Galanin inhibition of enteric cholinergic neurotransmission: guanosine triphosphate-binding protein interactions with adenylate cyclase.

BACKGROUND. The ability of galanin, a unique 29 amino acid peptide, to affect cholinergic neurotransmission was examined in the guinea pig myenteric plexus. METHODS. The effects of galanin on tritiated acetylcholine ([3H]ACh) release were studied with cultured guinea pig myenteric plexus neurons. Functional correlations were made with longitudinal strips of ileal smooth muscle with attached myenteric plexus for examination of isometric contraction. RESULTS. Galanin abolished potassium-stimulated [3H]ACh release (170% +/- 18% of basal vs 109% +/- 16%). Galanin inhibited [3H]ACh release stimulated by forskolin or cholera toxin. [3H]ACh release stimulated by cholecystokin octapeptide, calcitonin gene-related peptide, substance P, or vasoactive intestinal peptide was also suppressed by galanin (10(-8) mol/L). Inhibitory effects were reversed by pertussis toxin preexposure, indicating involvement of guanosine triphosphate-binding proteins. Galanin inhibited contractility of longitudinal smooth muscle strips exposed to cholecystokinin-8 (EC50 7.0 X 10(-9) mol/L for cholecystokinin alone vs 1.3 X 10(-8) mol/L for cholecystokinin-8 plus galanin) and abolished contractile responses to calcitonin gene-related peptide. CONCLUSIONS. Galanin inhibits cholinergic neurotransmission in myenteric plexus neurons. Inhibitory effects involve guanosine triphosphate-binding protein mediation.     

General Anesthetics Do Not Affect Release of the Neuropeptide Cholecystokinin from Isolated Rat Cortical Nerve Terminals

Background: General anesthetics inhibit evoked release of classic neurotransmitters. However, their actions on neuropeptide release in the central nervous system have not been well characterized.

Methods: The effects of representative intravenous and volatile anesthetics were studied on the release of sulfated cholecystokinin 8 (CCK8s), a representative excitatory neuropeptide, from isolated rat cerebrocortical nerve terminals (synaptosomes). Basal, elevated KCl depolarization-evoked and veratridine-evoked release of CCK8s from synaptosomes purified from rat cerebral cortex was evaluated at 35[degrees]C in the absence or presence of extracellular Ca2+. CCK8s released into the incubation medium was determined by enzyme-linked immunoassay after filtration.

Results: Elevation of extracellular KCl concentration (to 15-30 mm) or veratridine (10-20 [mu]m) stimulated Ca2+-dependent CCK8s release. Basal, elevated KCl- or veratridine-evoked CCK8s release was not affected significantly by propofol (12.5-50 [mu]m), pentobarbital (50 and 100 [mu]m), thiopental (20 [mu]m), etomidate (20 [mu]m), ketamine (20 [mu]m), isoflurane (0.6-0.8 mm), or halothane (0.6-0.8 mm).     

Dendritic outgrowth of myenteric plexus neurons in primary culture.

Myenteric plexus neurons derived from neonatal guinea pigs, when exposed to serum, demonstrated a characteristic pattern of growth, including a proliferating outgrowth zone of glial cells, peripheral extension of dendritic processes, and progressive dendritic growth. Serum effects upon dendritic growth, measured morphometrically, was strongly dose- and temporally dependent. Dendritic density was increased 10-fold (120 hr) by the addition of 6% serum, while mean dendritic length was increased 3-fold. Development of cholinergic function was reflected by release of [3H]ACh in response to cholecystokinin octapeptide, vasoactive intestinal peptide, substance P, and calcitonin gene-related peptide (10(-10) and 10(-8) M).     

