Studies on the differential response to glucagon concentration on gluconeogenesis in isolated hepatocytes containing high and low glycogen

Rat liver parenchymal cells were isolated with (a) collagenase alone and (b) with both collagenase and hyaluronidase. Addition of hyaluronidase significantly decreased intracellular glycogen content of cells from fed rats. Effects of various concentrations of glucagon on gluconeogenesis were also studied in isolated hepatocytes from fed and fasted rats. Glucagon at the concentration of 10^?12M to 10^?10M stimulated gluconeogenesis in fed rats. Higher concentrations (10^?8M) had no further stimulating effect. In fasted rats, glucagon at the concentrationsof 10^?12M had no effect whereas at 10^?10M to 10^?8M concentrations, it stimulated gluconeogenesis by 2 fold. These studies suggest that glucagon functions in gluconeogenesis both in the fed and fasted state.     

A reappraisal of the proposed metabolic and antioxidant actions of butylated hydroxytoluene (BHT) in the liver

Butylated hydroxytoluene (BHT) was investigated for its metabolic actions in the perfused rat liver. Contrary to what is expected from an uncoupler, BHT up to 500 μM did not stimulate oxygen uptake nor did it inhibit gluconeogenesis from lactate. Transformation of fructose into glucose was also not affected by BHT; only lactate production was slightly increased at the concentration of 100 μM. The uncoupling effect of BHT in isolated mitochondria was confirmed, but only at concentrations above 10 μM; uncoupling at lower concentrations, 10^?9 to 10^?6 M, could not be confirmed. BHT, however, increased reactive oxygen species (ROS) production in isolated mitochondria, starting at the concentration of 10^?8 M. This is the opposite of what can be expected from a compound with proven ex vivo antioxidant action. One cannot exclude the possibility that, in mitochondria, stimulation of ROS production rather than uncoupling could be the most significant effect of BHT.     

Gluconeogenesis in rat liver parenchymal cells in primary culture: Permissive effect of the glucocorticoids on glucagon stimulation of gluconeogenesis

Primary cultures of parenchymal cells isolated from adult rat liver by a collagenase perfusion procedure and maintained as a monolayer in a serum-free culture medium were used to study glucoeogenesis and the role that the glucocorticoids play in the control of this pathway. These cells carried out gluconeogenesis from three-carbon precursors (alanine and lactate) in response to glucagon and dexamethasone added alone or in combination. Maximum glucose production was observed with cells pretreated for several hours with dexamethasone and glucagon prior to addition of substrate and glucagon (8- to 12-fold increase over basal glucose production). Half-maximum stimulation of gluconeogenesis was seen with 3.6 × 10^?10 M glucagon and 3.6 × 10^?8 M dexamethasone. Maximum stimulation was oberved with 10^?7 M glucagon and 10^?6 M dexamethasone. The length of time of dexamethasone pretreatment was found to be important in demonstrating the effect of glucocorticoids on glucagon-stimulated gluconeogenesis. Treeatment of cells with dexamethasone for 2 hours did not result in an increase in glucose production over identical experimental conditions in the absence of dexamethasone, wherease pretreatment for 5 hours (1.2-fold increase) or 15 hours (1.7-fold increase) did result in an increase in glucose production. The results establish that the adult rat liver parenchymal cells in primary culture are a valid model system to study hepatic gluconeogenesis. In addition, we have established directly that the glucocorticoids amplify the glucagon stimulation of gluconeogenesis.     

Studies on the lack of glucagon response to clycogenolysis and gluconeogensis and decrease in protein synthesis in isolated hepatocytes from hypophysectomized animals.

Effect of various concentrations of glucagon on gluconeogenesis and glycogenolysis was studied in isolated hepatocytes obtained from normal and hypophysectomized animals. Addition of glucagon (10^?10 to 10^?6M) stimulated glycogenolysis and gluconeogenesis by 2–3 fold in normal hepatocytes. However, this concentration of glucagon had only a slight effect in isolated hepatocytes obtained from hypophysectomized animals. This lack of glucagon response was not due to reduction in glycogen levels in isolated hepatocytes obtained from hypophysectomized animals. Studies on the incorporation of¹⁴C-alanine,¹⁴C-leucine and¹⁴C-valine showed a 3–5 fold decrease in the incorporation of these amino acids into protein in hypophysectomized animals compared to normal controls.     

