Krebs-Ringer Buffer Research Articles

Effect of molecular charge on para- and transcellular access of horseradish peroxidase into rat bile.

The permeability pathway into the biliary tree for small inert molecules exhibits a charge selectivity. Using a method which distinguishes trans- from paracellular access, we have examined the charge selectivity of biliary access pathways for the 40-kD protein horseradish peroxidase (pI 7.5), which was derivatized to strongly anionic (pI less than 3.5) and strongly cationic (pI greater than 9.5) isoenzymes. Each isoenzyme was injected as a bolus into the perfusate of an isolated rat liver perfused in situ with a nonrecirculating Krebs-Ringer buffer. Bile was collected at intervals and horseradish peroxidase activity was measured. Its appearance allowed differentiation of paracellular from transcellular access, and the amount entering via each pathway was quantified. The species of enzyme entering bile was the same as that injected as determined by cation-exchange high-performance liquid chromatography of biliary horseradish peroxidase. Paracellular biliary access of anionic horseradish peroxidase was less than 50% that of neutral and cationic horseradish peroxidase both in the control state and when paracellular entry was augmented with 10(-10) M vasopressin. Transcellular access of anionic horseradish peroxidase was similarly restricted. To determine whether this restriction of anionic transcellular access was brought about by diminished hepatocellular uptake or augmented catabolism, we studied these parameters in 4-hr primary hepatocyte cultures. The uptake rates of all species were similar. Little or no degradation or efflux of any horseradish peroxidase species occurred over 30 min in the cultured cells. We conclude that access is charge selective for macromolecules and that this selectivity holds for trans- as well as for paracellular pathways.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain dialysis provides evidence for D 2-dopamine receptors modulating noradrenaline release in the rat frontal cortex

The frontal cortex of the rat receives dopaminergic and noradrenergic nerve terminals from the ventral tegmental area and the locus coeruleus, respectively, ct2-Adrenoceptors and D-2 dopamine (DA) receptors are present in this area, but their location and role are not clear. Peripheral noradrenergic nerve terminals have both a2-adrenoceptors and D-2 presynaptic receptors and the stimulation of either results in the inhibition of noradrenaline (NA) release (see Langer, 1980). The presence in the central noradrenergic nerve terminals of presynaptic a2-adrenoceptors modulating NA release has been shown in vitro (Galzin et al., 1982) and, more recently, in vivo by brain dialysis applied to the rat frontal cortex (L'Heureux et al., 1986). However, little is known of the presence of presynaptic D-2 receptors on noradrenergic nerve terminals in this area. We have used the technique of intracerebral dialysis in conscious freely moving rats in order to investigate if D-2 receptors other than ct2-adrenoceptors might control the release of NA from nerve terminals in the frontal cortex. Brain microdialysis was performed in male Sprague-Dawley CD 350-400 g (Charles River, Italy) freely moving rats essentially as described by Imperato and Di Chiara (1984), using bicarbonate Krebs-Ringer buffer pH 7.4 as perfus-

Effect of chronic exercise on glucose uptake and activities of glycolytic enzymes measured regionally in rat heart.

Regional glucose uptake in perfused hearts, and the activities of several glycolytic enzymes contributing to the glucose metabolism in perfused and nonperfused hearts were studied in male and female rats after 8-9 weeks of swimming training. The left ventricular glucose uptake showed a transmural gradient in the sedentary animals, the subendocardial uptake being 30% and 12% higher than that of the subepicardial layer in the males and females, respectively. Swimming exercise abolished the left ventricular glucose uptake gradient in male rats, and in female rats an opposite gradient was found, the subepicardial uptake being 23% higher than the subendocardial uptake. The activities of phosphofructokinase and 3-phosphoglyceraldehyde dehydrogenase also showed transmural gradients in the left ventricles. Training did not abolish these gradients. Training-induced changes in the activities of phosphofructokinase, 3-phosphoglyceraldehyde dehydrogenase, pyruvate kinase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, citrate synthase, and malate dehydrogenase were found in certain sites of the myocardium. Perfusion of isolated hearts for 50 min with insulin-containing Krebs-Ringer buffer especially affected the activities of phosphofructokinase, lactate dehydrogenase, and citrate synthase, increasing these activities in the left ventricles and decreasing them in the atria. These results indicate that there are regional differences between male and female rats in the cardiac glucose uptake rate after swimming training.