General anesthetics do not affect release of the neuropeptide cholecystokinin from isolated rat cortical nerve terminals

BACKGROUND: General anesthetics inhibit evoked release of classic neurotransmitters. However, their actions on neuropeptide release in the central nervous system have not been well characterized. METHODS: The effects of representative intravenous and volatile anesthetics were studied on the release of sulfated cholecystokinin 8 (CCK8s), a representative excitatory neuropeptide, from isolated rat cerebrocortical nerve terminals (synaptosomes). Basal, elevated KCl depolarization-evoked and veratridine-evoked release of CCK8s from synaptosomes purified from rat cerebral cortex was evaluated at 35 degrees C in the absence or presence of extracellular Ca2+. CCK8s released into the incubation medium was determined by enzyme-linked immunoassay after filtration. RESULTS: Elevation of extracellular KCl concentration (to 15-30 mM) or veratridine (10-20 microm) stimulated Ca2+ -dependent CCK8s release. Basal, elevated KCl- or veratridine-evoked CCK8s release was not affected significantly by propofol (12.5-50 microm), pentobarbital (50 and 100 microm), thiopental (20 microm), etomidate (20 microm), ketamine (20 microm), isoflurane (0.6-0.8 mM), or halothane (0.6-0.8 mMm). CONCLUSIONS: Clinically relevant concentrations of several classes of general anesthetics did not affect basal, KCl-evoked, or veratridine-evoked CCK8s release from isolated rat cortical nerve terminals. This is in contrast to the demonstrable effects of certain general anesthetics on the release of amino acid and catecholamine transmitters. These transmitter-specific presynaptic effects of general anesthetics suggest that anesthetic-sensitive presynaptic targets are not common to all transmitter classes.     

Leptin increases small intestinal response to cholecystokinin in leptin-deficient obese mice

INTRODUCTION: Leptin receptors are present in the jejunum, ileum, and vagal neurons. Leptin increases duodenal secretion of cholecystokinin (CCK) and acts with CCK on vagal mechanoreceptors in the regulation of small intestinal motility. We have demonstrated that leptin-deficient (Lepob) obese mice have increased jejunal and normal ileal responses to CCK. Therefore, we hypothesized that leptin administration alters small intestinal motility observed in leptin-deficient obese mice. MATERIALS AND METHODS: Twelve-week-old female leptin-deficient (Lepob) obese mice received either saline (n=12) or 5 microg/g leptin ip (n=12) injections daily. After 4 weeks, jejunal and ileal segments were harvested, mounted in an organ bath, and reacted with acetylcholine (ACh, 10(-5)M) and CCK (10(-8,-7,-6)M). Data were expressed as N/cm2 and compared by ANOVA and Student's t test. RESULTS: The average body weights in the leptin-treated group were significantly decreased compared to those of the saline-treated group (34 versus 49 g, P <0.01). Jejunal responses to ACh within each group were significantly decreased (P <0.05) when compared to ileal responses. No significant differences in responses to ACh were observed between groups. Jejunal responses to 10(-7,-6)M CCK in the leptin-treated group were significantly greater than those in the saline-treated group. Ileal responses in the leptin group were similarly increased at all CCK concentrations. CONCLUSIONS: These data suggest that daily leptin administration for 4 weeks in leptin-deficient (Lepob) obese mice increases jejunal and ileal responses to CCK and does not alter responses to ACh. Therefore, we conclude that regulation of small intestinal motility may be influenced by synergistic action of cholecystokinin and leptin.     

Halothane and Isoflurane Differentially Affect the Regulation of Dopamine and Gamma-aminobutyric Acid Release Mediated by Presynaptic Acetylcholine Receptors in the Rat Striatum

Background: General anesthetics are thought to produce their hypnotic effects mainly by acting at ligand-gated ionic channels in the central nervous system (CNS). Although it is well established that volatile anesthetics significantly modify the activity of the acetylcholine nicotinic receptors of the neuromuscular junction, little is known about their actions on the acetylcholine receptors in the CNS. In this study, the effects of halothane and isoflurane on the regulation of dopamine (DA) (gamma-aminobutyric acid [GABA]) depolarization-evoked release mediated by nicotinic (muscarinic) presynaptic receptors were studied in the rat striatum.

Methods: Assay for GABA (dopamine) release consisted of3 H-GABA (sup 3 H-DA)-preloaded synaptosomes with artificial cerebrospinal fluid (0.5 ml/min, 37 degrees Celsius) and measuring the radioactivity obtained from 1-min fractions for 18 min, first in the absence of any treatment (spontaneous release, 8 min), then in the presence of depolarizing agents combined with vaporized halothane and isoflurane (0.5-5%, 5 min), and finally with no pharmacologic stimulation (5 min). The depolarizing agents were potassium chloride (KCl; 9 mM) alone or with acetylcholine (10 sup -6 - 10 sup -4 M) and/or atropine (10 sup -5 M) for experiments with3 H-GABA, and KCl (15 mM) and nicotine (10 sup -7 - 5 x 10 sup -4 M) alone or with mecamylamine (10 sup -5 M) for experiments with3 H-DA.