Interaction of thyroid hormones and cyclic AMP in the stimulation of hepatic gluconeogenesis

The possible interaction of l-3,3′,-5-triiodthyronine (T₃) and cycli AMP on hepatic gluconeogenesis was investigated in perfused livers isolated from hypothyroid rats starved for 24 h. T₃ (1·10^?6) and cyclic AMP (2·10^?4 M) increased hepatic gluconeogenesis from alanine within 30–60 min perfusion time (+85%/ + 90%), both were additive in their action (+191%). Concomitantly, α-amino[¹⁴C]isobutyric acid as well as net alanine uptake and urea production were elevated by T₃ and by cyclic AMP. T₃ increased the oligomycin-sensitive O₂ consumption and the tissue ‘overall’ ATP/ADP ratio, whereas cyclic AMP showed only a minor effect on cellular energy metabolism. As was observed recently for cyclic AMP, the stimulating action of T₃ on hepatic gluconeogenesis was independent of exogenous Ca²⁺ concentration. T₃ by itself affected neither the total nor the protein-bound hepatic cyclic AMP contents, pyruvate kinese (v:0.15 mM) activation nor the tissue levels of gluconeogenic intermediates. In contrast, cyclic AMP itself — although less effective than in euthyroid livers — decreased pyruvate kinase activity in hypothyroid livers with a concomitant increase in hepatic phosphoenolpyruvate concentration. This resulted in a ‘crossover’ between pyruvate and phosphoenolpyruvate. Cyclic AMP action was not affected by the further addition of T₃. Glucagon (1·10^?8 M) was less effective in hypo-than in euthyroid livers in increasing endogenous cyclic AMP content, deactivating pyruvate kinase and stimualting glucose production; this is normalized by the further addition of 1-methyl-3-isobutylxanthine (50 μM). It is concluded that T₃ stimulats hepatic gluconeogenesis by a cyclic-AMP-independent mechanism. In addition, the stimulatory action of cyclic AMP and glucagon with respect to hepatic gluconeogenesis is reduced in hypothyroidism. This may be explained by an increase in hepatic phosphodiesterase activity.     

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 action of angiotensin on the isolated perfused cat heart

The effects of angiotensin I (AI) and angiotensin (AII) on myocardial contractility, heart rate and coronary perfusion were observed in the isolated perfused cat heart. AII (10^?11 mol) and AI (10^?10 mol) both caused slowly developing sustained increases in systolic pressure of approximately 55%. There were inconsistent small increases in heart rate. Coronary perfusion was initially diminished, but later increased to above control values during the positive inotropic effect. The actions of both AI and AII were blocked by 1 sar 8 ile AII (10^?9 mol). The converting enzyme inhibitors SQ 20881 (10^?8 mol) and SQ 14225 (10^?8 mol) blocked the effect of AI. The actions of AII or AI were not blocked by α or β adrenergic blockade or by prior treatment with reserpine.     

1-substituted tetrahydroisoquinoline analogs: beta adrenoceptor blocking agents in the isolated perfused rabbit heart.

The 1-benzyl and 1-methyl congeners of trimetoquinol were tested for antagonism of receptors which mediate inotropy and chronotropy in the isolated perfused rabbit heart. 1-Benzyltrimetoquinol was found to be a blocker of resting, isoproterenol- and dobutamine-stimulated inotropy at concentrations (10^?7?10^?5M) which did not significantly affect chronotropy ( > 10^?5M). 1-Methyltrimetoquinol was found to be a partial agonist in the resting myocardium, weakly blocking inotropy and chronotropy at doses of 10^?7?10^?5M. At a concentration of 10^?4M, 1-methyltrimetoquinol was an agonist of both chronotropy and inotropy. These stimulatory properties appear to be direct (not affected by prior reserpinization) and antagonized by propranolol. In the isoproterenol-stimulated heart, 1-methyltrimetoquinol was a specific negative inotropic agent at doses (10^?7?10^?5M) above which agonist properties were manifest. At 10^?4M, 1-methyltrimetoquinol acted synergistically with isoproterenol to produce positive inotropy and chronotropy significantly greater than that of isoproterenol alone. Currently, it is believed that the receptors which mediate inotropy and chronotropy are $\text{beta}$ adrenergic in nature. Thus, it would appear that 1-benzyltrimetoquinol is a specific antagonist of those $\text{beta}$-receptors which mediate inotropy, while 1-methyltrimetoquinol is a partial agonist of both inotropic and chronotropic $\text{beta}$-receptors. Further, the response to these compounds does not appear to be proportionate in various regions of the myocardium.     