Leukotriene D4 inhibits atrial natriuretic factor-dependent cGMP production in rat glomerulus

The effects of leukotriene D4 (LTD4) on guanosine 3',5'-cyclic monophosphate (cGMP) production induced by atrial natriuretic factor (ANF) or sodium nitroprusside (SNP) were investigated in isolated rat glomeruli. First, the isolated glomeruli were preincubated in Krebs-Ringer buffer containing 3-isobutyl-1-methylxanthine for 10 min, then incubated with varying concentrations of ANF, SNP, and LTD4 for 3 min. cGMP content was determined by radioimmunoassay. cGMP production (fmol.glomerulus-1.3 min-1) was not increased by LTD4. cGMP accumulation was increased by ANF (1 microM) and SNP (100 microM) remarkably from 0.19 +/- 0.09 to 3.24 +/- 0.21 (n = 7, mean +/- SE), and from 0.19 +/- 0.09 to 3.67 +/- 0.47 (n = 4), respectively. The increase in cGMP production by ANF was significantly and dose-dependently inhibited by LTD4 (P less than 0.05). Maximum suppression was observed at the concentration of 100 nM (from 3.06 +/- 0.33 to 1.43 +/- 0.09; P less than 0.05). However, LTD4 caused no significant change in the level of cGMP production induced by SNP. Second, particulate or soluble guanylate cyclase from glomeruli were similarly investigated. ANF selectively activated particulate guanylate cyclase and the response was selectively inhibited by LTD4 from 9.07 +/- 1.79 fmol.micrograms-1.3 min-1 (n = 6) to 4.70 +/- 0.70 (n = 6, P less than 0.05). These results suggest the site where LTD4 has effect on cGMP production induced by ANF was the particulate guanylate cyclase system, but not the soluble guanylate cyclase system.

Characterisation of a Na +/K +/Cl − cotransporter in alkylating agent-sensitive L1210 murine leukemia cells

The mode of influx of 86Rb +, a K + congener, to exponentially proliferating L1210 murine leukemia cells, incubated in a Krebs-Ringer buffer, has been characterised. The influx was composed of a ouabain-sensitive fraction (approx. 40%), a loop diuretic-sensitive fraction (approx. 40%) and a fraction which was insensitive to both types of inhibitor (approx. 15%). The fraction of ouabain-insensitive 86Rb + influx, which was fully inhibited by furosemide (1 mM) or bumetanide (100 μM), was completely inhibited when Cl − was completely substituted by nitrate or gluconate ions, but was slightly (29±12%) stimulated if the Cl − was substituted by Br −. The substitution of Na + by Li +, choline or tetramethylammonium ions inhibited the loop diuretic-sensitive fraction of 86Rb + uptake. These results suggested that a component of 86Rb + influx to L1210 cells was mediated via a Na +/K +/Cl − cotransporter. 86Rb + efflux from L1210 cells which had been equilibrated with 86Rb + and incubated in the presence or absence of 1 mM ouabain, was insensitive to the loop diuretics. Additionally, efflux rates were found to be independent of the external concentration of K +, suggesting that efflux was not mediated by K +K + exchange. The initial rate of 86Rb + influx to L1210 cells in the plateau phase of growth was reduced to 44% of that of exponentially dividing cells, the reduction being accounted for by significant decreases in both ouabain- and loop diuretic-sensitive influx; these cells were reduced in volume compared to cells in the exponential phase of cell growth. In cells which had been deprived of serum for 18 h, and which showed an increase of the proportion of cells in the G 1 phase of the cell cycle, the addition o serum stimulated an immediate increase in the furosemide-sensitive component of 86Rb + influx. Diuretic-sensitive 86Rb + influx was not altered by the incubation of the cells with 100 μM dibutyryl cyclic AMP, but was inhibited by 10 μM of the cross-linking agent nitrogen mustard (bis(2-chloroethyl)methylamine, HN2).

3H]arachidonic acid metabolism in rat brain minces: effects of nucleotide triphosphates, CDPcholine and CMP.