Results: Potassium chloride induced a significant, Ca2+ -dependent release of both3 H-GABA and3 H-DA. Nicotine produced a concentration-related, mecamylamine-sensitive3 H-DA release that was significantly attenuated by nicotine (10 sup -7 M) preincubation. Acetylcholine elicited a dose-dependent, atropine-sensitive reduction of the KCl-evoked3 H-GABA release. Halothane and isoflurane significantly decreased the nicotine-evoked3 H-DA release but had only limited depressant effects on the KCl-stimulated3 H-DA and no action on the KCl-induced3 H-GABA release. The effects of acetylcholine on3 H-GABA release were reversed by halothane but not by isoflurane.     

Anesthetics Affect the Uptake but Not the Depolarization-evoked Release of GABA in Rat Striatal Synaptosomes

Background: Numerous classes of anesthetic agents have been shown to enhance the effects mediated by the postsynaptic gamma-aminobutyric acid A (GABAA) receptor-coupled chloride channel in the mammalian central nervous system. However, presynaptic actions of anesthetics potentially relevant to clinical anesthesia remain to be clarified. Therefore, in this study, the effects of intravenous and volatile anesthetics on both the uptake and the depolarization-evoked release of GABA in the rat stratum were investigated.

Methods: Assay for specific GABA uptake was performed by measuring the radioactivity incorporated in purified striatal synaptosomes incubated with3 H-GABA (20 nM, 5 min, 37 degrees Celsius) and increasing concentrations of anesthetics in either the presence or the absence of nipecotic acid (1 mM, a specific GABA uptake inhibitor). Assay for GABA release consisted of superfusing3 H-GABA preloaded synaptosomes with artificial cerebrospinal fluid (0.5 ml *symbol* min sup 1, 37 degrees Celsius) and measuring the radioactivity obtained from 0.5 ml fractions over 18 min, first in the absence of any treatment (spontaneous release, 8 min), then in the presence of either KCl alone (9 mM, 15 mM) or with various concentrations of anesthetics (5 min), and finally, with no pharmacologic stimulation (5 min). The following anesthetic agents were tested: propofol, etomidate, thiopental, ketamine, halothane, enflurane, isoflurane, and clonidine.

Results: More than 95% of3 H-GABA uptake was blocked by a 10 sup 3 -M concentration of nipecotic acid. Propofol, etomidate, thiopental, and ketamine induced a dose-related, reversible, noncompetitive, inhibition of3 H-GABA uptake: IC50 = 4.6 plus/minus 0.3 x 105 M, 5.8 plus/minus 0.3 x 10 sup -5 M, 2.1 plus/minus 0.4 x 10 sup -3 M, and 4.9 plus/minus 0.5 x 10 sup -4 M for propofol, etomidate, thiopental, and ketamine, respectively. Volatile agents and clonidine had no significant effect, even when used at concentrations greater than those used clinically. KCl application induced a significant, calcium-dependent, concentration-related, increase from basal3 H-GABA release, +34 + 10% (P < 0.01) and +61 plus/minus 13% (P < 0.001), respectively, for 9 mM and 15 mM KCl. The release of3 H-GABA elicited by KCl was not affected by any of the anesthetic agents tested.     

Cholecystokinin-induced alterations in beta-cell sensitivity. Duration, specificity, and involvement of phosphoinositide metabolism