Enkephalins modulate the responsiveness of rat atria in vitro to norepinephrine

Potential interactions between opiate peptides and catecholamines in mammalian heart were examined using isolated spontaneously beating rat atria as a test system. Methionine-enkephalin (ME), leucine-enkephalin (LE), phe-met-arg-phe amide (FMRFamide), D-ala², N-methyl-phe⁴, met (O)⁵-ol-enkephalin (FK 33-834), methionine-enkephalin arg⁶ arg⁷ (ME arg⁶ arg⁷) and β-endorphin had no effect on basal beating rate of isolated atria at all concentrations up to 10^?5 M. The positive chronotropic effect of norepinephrine (NE) on atrial rate is, however, significantly attenuated by enkephalin peptides. Thus, the maximal chronotropic effect of NE (an increase from 317±7.0 to 473±7.3 beats per minute (bpm) in 250 gm rats at a dose of 10^?5 M NE) is decreased by 42% in the presence of 10^?7 m ME. The action of ME is completely blocked by addition of 10^?7 M naloxone, which by itself has no effect on NE-induced positive chronotropy or basal beating rate. The dose-effect curve for ME attenuation of NE-induced positive chronotropy is bell-shaped, i.e., both 10^?8 M and 10^?5 M ME have no significant effect on NE positive chronotropy. Other enkephalin peptides acted in a similar manner to ME; LE (10^?7 M) and FK 33-834 (10^?8 M) decreased maximal NE-induced positive chronotropy 42 and 27%, respectively. The molluscan cardioexcitatory peptide FMRFamide (10^?7 M) also decreased maximal NE positive chronotropy, about 30%. In contrast, β-endorphin did not significantly affect NE stimulation of atrial rate. We conclude that enkephalins can modulate the noradrenergic responsiveness of rat atria in vitro. The possible physiological relevance of this interaction is discussed.     

Studies on the effects of insulin and acetylcholine on activation of glycogen synthase and on glycogenesis in hepatocytes

The addition of insulin (4.0 × 10^?11 M) or acetylcholine (10^?6 M) to isolated hepatocytes stimulated glycogen accumulation and this stimulation was more pronounced when the medium glucose was raised from 50 to 300 mg percent. Studies with [¹⁴C]-glucose showed a two-fold stimulation in glycogen synthesis by the addition of insulin (4.0 × 10^?11 M) or acetylcholine (10^?6 M). A sixteen percent increase in the activity of glycogen synthase was observed in cells incubated for 10 minutes with insulin (4.0 × 10^?11 M) or acetylcholine (10^?6 M), whereas at one hour incubation a 40 percent increase in activity was observed with the same concentration of insulin or acetylcholine. The effects of insulin and acetylcholine were not additive.     

An inhibitory effect of arachidonic acid on subsequent responses of canine cerebral arteries to serotonin and other agonists

Arachidonic acid (AA) at 10^?4M and 10^?3M produced a phasic contraction in isolated canine basilar arteries that peaked rapidly and then slowly declined. This contraction was evidently due to the conversion of AA to prostanoids because it was blocked by cyclooxygenase inhibitors and because 11, 14, 17 eicosatrienoic acid (10^?3M), which is not a cyclooxygenase substrate, failed to produce a contraction. When the artery was exposed to 10^?3M AA for 20 min and washed, subsequent contractile responses to 10^?6M serotonin (5-HT) were only 10% of control. Contractions produced by prostaglandin E₂ (10^?5M), uridine triphosphate (10^?4M) and potassium (5.5×10^?4M) were inhibited to a lesser degree than 5-HT, the response to potassium being the least affected (66% of control). This damaging effect of 10^?3M AA did not occur if the artery was washed at peak contraction nor with 10^?4M AA. Autooxidation products were evidently not responsible for the damage because prior oxygenation (90 min) of 10^?4M AA had no such effect. Pretreatment with superoxide dismutase or ascorbate did not prevent the inhibition, suggesting that free radical reactions were not involved. Pretreatment with indomethacin (3×10^?4M) or meclofenamate (10^?4M) also failed to prevent the inhibitory phenomenon. Saponin, a detergent, produced similar inhibitory effects but 11, 14, 17 eicosatrienoic acid or oleate (10^?3M) did not. The arteries partially recovered from the inhibition with time. In conclusion, AA produced contraction in basilar arteries by inducing prostaglandin synthesis but can produce secondarily by an unidentified mechanism an inhibition of the contractile responses evoked by various agonists that is both time and concentration dependent.     