Rat brain minces were used to investigate the effects of nucleotides on the metabolism of arachidonic acid in nerve tissue. Brain free fatty acids, neutral lipids and phospholipids, were radiolabeled in vivo following intracerebral injection of [3H]arachidonic acid. Minces were prepared from the radiolabeled cerebra and were incubated in a modified Krebs-Ringer buffer with and without various nucleotides. The incubation-induced accumulation of unesterified [3H]arachidonate was reduced in the presence of CDPcholine, ATP, CTP, GTP, and UTP. These nucleotides inhibited choline and inositol glycerophospholipid hydrolysis. They also reduced the amount of labeled diglycerides. However, CDPethanolamine had no effect on arachidonic acid metabolism in the mince preparation and CMP appeared to stimulate further hydrolysis of choline glycerophopholipids, resulting in increased accumulation of [3H]arachidonic acid and labeled diglycerides. We suggest that the production of unesterified [3H]arachidonate and labeled diglycerides is due to the involvement of more than one catabolic reaction, since the high energy nucleotides had similar effects on fatty acid accumulation, but different effects on phospholipid labeling.

Modulatory effect of glucose on VIP-induced gastric somatostatin release.

The present study was designed to examine the effect of increasing perfusate glucose concentrations on vasoactive intestinal peptide (VIP)-induced somatostatin (SLI) release from the isolated rat stomach. The stomach of overnight-fasted rats was perfused with Krebs-Ringer buffer containing 100, 150, or 200 mg/dl glucose, respectively. VIP was administered at 10(-12), 10(-11), 10(-9), and 10(-8) M. At a normal glucose concentration of 100 mg/dl, VIP at doses of 10(-12), 10(-11), and 10(-9) M elicited a small inhibitory effect on SLI release by 200-300 pg/min (P less than 0.01). As reported previously at 10(-8) M, VIP stimulated gastric SLI secretion by 500 pg/min (P less than 0.01). Increasing perfusate glucose to 150 mg/dl resulted in a stimulation of SLI release by all four concentrations of VIP with a maximal effect at 10(-9) M. During 200 mg/dl glucose, VIP had no effect in concentrations below 10(-9) M, and only the two highest doses (10(-9) and 10(-8) M) stimulated SLI release significantly. In the absence of VIP, glucose had no effect on gastric SLI release. In conclusion, the present data demonstrate for the first time that at normal glucose levels VIP has not only stimulatory but also inhibitory effects on gastric SLI, and second, a modest elevation of glucose has a modulatory effect on gastric D-cell function.

Ubiquitin‐Directed Antibodies Inhibit Neuronal Transporters in Rat Brain Synaptosomes

Affinity-purified antibodies specific for ubiquitin were found to inhibit the sodium-dependent uptake of [3H]choline, gamma-[3H]aminobutyric acid [( 3H]GABA), [3H]glutamate, [3H]norepinephrine, [3H]aspartate, and [3H]serotonin in rat cerebral cortical synaptosomes at a low concentration (10 micrograms/ml). These antibodies (termed anti-Ub) had no effect on the sodium-independent uptake of these substances or their calcium-dependent efflux. Synaptosomal [3H]deoxyglucose uptake was not affected in normal Krebs Ringer buffer containing 10 mM glucose, but was inhibited in glucose-free medium. Other nonneuronal sodium-dependent transport processes were found to be unaffected by 10 micrograms/ml anti-Ub, suggesting that anti-Ub does not bind indiscriminantly to sodium-binding sites on sodium-dependent organic solute transporters. Finally, anti-Ub inhibited sodium-dependent [3H]GABA and [3H]glutamate uptake in plasma membrane ghosts, devoid of membrane potential, which were derived from rat cerebral cortical synaptosomes. These results suggest that neuronal transporters or sites proximal to them may be ubiquitinylated on the plasma membrane surface.

Stimulation of luteinizing hormone-releasing hormone release from perifused hypothalamic fragments by phospholipase A2.