Prior exposure of isolated perifused rat islets to the sulfated gut hormone cholecystokinin-8 (CCK-8S) dramatically increased their insulin secretory response to 7.5 mM glucose, 10 mM arginine, and 10 mM alpha-ketoisocaproate. In the case of glucose, the heightened secretory response was still apparent 60-80 min after CCK-8S removal from the perifusion medium. Prior exposure of perifused islets to arginine (10 mM), tolbutamide (25 microM), or forskolin (1.0 microM) did not sensitize them to 7.5 mM glucose. CCK-8S exposure increased 3H efflux from islets prelabeled with [3H]inositol, and the increase in 3H efflux was sustained after CCK-8S removal from the perifusion medium. The duration of this increase in 3H efflux paralleled the temporal characteristics of this sensitization process and was significantly attenuated by 25 microM asperlicin, a competitive antagonist of CCK binding to its membrane receptor. Arginine, tolbutamide, or forskolin treatment of islets did not increase 3H efflux from [3H]inositol-prelabeled islets. The results suggest that the turnover of membrane phosphoinositides induced by CCK-8S is largely responsible for this heightened state of secretory responsiveness to various stimulants. Second-messenger molecules generated during phosphoinositide turnover may be responsible for the phenomenon of sensitization displayed by islet tissue to CCK-8S addition.     

Riluzole Blocks Dopamine Release Evoked by N-methyl-D-aspartate, Kainate, and Veratridine in the Rat Striatum

Background: Dopamine (DA) is released in large amounts during cerebral ischemia and may exacerbate tissue damage. Riluzole (54274 RP) is a recently developed agent that depresses glutamate neurotransmission in the central nervous system (CNS) and that may protect against ischemic injury in some animal models. Because glutamate stimulates the release of DA in the striatum, the authors hypothesized that riluzole could antagonize DA release in this structure.

Methods: Assay for DA release consisted of superfusing3 H-DA preloaded synaptosomes with artificial cerebrospinal fluid (1 ml/min, 37 [degree sign] Celsius) and measuring the radioactivity obtained from 1-min fractions over 22 min, first in the absence of any treatment (spontaneous release, 8 min), then in the presence of depolarizing agents combined with riluzole (0.1-100 micro Meter, 5 min), and finally with no pharmacologic stimulation (9 min). The following depolarizing agents were tested: KCl (9, 15 mM), veratridine (0.01-1 micro Meter), N-methyl-D-aspartate (NMDA, 0.1-1 mM), kainate (0.1-1 mM), and nicotine (0.01-0.5 mM). Assay for DA uptake was performed by measuring the radioactivity incorporated in synaptosomes incubated with3 H-DA (44 nM; 5 min; 37 [degree sign] Celsius).

Results: All depolarizing agents produced a significant, concentration-related increase from basal3 H-DA release. Riluzole was found to decrease the release induced by veratridine (1 micro Meter), NMDA (1 mM), and kainate (1 mM) in a significant, concentration-related manner (IC50 = 9.5 micro Meter, 1.6 micro Meter, and 5.8 micro Meter for veratridine, NMDA, and kainate, respectively). In contrast, it did not affect the release elicited by either KCl or nicotine. Riluzole had no significant effect on the specific3 H-DA uptake.     

Stimulation of insulin secretion from isolated rat islets by SaRI 59-801

Oral administration of SaRI 59-801 (DL-alpha-[dimethylaminomethyl]-2-[3-ethyl-5-methyl-4-isoxazolyl]-1H- indole-3-methanol) has been reported to decrease blood glucose in several species and to elevate plasma insulin in rats and mice. In studies with isolated rat pancreatic islets incubated 1 h with 3 mM glucose, 0.05 mM 59-801 produced a significant increase in insulin secretion, and 0.3 mM produced maximum release. 59-801 (0.3 mM) stimulated insulin release 4-5-fold from islets incubated with 0, 3, or 5 mM glucose but had little effect on the high rates of release obtained at 10 or 20 mM glucose. Ten millimolar mannoheptulose, which inhibits phosphorylation of glucose and blocks glucose-stimulated insulin release, had little effect on the stimulation of insulin release by 0.3 mM 59-801 from islets incubated with 3 mM glucose. Stimulation of insulin release in the absence of glucose or in the presence of 3 mM glucose plus 10 mM mannoheptulose suggests that glucose metabolism is not required for the action of 59-801. The rate of conversion of 5 mM [5(-3)H]-glucose to 3H2O by islets, a measure of the rate of glycolysis, was not affected by 59-801. The potency, dependency on glucose concentration, lack of inhibition by mannoheptulose, and lack of effect on glycolysis of 59-801 were similar to that of tolbutamide. However, proinsulin synthesis by islets incubated with 5.55 mM glucose was not affected by 0.5 mM 59-801, but was inhibited 72% and 67% by 0.5 mM tolbutamide and 0.1 mM glibenclamide, respectively.     

