and FSH in the gland was observed. PGE₁ in a 6.5 hour incubation increased the storage of LH within the gland in the absence of LRH. In a 1.5 hour incubation in the presence of LRH, storage of LH was also increased. PGE₁ had no effect on LH and FSH release; however, in 1 of 2 experiments it stimulated prolactin release in the absence of LRH. Prostynoic acid stimulated LH and FSH release but did not synergize with LRH action in the same tissue. Cycloheximide did not affect LH release during the first 30 minutes of incubation; however, the release during the subsequent 1 hour was significantly inhibited. Similar tissue also exposed to cycloheximide was still responsive to LRH during the latter 1 hour incubation period. Cycloheximide had no effect on prolactin storage and release from the same tissue.     

Vasoactive intestinal polypeptide: increased tone, enhancement of acetylcholine release, and stimulation of adenylate cyclase in intestinal smooth muscle

Vasoactive intestinal polypeptide (VIP) was examined $\text{in vitro}$ for effects on tone and neuronal release mechanisms in intestinal smooth muscle since this is a site of high peptide concentration. VIP contracted the guinea pig ileum and rabbit jejunum in concentrations ranging from 10^?9 to 10^?7 M. Increased tone in the guinea pig ileum was partially antagonized by the anticholinergic agent, atropine (4.38 × 10^?6 M) suggesting that one component of the contractile response was due to the indirect release of acetylcholine. The H₁ receptor antagonist, pyrilamine, did not alter the increased tone produced by VIP indicating that histamine release did not contribute to the ileal contractile response and that VIP exerted a selective effect to enhance neuronal release of acetylcholine. The ability of VIP to modulate acetylcholine release was confirmed in field stimulated ileal preparations where VIP increased the force developed to endogenously released acetylcholine without altering the direct response to acetylcholine. In rabbit jejunum and ileal smooth muscle, VIP related cyclic AMP levels. However, inhibition of phosphodiesterase with papaverine did not potentiate either the VIP-induced ileal contraction or enhancement of the field stimulated response. This raises the possibility that increases in intestinal cyclic AMP may be involved more in VIP-induced alterations in ion transport or secretory phenomenon than in intestinal motility. These studies describing the ability of VIP to modulate acetylcholine release and to increase ileal tone are consistent with the proposed role of VIP in intestinal patholgies involving excessive mucous secretion and motility.     

Enterotoxigenicity of diarrhoeal isolates of Salmonella bareilly

Whole cell cultures, cell-free supernatants, and cell sonicates from ten strains of Salmonella bareilly induced fluid accumulation in ligated rabbit and rat ileal loops. All strains had an intracellular vascular permeability factor, half were suckling mouse positive indicating the presence of a heat-stable type of activity. The toxin(s), however, were Immunologically distinct from the heat-labile toxin of Escherichia coll LT and cholera toxin. Besides enterotoxigenicity, all strains exhibited potential Invasive character.The authors are with the Department of Microbiology, Panjab University, Chandigarh, India 160 014. M. Saxena is the corresponding author.     

Interleukin-1 and tumor necrosis factor are not synergistic for human synovial fibroblast PLA2 activation and PGE2 production

Patterns of arachidonic acid release and metabolism were altered in human synovial fibroblasts following exposure to cytokines. Recombinant interleukin-1 induced an approximate 3-fold in crease in [³H]-AA release, a 7-fold increase in PGE₂ production and a 2-fold increase in PLA₂ activity in human synovial fibroblasts. Recombinant tumor necrosis factor induced similar responses, however, the magnitude was less than that mediated by interleukin-1. A combination of the two cytokines had an additive effect on [³H]-AA release and PLA₂ activity while PGE₂ production was similar to that detected using interleukin-1 alone. [³H]-AA, was released in substantial amounts when sodium fluoride was used as a stimulus but PGE₂ was not. These data show that tumor necrosis factor and interleukin-1 can both activate synovial cell PLA₂ and induce generation of PGE₂, but act in an additive rather than a synergistic fashion. Furthermore, the data show that PGE₂ production is not always concordant with [³H]-AA release, suggesting that appropriate enzyme(s) must be activated.     

Metabolism of [C] arachidonic acid in the isolated rabbit spleen

Infusion of [¹⁴C] arachidonic acid (AA) into the isolated, Tyrode perfused rabbit spleen resulted in the release of a substance into the venous effluent with the musculotropic activity and chromatographic properties of prostaglandin (PG)E₂. Smaller amounts of radioactive materials with the chromatographic properties of PGF_2α, 6-keto-PGF_1α, and PGD₂ were also released. The radiolabeled material released in largest amounts from the spleen was identified as PGE₂ on the basis of: 1) Co-chromatography with PGE₂ in three solvent systems, 2) Conversion of the radioactive material and of authentic [³H] PGE₂ to similar products by treatment with sodium borohydride and with potassium hydroxide, and 3) Stability of the musculotropic activity in Tyrode solution at 37°C. Release of the major and minor radioactive products was inhibited by pretreatment of the spleen with either indomethacin or 5,8,11,14-eicosatetraynoic acid.