LHRH release is dependent on the availability of calcium, and prostaglandin E2 is a potent releaser of LHRH. Therefore, we investigated the role of phospholipase A2 (PLA2) on the release of LHRH from the hypothalamus. Four rat hypothalami were perifused with Krebs-Ringer buffer, and after a 60-min preincubation period, PLA2 was applied during 10 min. The LHRH response was determined by RIA of 10-min fractions collected for the next 60 min. PLA2 induced LHRH release in a dose-related manner at amounts of 2, 10, and 50 U. Omission of Ca++ from the medium using EGTA eliminated the PLA2 effect. Indomethacin treatment increased rather than diminished the PLA2 stimulation. Perifusion with melittin, an activator of PLA2, also increased LHRH release. These results are interpreted as a demonstration that PLA2 has a role in the release of LHRH and that a different route of the cyclooxygenase may be involved besides the well known mediation of prostaglandin E2.

The lipolytic effects of mouse placental lactogen II, mouse prolactin, and mouse growth hormone on adipose tissue from virgin and pregnant mice.

The lipolytic activities of three structurally related mouse hormones, placental lactogen II (mPL-II), GH (mGH), and PRL (mPRL), and human PL (hPL) were investigated. Adipose tissue was obtained from virgin and day 12 and day 16 pregnant mice. Lipolytic activity was assessed by the ability of the hormones to stimulate glycerol release from fat explants in the presence of dexamethasone and by the ability of the hormones to sensitize adipose tissue to the lipolytic stimulus theophylline. In the first experiment, adipose tissue explants were incubated in Krebs-Ringer buffer with 0.0, 0.1, 0.5, 1.0, 5.0, and 10.0 micrograms/ml hormone for 4 h. mGH was lipolytic at a concentration of 0.5 micrograms/ml or greater in adipose tissue from both virgin and pregnant mice. mPRL was lipolytic at a concentration of 5.0 micrograms/ml or greater in adipose tissue from virgin mice. In adipose tissue from pregnant mice mPRL was not lipolytic in day 12 tissue, but it was lipolytic at a concentration of 5.0 micrograms/ml in day 16 tissue. mPL-II and hPL did not stimulate glycerol release in mouse adipose tissue from virgin or pregnant mice. In the second experiment preincubating adipose tissue from virgin mice in the presence of 0.5 or 5.0 micrograms/ml mGH significantly increased the ability of the tissue to respond to theophylline; however, mGH did not induce this response in adipose tissue from pregnant mice, mPRL, mPL-II, and hPL did not increase theophylline-induced lipolysis in adipose tissue from either virgin or pregnant mice. These results indicate that two lipolytic mechanisms are activated in adipose tissue from mice; mGH can activate both mechanisms, whereas mPRL can activate only one.

The metabolite ratio as a function of chloral hydrate dose and intracellular redox state in the perfused rat liver.

Chloral hydrate (CH), an intermediate metabolite of trichloroethylene, is reduced to trichloroethanol (TCE) by alcohol dehydrogenase and aldehyde reductase, and is also oxidized to trichloroacetic acid (TCA) by the nicotinamide adenine dinucleotide (NAD)-dependent enzyme, CH dehydrogenase. Alcohol dehydrogenase requires reduced NAD (NADH), aldehyde reductase requires reduced nicotinamide adenine dinucleotide phosphate (NADPH) and CH dehydrogenase requires NAD to complete the reaction. It is unclear which reaction is predominant at the physiological redox level in intact liver cells. To study this question, we perfused the livers of well-fed rats with Krebs-Ringer buffer solution containing 0.1 mM pyruvate/1.0 mM lactate. The levels of TCE and TCA in the effluent were measured by gas chromatography, and the fluorescence of reduced pyridine nucleotides was measured with a surface fluorometer. When a low concentration (below 0.25 mM) of CH was administered, more TCA than TCE was produced. When a high concentration of CH was administered (over 0.5 mM), TCE production was greater. Reduced pyridine nucleotides decreased inversely with the CH concentration. Even at low CH concentrations, pyridine nucleotides were not reduced. When 10 mM lactate was added to the perfusate in order to reduce the pyridine nucleotides in the liver cells, the TCE/TCA ratio increased. On the other hand, the TCE/TCA ratio tended to fall following the addition of 5.0 mM pyruvate. In conclusion, the TCE/TCA ratio was altered according to the concentration of CH, and to the redox level of pyridine nucleotides in the liver.

Short-acting novel MAO inhibitors: in vitro evidence for the reversibility of MAO inhibition by moclobemide and Ro 16-6491.