Gallbladder myocytes are short and cholecystokinin-resistant in obese diabetic mice

BACKGROUND: Obesity is associated with diabetes and gallstone formation. Obese leptin-deficient (Lepob) and leptin-resistant (Lepdb) mice are hyperglycemic and have enlarged gallbladders with diminished response in vitro to cholecystokinin (CCK) and acetylcholine (ACh). Whether this phenomenon is secondary to hyperosmolar myocytes and/or decreased neuromuscular transmission remains unclear. We hypothesize that myocytes from Lepob and Lepdb obese mice would not respond normally to neurotransmitters. METHODS: Cholecystectomy was performed on 39 lean, 19 Lepob, and 20 Lepdb 12-week-old female mice. The gallbladder was divided and enzymatically digested. Half of each gallbladder's myocytes had contraction induced by CCK (10(-8) mol/L, n = 38) or ACh (10(-5) mol/L, n = 40). RESULTS: Body weights, gallbladder volumes, and serum glucoses were greater for Lep(ob) and Lepdb mice compared to controls (P < .001). Resting myocyte lengths from Lepob and Lepdb mice were 93% and 91% of the length of controls (P < .001). In response to CCK, lean myocytes shortened 6% (P < .01), while myocytes from obese mice demonstrated no shortening. None of the myocytes demonstrated significant shortening with ACh. CONCLUSIONS: These data suggest that gallbladder myocytes from obese mice are (1) foreshortened and (2) have a diminished response to cholecystokinin. We conclude that altered leptin and/or increased glucose may foreshorten myocytes and decrease response to cholecystokinin.     

Pioglitazone increases gallbladder volume in insulin-resistant obese mice

BACKGROUND: Both obesity and diabetes are associated with an increased incidence of gallstones. Recent animal and human data from our laboratory suggest that insulin resistance is associated with increased gallbladder volume and/or impaired gallbladder emptying. Pioglitazone is a thiazolidinedione that has been shown to improve insulin resistance. Therefore, we tested the hypothesis that pioglitazone would improve insulin resistance, decrease resting gallbladder volume and improve gallbladder response to neurotransmitters in insulin-resistant obese mice fed a 25% carbohydrate diet. MATERIALS AND METHODS: Twenty eight-week-old insulin-resistant obese (Lep(ob)) mice fed a 25% carbohydrate diet for 4 weeks. Half of the animals had 0.3 g/kg pioglitazone added to their diet. At 12 weeks all animals were fasted and underwent cholecystectomy. Gallbladder volume and weight were measured, and fresh gallbladders were placed in a muscle bath to assess response to acetylcholine (ACh 10(-5)M), neuropeptide Y (NPY 10(-8,-7,-6)M) and cholecystokinin (CCK 10(-10,-9,-8,-7)M). Serum glucose and insulin were measured, and HOMA Index, a measure of insulin resistance, was calculated. RESULTS: Fasting serum insulin and HOMA Index were significantly decreased (P < 0.03), but gallbladder volume was significantly increased (P < 0.03) in the pioglitazone treated group. Pioglitazone did not alter gallbladder weight or response to ACh, NPY, or CCK. CONCLUSION: These data suggest that in insulin-resistant obese mice pioglitazone 1) lowers insulin-resistance, 2) increases resting gallbladder volume, and 3) does not alter gallbladder response to neurotransmitters. Therefore, we conclude that pioglitazone, while improving insulin resistance, paradoxically increases gallbladder volume and, thereby, may increase the propensity for gallstone formation by enhancing gallbladder stasis.     