Purification and properties of UDP-glucose: coniferyl alcohol glucosyltransferase from suspension culturesof Paul's scarlet rose.

UDP-glucose:coniferyl alcohol glucosyltransferase was isolated from 10-day-old, darkgrown cell suspension cultures of Paul's scarlet rose. The enzyme was purified 120-fold by (NH₄)₂SO₄ fractionation and chromatography on DEAE-cellulose, hydroxyapatite, and Sephadex G-100. The enzyme has a pH optimum of 7.5 in Tris-HCl buffer, required an -SH group for activity, and is inhibited by ?-chloromercuribenzoate and EDTA. Its molecular weight is estimated to be 52,000. The enzyme is specific for the glucosylation of coniferyl alcohol (K_m 3.3 × 10^?6 M) and sinapyl alcohol (K_m 5.6 × 10^?6 M). With coniferyl alcohol as substrate the apparent K_m value for UDP-glucose is 2 × 10^?6m. The enzyme activity can be detected in a number of callus-tissue and cell-suspension cultures. The role of this enzyme is believed to be to catalyze the transfer of glucose from UDPG to coniferyl (or sinapyl) alcohol as storage intermediates in lignin biosynthesis.     

Effects of prostacyclin on the canine isolated basilar artery

Prostacyclin (PGI₂) produced a biphasic response in canine isolated basilar arteries. In low doses (1 × 10^?8M?1 × 10^?7M) PGI₂ caused a slight but consistent relaxation of resting muscle tone. In low concentrations (1 × 10^?8M?1 × 10^?6M) PGI₂ antagonized muscle contractions caused by serotonin or prostaglandin (PG) F_2α. This relaxant effect with low doses of PGI₂ on the isolated cerebral artery contrasts with findings obtained with other PGs and supports the hypothesis that PGI₂ is a mediator of vasodilatation. However, in 1 × 10^?5M concentrations PGI₂ contracted the arterial muscle and did not antagonize contractions induced by serotonin or PGF_2α.     

The Effect of Hydrogen Peroxide on the Growth of Microscopic Mycelial Fungi Isolated from Habitats with Different Levels of Radioactive Contamination

The effect of hydrogen peroxide (10^?9–10^?1 M) on the mycelial growth of the fungi Alternaria alternata, Cladosporium cladosporioides, Mucor hiemalis, and Paecilomyces lilacinus has been studied. The growth of fungi isolated from habitats with a background level of radioactive contamination was stopped by H₂O₂ concentrations equal to 10^?3 and 10^?2 M, whereas the growth of fungi that were isolated from habitats with high levels of radioactive contamination was only arrested by 10^?1 M H₂O₂. The response of the different fungi to hydrogen peroxide was of three types: (1) a constant growth rate of fungal hyphae at H₂O₂ concentrations between 10^?9 and 10^?4 M and a decrease in this rate at 10^?3 M H₂O₂, (2) a gradual decrease in the growth rate as the H₂O₂ concentration was increased, and (3) an increase in the growth rate as the H₂O₂ concentration was increased from 10^?6 to 10^?5 M. The melanin-containing species A. alternata and C. cladosporioides exhibited all three types of growth response to hydrogen peroxide, whereas the light-pigmented species M. hiemalis and P. lilacinus showed only the first type of growth response. A concentration of hydrogen peroxide equal to 10^?1 M was found to be lethal to all of the fungi studied. The most resistant to hydrogen peroxide was found to be the strain A. alternata 56, isolated from the exclusion zone of the Chernobyl Nuclear Power Plant.     