The inhibition of monoamine oxidase (MAO) in rat liver and brain by the short-acting MAO-A inhibitors moclobemide (Ro 11-1163 = p-chloro-N-[2-morpholinoethyl]benzamide) and brofaremine and by the short-acting MAO-B inhibitors Ro 16-6491 (N-[2-aminoethyl]-p-chloro-benzamide) and almoxatone, administered p.o. at roughly equieffective doses 2 h before decapitation, was investigated for its reversibility under various in vitro conditions. MAO A activity in liver homogenates, inhibited by moclobemide (300 mumol/kg) to approx. 15% of control, time dependently recovered during 0.5 to 2 h of incubation at 37 degrees C, irrespective of whether the homogenates were prepared and incubated in distilled water or Krebs-Ringer buffer (KRB). Dialysis of such homogenates for 4 h in distilled water at 37 degrees C (but not at 13 degrees C) led to a complete return of the MAO activity. In liver homogenates from rats pretreated with brofaremine (30 mumol/kg), dialysis for 4 h at 37 degrees C against distilled water caused only little recovery of the MAO activity. Likewise, MAO-B inhibited by Ro 16-6491 (30 mumol/kg) to approx. 4% of control returned to almost control activity after 4 h of dialysis at 37 degrees C, while inhibition induced by almoxatone (30 mumol/kg) was little or not reversed at all. In brain homogenates prepared in, and dialysed against, distilled water or KRB at 37 degrees C (but not at 13 degrees C), MAO-A inhibited by moclobemide (100-300 mumol/kg) to approx. 15% of control, partially (KRB) or almost completely (dist. water) returned to control activity after 4 h of dialysis.(ABSTRACT TRUNCATED AT 250 WORDS)

IN VITRO SIMULTANEOUS RELEASE OF ENDOGENOUS SOMATOSTATIN AND GROWTH HORMONE FROM HUMAN ADENOMATOUS PITUITARY CELLS

Dispersed cells obtained from 10 GH-secreting adenomas were studied in perifusion columns, eluted with Krebs Ringer buffer. Fractions were collected each 5 min for 5 hours and assayed by RIA for GH and SRIF contents. Basal GH secretion was variable (10 to 94 ng/ml/ 106 cells). Within the first 15 minutes of the perifusion SRIF was detected in 5 cases (5 to 41 pg/ml/106 cells). However in 5 cases a great release of SRIF was only observed after 90 minutes or more (7 to 25 pg/ml/ 1O6 cells). In 2 cases, during the perifusion, the variations of GH and SRIF levels were significantly negatively correlated (p<0.001). Conclusions: 1) SRIF is released in vitro from human pituitary GH-secreting cells, 2) origin of SRIF release: internalization or synthesis ? 3) the inverse correlation between SRIF and GH levels suggests a role of the intracellular SRIF in the regulation of GH secretion.

Characterization of tetanus toxin binding to rat brain membranes. Evidence for a high-affinity proteinase-sensitive receptor.

Binding of 125I-labelled tetanus toxin to rat brain membranes in 25 mM-Tris/acetate, pH 6.0, was saturable and there was a single class of high-affinity site (KD 0.26-1.14 nM) present in high abundance (Bmax. 0.9-1.89 nmol/mg). The sites were largely resistant to proteolysis and heating but were markedly sensitive to neuraminidase. Trisialogangliosides were effective inhibitors of toxin binding (IC50 10 nM) and trisialogangliosides inserted into membranes lacking a toxin receptor were able to bind toxin with high affinity (KD 2.6 nM). The results are consistent with previous studies and the hypothesis that di- and trisialogangliosides act as the primary receptor for tetanus toxin under these conditions. In contrast, when toxin binding was assayed in Krebs-Ringer buffer, pH 7.4, binding was greatly reduced, was non-saturable and competition binding studies showed evidence for a small number of high-affinity sites (KD 0.42 nM, Bmax. 0.90 pmol/mg) and a larger number of low-affinity sites (KD 146 nM, Bmax. 179 pmol/mg). Treatment of membranes with proteinases, heat, and neuraminidase markedly reduced binding. Trisialogangliosides were poor inhibitors of toxin binding (IC50 11.0 microM), and trisialogangliosides inserted into membranes bound toxin with low affinity. The results suggest that in physiological buffers tetanus toxin binds with high affinity to a protein receptor, and that gangliosides represent only a low-affinity site.