Increased beta-cell secretory responsiveness to ceruletide and TPA in streptozocin-induced mildly diabetic rats

We examined the effects of various stimuli on immunoreactive insulin (IRI) and glucagon (IRG) release from perfused pancreases isolated from control and streptozocin-induced diabetic (STZ-D) rats. Diabetes was induced by injecting 30 mg/kg STZ into rats fasted for 16-18 h 12-17 days before our experiments. Glucose (11.1 mM) caused a distinct biphasic pattern of IRI release from the control pancreas, whereas the first phase was marginal and the second phase was absent in the diabetic pancreas. Arginine (20 mM)-induced IRI release was similar in both groups, whereas IRG release was greater in the control rats than in the diabetic rats. Thus, this model of STZ-D simulates a certain class of non-insulin-dependent diabetes mellitus (NIDDM). In these diabetic animals, the cholecystokinin (CCK) analogue ceruletide (620 pM) caused a significantly greater increase in IRI release in the presence of 5.6 mM glucose than in the control rats, but ceruletide caused a similar IRG release in both groups. Because CCK and ceruletide stimulate phosphoinositide turnover in pancreatic islets, we examined the effects of carbachol and phorbol ester TPA on IRI release in the presence of 5.6 mM glucose. Carbachol (10 microM), which is thought to generate similar second messengers as ceruletide, induced greater IRI release in diabetic than in control rats. TPA (100 nM) caused a significantly greater increase in IRI release from the diabetic than the control pancreas. Our results demonstrate that the insulin-releasing mechanism involved in protein kinase C activation is enhanced in this model of NIDDM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulatory effects of cholecystokinin on isolated perifused islets inhibited by potent and specific antagonist L 364718 [corrected

The influence of L 364718 on islet responsiveness to sulfated cholecystokinin (CCK-8S) was investigated. In islets whose inositol-containing phospholipids were prelabeled during a 2-h incubation period, subsequent exposure to L 364718 (1 nM) significantly impaired the secretion of insulin usually noted in response to 200 nM CCK-8S in the simultaneous presence of 7 mM glucose. A higher level of the antagonist (10 nM) completely abolished insulin secretion. L 364718 (1-10 nM) reduced the efflux of 3H from myo-[2-3H]-inositol prelabeled islets in parallel with the reduction in secretion. L 364718 (10 nM) significantly reduced the accumulation of 3H-containing inositol phosphates usually noted with CCK-8S addition. L 364718, at levels 10- to 100-fold greater than those necessary to attenuate CCK-8S-induced insulin secretion, had no adverse effect on the insulin secretory response of freshly isolated islets to 10 mM glucose alone, 5 mM D-glyceraldehyde, 15 mM alpha-ketoisocaproate, or 50 ng/ml gastric inhibitory polypeptide. L 364718 (1000 nM) had no adverse influence on carbamylcholine (1 mM)-induced phosphoinositide hydrolysis. These results establish L 364718 as a potent and highly selective antagonist of cholecystokinin's stimulatory actions on beta-cells. Because of its potency, selectivity, and oral effectiveness, in vivo studies with L 364718, aimed at unraveling the pleiotropic effects of CCK-8S on glucose and insulin homeostasis, seem feasible.     

The effects of general anesthetics on norepinephrine release from isolated rat cortical nerve terminals

Intravenous and volatile general anesthetics inhibit norepinephrine (NE) release from sympathetic neurons and other neurosecretory cells. However, the actions of general anesthetics on NE release from central nervous system (CNS) neurons are unclear. We investigated the effects of representative IV and volatile anesthetics on [(3)H]NE release from isolated rat cortical nerve terminals (synaptosomes). Purified synaptosomes prepared from rat cerebral cortex were preloaded with [(3)H]NE and superfused with buffer containing pargyline (a monoamine oxidase inhibitor) and ascorbic acid (an antioxidant). Basal (spontaneous) and stimulus-evoked [(3)H]NE release was evaluated in the superfusate in the absence or presence of various anesthetics. Depolarization with increased concentrations of KCl (15-20 mM) or 4-aminopyridine (0.5-1.0 mM) evoked concentration- and Ca(2+)-dependent increases in [(3)H]NE release from rat cortical synaptosomes. The IV anesthetics etomidate (5-40 microM), ketamine (5-30 microM), or pentobarbital (25-100 microM) did not affect basal or stimulus-evoked [(3)H]NE release. Propofol (5-40 microM) increased basal [(3)H]NE release and, at larger concentrations, reduced stimulus-evoked release. The volatile anesthetic halothane (0.15-0.70 mM) increased basal [(3)H]NE release, but did not affect stimulus-evoked release. These findings demonstrate drug-specific stimulation of basal NE release. Noradrenergic transmission may represent a presynaptic target for selected general anesthetics in the CNS. Given the contrasting effects of general anesthetics on the release of other CNS transmitters, the presynaptic actions of general anesthetics are both drug- and transmitter-specific. IMPLICATIONS: General anesthetics affect synaptic transmission both by altering neurotransmitter release and by modulating postsynaptic responses to transmitter. Anesthetics exert both drug-specific and transmitter-specific effects on transmitter release: therapeutic concentrations of some anesthetics stimulate basal, but not evoked, norepinephrine release, in contrast to evoked glutamate release, which is inhibited.     