A 31P-NMR study of some metabolic and functional effects of the inotropic agents epinephrine and ouabain,and the ionophore R02-2985 (X537A) in the isolated,perfused rat heart

1. Some metabolic effects of increased mechanical activity by the Langendorff-perfused rat heart have been characterized using ³¹P-NMR. Mechanical activity was increased by infusion of ouabain (0.9?7.0·10^?5 M), the ionophore R02-2985 (1·10^?5 M) or epinephrine (5·10^?8 M). 2. Similar metabolic changes accompanied infusion of each of the positive inotropic agents into hearts perfused with buffer containing 11 mM glucose as the substrate. In each case phosphocreatine concentrations decreased. During the period of epinephrine infusion the phosphocreatine began to recover its original concentration, although there were no significant changes in mechanical activity. 3. Comparisons of the metabolic changes accompanying the positive inotropic and chronotropic effects of epinephrine were made between hearts perfused with either glucose (11 mM), acetate (5 mM) or lactate (5 mM). A time-dependent decrease in phosphocreatine concentrations also accompanied infusion of epinephrine into hearts perfused with lactate as the sole exogenous substrate, but no statistically significant metabolite changes were observed after identical epinephrine infusions with acetate as the substrate. 4. Calculation of the concentration of free ADP assuming equilibrium in the creatine phosphokinase reaction allows estimation of the cytosolic phosphate potential ($\text{[}\text{ATP}\text{]}\text{[ADP][P}{}_{\mathrm{<mtext>i</mtext>}}\text{]}$), which appears to be dependent on a number of factors, including the nature of the exogenous substrate and the level of mechanical activity. 5. Thus, we conclude that there is no general correlation between the phosphate potential and the mitochondrial respiratory rate in the perfused rat heart.     

Acetylcholine potentiation of field stimulated rat vas deferens: A pre-synaptic muscarinic mechanism?

Acetylcholine (ACh) was found to markedly enhance the nerve stimulation induced twitch response of isolated, field-stimulated rat vas deferens (RVD). The ED₂₀₀ (concentration which enhances the twitch response to 200% of control) for this potentiation was 6 × 10^?6M with the maximum twitch response being increased by more than 3 fold (325 ± 30%). Carbachol (ED₂₀₀ = 8.5 × 10^?7M) showed identical results. With each drug the potentiation was competitively antagonized by atropine (10^?7?10^?5M). Physostigmine 10^?8?10^?6M) both enhanced the basal twitch response (215 ± 8% of control at 10^?5M) and the sensitivity of the RVD to ACh (ED₂₀₀ = 3.3 × 10^?7M) but not to carbachol. Atropine, on the other hand reduced the basal twitch response by 18 ± 3% at 10^?5M. Hemicholinium (10^?4M) also reduced the basal twitch responses by 23 ± 5%. ACh (10^?7M?10^?5M) did not modify the responses of unstimulated RVD to norepinephrine or KCl suggesting a pre-synaptic site of action. Taken together these results are compatible with the presence of a pre-junctional, excitatory muscarinic mechanism in the field stimulated RVD. That this cholinergic system may be of physiological significance is supported by the observations that atropine and hemicholinium depress while physostigmine enhances the twitch response in the absence of exogenous ACh.     

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.     

Effect of melanostatin and thyroliberin on the biosynthesis and release of dopamine by rat brain striatal P2 fractions

The effect of melanostatin (MIF) and thyroliberin (TRH) on dopamine biosynthesis and release has been studied in striatal P₂ fractions from rat brain. Dopamine synthesis was not affected by either MIF (10^?4M) or TRH (10^?4M) in the presence of 2 mM EDTA. In contrast, tyramine (10^?4M) and (+)-amphetamine (10^?4M) both stimulated dopamine biosynthesis, in a manner inversely proportional to the rate of dopamine biosynthesis, in the absence of drugs. MIF (10^?3M) or TRH (10^?3M) stimulated the release of dopamine in the presence of 1.25 mM calcium chloride but not in the presence of 2 mM EDTA. In contrast (+)-amphetamine 10^?4M) stimulated release of dopamine under both conditions. These results are discussed in relation to the hypothesis that MIF and TRH stimulate the release of dopamine from vesicular stores by a calcium dependent mechanism, whereas amphetamine stimulates release of dopamine from both vesicular stores and a cytoplasmic pool which regulates tyrosine hydroxylase activity.