Rapid Selection of Viable Transplantable Human Fetal Pancreatic Islets by Trypan Blue Exclusion

A rapid method of separating viable from nonviable human fetal pancreatic islets prior to transplantation is needed to quantify grafted tissue and optimize the possibility of successful treatment of diabetes mellitus in the recipient. After incubation with 0.04% trypan blue in isotonic Krebs-Ringer buffer solution for 15 min, the percentage of islets that excluded trypan blue was found to correlate well with fractional stimulated insulin secretion rates. The incubation procedure did not alter the subsequent insulin secretory capacity of the islets. Trypan blue exclusion rapidly and reliably identifies viable functional islet tissue prior to transplantation.

Enzyme release from the perfused rat heart. The functions of the cytoskeleton under cell-pathological conditions.

The mechanism of enzyme release from Langendorff-perfused rat hearts was studied under the injury conditions of the Ca2+ paradox and 2,4-dinitrophenol poisoning. During perfusion with Krebs-Ringer buffer or in buffered sucrose sarcoplasmic enzymes were massively released when Ca2+ was reintroduced to the perfusion medium (Ca2+ paradox). Mitochondrial matrix enzymes were released to a very small extent. Only the cytoplasmic isoenzyme of the bilocular enzyme malate dehydrogenase was released. The release kinetics of various enzymes with greatly differing molecular weights showed no significant differences. Qualitatively the same results were obtained under 2,4-dinitrophenol poisoned conditions in Ca2+ -free sucrose media. Sarcoplasmic enzymes were massively released, mitochondrial enzymes did not appear in the perfusate. 2,4-Dinitrophenol poisoning alone was not sufficient to cause enzyme release. An additional swelling under these conditions was necessary. ATP from the extracellular space was able to enhance the enzyme release, which was brought about by 2,4-dinitrophenol and cell swelling. A hypothesis is presented that enzyme release is produced by initiating a membrane blebbing process. An elevated intracellular Ca2+ concentration is a necessary prerequisite. In the presence of ATP, active membrane blebbing is caused by contractions of the membrane-anchored cytoskeleton. In the absence of ATP passive membrane blebbing is induced by cell swelling, provided that the cytoskeleton has been crosslinked by Ca2+.

Effect of vasoactive intestinal peptide, peptide histidine isoleucine and growth hormone-releasing factor-40 on bombesin-like immunoreactivity, somatostatin and gastrin release from the perfused rat stomach

Bombesin-like immunoreactivity (BLI) has been demonstrated in neurons of the gastrointestinal tract and gastric BLI secretion can be demonstrated in response to the classical neurotransmitter acetylcholine. Since structurally related peptides VIP, PHI and GRF have to be considered as peptidergic neurotransmitters it was of interest to determine their effect on gastric BLI secretion. Additionally, somatostatin (SLI) and gastrin secretion was examined. The isolated stomach of overnight fasted rats was perfused with Krebs-Ringer buffer via the celiac artery and the effluent was collected via the portal vein. The gastric lumen was perfused with isotonic saline at pH7 or pH2. All four peptides were tested at a dose of 10 −11 M and 10 −8 M at both pH levels and in addition the effect of VIP and PHI was examined at 10 −14 M and 10 −12 M during luminal pH2. At luminal pH7 VIP and PHI stimulated SLI release at 10 −8 M but had no effect on BLI or gastrin secretion. rGRF and hpGRF were both ineffective on SLI and gastrin release while rGRF inhibited and hpGRF stimulated BLI secretion. This effect was not dose related. At luminal pH2 all four peptides stimulated BLI secretion. Stimulation by PHI was already observed at a dose of 10 −14 M while VIP elicited a stimulatory effect at 10 −12 M. PHI at the two lowest concentrations of 10 −14 and 10 −12 M elicited a stimualtion of SLI and gastrin release while the same doses of VIP and the higher doses of all four peptides had no effect on SLI and gastrin secretion at an acidic intraluminal pH. The present data demonstrate that at a low intragastric pH BLI secretion is stimulated predominantly by putative peptidergic neurotransmitters such as PHI and VIP and possibly GRF in contrast to the previously reported cholinergic stimulation at neutral pH. The effect of low dose PHI on gastrin secretion suggests that this peptide might participate in the previously observed vagally-induced gastrin secretion during low intragastric pH. On the other hand, VIP and PHI might contribute to postprandial stimulation of somatostatin release during meal-induced increase of the intragastric pH.