Induction of resistance to endothelin-1's biochemical actions by elevated glucose levels in retinal pericytes.

Because retinal pericytes have contractile properties and are affected by diabetes, we have studied the responsiveness of pericytes to ET-1, a potent vasoconstrictor, in the presence of various concentrations of glucose. Cultured calf retinal pericytes were exposed to glucose levels of 5.5 or 25 mM for up to 8 days. Radioreceptor studies that used [125I]ET-1 showed that pericytes contained high-affinity binding sites with Kd of 3 x 10(-10) M, and these binding affinities were unaffected by glucose concentration. Receptor number appears to be elevated, but this increase was NS. Responsiveness of pericytes to ET-1 was studied with respect to stimulation of DAG and IP3 levels and PKC activities. In contrast to receptor binding, exposure to 25 mM glucose for > 6 days blunted pericyte responsiveness to ET-1. The time course of ET-1 stimulation as measured by [3H]glycerol labeling, and IP3 level showed a 98% increase in [3H]DAG at 10 min and a fourfold increase for IP3, respectively. Cells exposed to 25 mM glucose only had a 32% increase for DAG, and no increase for IP3 was observed. Dose-response studies on the stimulation of [3H]DAG increase showed the range of ET-1's effect to be between 10(-9) and 10(-7) M. At maximum, cells exposed to 5.5 mM glucose had a 70% increase versus only a 30% increase in those exposed to 25 mM glucose. Similarly, ET-1 only increased the total DAG levels in pericytes exposed to 5.5 mM glucose by 41%. PKC activity also was measured because DAG is one of its cellular activators.(ABSTRACT TRUNCATED AT 250 WORDS)     

The combined effect of perioperative donor spleen cells or KCl-extracted antigen and cyclosporine on renal allograft survival in the rat

The effects of using perioperative cyclosporine in conjunction with pretreatment with donor spleen cells or 3M KCl solubilized extracts of donor antigen were investigated in a LEW-to-DA rat renal allograft model. Cyclosporine given orally in a dose of 10 mg/kg/day around the time of transplantation (days -1, 0, +1), did not prolong renal allograft survival (median survival time [MST]--10 days). However when used in combination with pretreatment with either 10(8) donor spleen cells (1 day before transplantation), or 10(5) donor spleen cells (7 days before transplantation), pretreatment regimens that were in themselves ineffective, DA recipients accepted Lewis renal allografts indefinitely (MST greater than 100 days). Soluble antigen was prepared by 3M KCl extraction from donor spleen cells. Absorption assays were used to quantify the amount of class I major histocompatibility complex antigen in the preparation, and amounts of antigen equivalent to that expressed by 10(6)-10(8) donor spleen cells were used for pretreatment. These soluble antigen preparations given either 1 or 7 days before transplantation with or without perioperative cyclosporine did not prolong allograft survival of either homozygous or heterozygous donors (MST 10 days).     

Effect of Bupivacaine and Levobupivacaine on Exocytotic Norepinephrine Release from Rat Atria

Background: The cardiotoxic mechanism of local anesthetics may include interruption of cardiac sympathetic reflexes. The authors undertook this investigation to determine if clinically relevant concentrations of bupivacaine and levobupivacaine interfere with exocytotic norepinephrine release from cardiac sympathetic nerve endings.

Methods: Rat atria were prepared for measurements of twitch contractile force and 3[H]-norepinephrine release. After nerve endings were loaded with 3[H]-norepinephrine, the tissue was electrically stimulated in 5-min episodes during 10 10-min sampling periods. After each period, a sample of bath fluid was analyzed for radioactivity and 3[H]-norepinephrine release was expressed as a fraction of tissue counts. Atria were exposed to buffer alone during sampling periods 1 and 2 (S1 and S2). Control atria received saline (100 [mu]l each, n = 6 atria) in S3-S10. Experimental groups (n = 6 per group) received either bupivacaine or levobupivacaine at concentrations (in [mu]M) of 5 (S3-S4), 10 (S5-S6), 30 (S7-S8), and 100 (S9-S10).