The effect of prednisone and hydrocortisone on the plasma protein binding of prednisolone in man.

The protein binding of prednisolone was studied in plasma obtained from healthy volunteers in the presence of added amounts of prednisone and hydrocortisone. The plasma protein binding was determined using in vitro equilibrium dialysis for 16 h at 37 degrees C against isotonic Krebs-Ringer buffer using radioactive prednisolone. Prednisone appeared to have no effect on prednisolone binding. This surprising result was observed even when prednisone concentrations were more than 35 fold greater than prednisolone concentrations. In contrast, a marked competition between hydrocortisone and prednisolone was observed. These binding data were fit using a nonlinear least squares regression computer program and the capacity and affinity constants for the binding of prednisolone to transcortin and albumin were estimated including the competition for binding sites between prednisolone and hydrocortisone. The results from these studies compare favorably with recent parameter calculations, and our previous work where differences in binding were noted between cushingoid and noncushingoid patients.

The effect of pentagastrin on the gastric secretion by the totally isolated vascularly perfused rat stomach.

Gastric acid and pepsin secretion by the totally isolated vascularly perfused rat stomach was studied. Rat stomachs were vascularly perfused with Krebs-Ringer buffer gassed with 96% O2-4% CO2 and containing 5 mM glucose, 5 mM pyruvate, 4% bovine serum albumin, 10% ovine erythrocytes, and various concentrations of pentagastrin. The gastric lumen was perfused with distilled water gassed with 100% O2. To remove preformed juice and to enable preformed pepsin to be washed out, the initial 20-min gastric juice was discarded. The gastric effluent was then collected continuously for six 10-min periods, and the H+ concentration was determined by titration and the pepsin concentration estimated by a hemoglobin digestion method. Inclusion of pentagastrin in the perfusion buffer increased the acid output dose-dependently from 2.3 +/- 0.4 mueq/h during the control perfusion to a maximum of 12.7 +/- 2.1 mueq/h (p less than 0.01). With the higher doses of pentagastrin and concomitant high acid secretion, gastric acid secretion faded during the collection period. In contrast, gastric pepsin secretion was virtually unaffected by pentagastrin. The present model should be useful for studying the regulation of gastric acid and pepsin secretion.

Glucostatic regulation of (+)-[3H]amphetamine binding in the hypothalamus: correlation with Na+,K+-ATPase activity.

Preincubation of rat hypothalamic slices in glucose-free Krebs-Ringer buffer (37 degrees C) resulted in a time-dependent decrease in specific (+)-[3H]amphetamine binding in the crude synaptosomal fraction prepared from these slices. The addition of D-glucose resulted in a dose- and time-dependent stimulation of (+)-[3H]amphetamine binding, whereas incubation with L-glucose, 2-deoxy-D-glucose, or 3-O-methyl-D-glucose failed to increase the number of (+)-[3H]amphetamine binding sites. Ouabain potently inhibited the glucose-induced stimulation of (+)-[3H]amphetamine binding, suggesting the involvement of Na+,K+-ATPase. Preincubation of hypothalamic slices with glucose also resulted in an increase in Na+,K+-ATPase activity and the number of specific "high-affinity" binding sites for [3H]ouabain, and a good correlation was observed (r = 0.89; P less than 0.02) between the glucose-stimulated increase in (+)-[3H]amphetamine and [3H]ouabain binding. Similar increases in (+)-[3H]amphetamine binding, [3H]ouabain binding, and Na+,K+-ATPase activity were observed in the hypothalamus after parenteral administration of glucose to rats. The administration of anorectic doses of amphetamine (0.1-5.0 mg/kg of body weight) also increased Na+,K+-ATPase activity in the hypothalamus. These data suggest that the (+)-[3H]amphetamine binding site in hypothalamus, previously linked to the anorectic actions of various phenylethylamines, is regulated both in vitro and in vivo by physiological concentrations of glucose. Glucose and amphetamine appear to interact at common sites in the hypothalamus to stimulate Na+,K+-ATPase activity, and the latter may be involved in the "glucostatic" regulation of appetite.