Results: Bupivacaine and levobupivacaine decreased stimulation-evoked fractional 3[H]-norepinephrine release with inhibitory concentration 50% values of 5.1 +/- 0.5 and 6.1 +/- 1.3 [mu]m. The inhibitory effect of both local anesthetics (~70%) approached that of tetrodotoxin. Local anesthetics abolished the twitch contractions of atria with inhibitory concentration 50% values of 12.6 +/- 5.0 [mu]m (bupivacaine) and 15.7 +/- 3.9 [mu]m (levobupivacaine). In separate experiments, tetrodotoxin inhibited twitch contractile force by only 30%.     

Adenosine-induced modulation of excitatory amino acid transport across isolated brain arterioles

OBJECT: Excitatory amino acid (EAA) uptake by neurons and glia acts synergistically with stereoselective transport across the blood-brain barrier (BBB) to maintain EAA homeostasis in the brain. The endogenous neuroprotectant adenosine counteracts many aspects of excitotoxicity by increasing cerebral blood flow and by producing pre- and postsynaptic actions on neurons. In the present study, the authors explored the effect of adenosine on EAA transport across the BBB. METHODS: The effects of adenosine on the permeability of the BBB and transport of aspartate and glutamate across the BBB were studied in a well-characterized isolated penetrating cerebral arteriole preparation suitable for simultaneous investigations of changes in diameter and permeability. At concentrations within the physiological to low pathophysiological range (10(-7)-10(-6) M), the net vectorial transport of [3H]L-glutamate or [3H]L-aspartate from blood to brain was significantly attenuated, whereas there was no effect of adenosine on paracellular BBB permeability to [14C]sucrose or [3H]D-aspartate. With higher concentrations of adenosine (10(-4) M and 10(-3) M) the net vectorial transport of [3H]L-glutamate and [3H]Laspartate returned toward baseline. At 10(-3) M, the permeability to [14C]sucrose was significantly altered, indicating a breakdown in the BBB. The effect of adenosine (10(-6) M) was blocked by theophylline, a blocker of the A1 and A2 receptors of adenosine. CONCLUSIONS: Adenosine-mediated modulation of glutamate and aspartate transport across the BBB is a novel physiological finding.     

Cytochalasin B binding to rabbit proximal tubular basolateral membranes

Cytochalasin B binds to the Na+-independent D-glucose transporter in non-renal tissues. We have shown previously that the Na+-independent D-glucose transporter of the rabbit renal proximal tubular cell is localized exclusively in the basolateral membrane. To determine whether cytochalasin B binds to this renal transporter we measured binding of [3H]cytochalasin B to proximal tubular basolateral membranes isolated from rabbit kidneys. A steady state of binding is reached by 15 minutes at 20 degrees C over a concentration range of 0.01 to 50 microM. Non-linear regression analysis of cytochalasin B binding from 0.01 to 20 microM plotted according to Scatchard reveals two classes of binding sites with Kd 5.88 x 10(-8) M, Bmax 16.1 pmol/mg protein; and Kd 5.62 x 10(-5) M, Bmax 2816 pmol/mg protein. [3H]cytochalasin B (0.1 microM) binding to basolateral membranes is a reversible process; it is displacable by excess unlabeled cytochalasin B with a time course similar to binding of [3H]cytochalasin B. Binding of [3H]cytochalasin B is inhibited by 500 mM D-glucose (21%), 2-deoxy-D-glucose (57%) and 3-O-methyl-D-glucose (64%), but not by L-glucose. [3H]cytochalasin B binding is reduced 71% by 0.1 mM phloretin, but only 26% by 0.1 mM phlorizin. Such substrate specificity and inhibitor sensitivity are similar to those previously demonstrated in non-renal tissues by others as well as in rabbit renal proximal tubular basolateral membranes by us. Our data suggest that cytochalasin B binds to the Na+-independent D-glucose transporter or a component of the transporter in the renal proximal tubular basolateral membrane.