Cyclic nucleotide function in trachealis muscle of dogs with and without airway hyperresponsiveness

We examined basal adenosine 3',5'-cyclic monophosphate (cAMP) levels, isoproterenol (ISO)-stimulated cAMP responses, basal cAMP, and guanosine 3',5'-cyclic monophosphate (cGMP) phosphodiesterase (PDE) activities and protein-kinase (PK) activities in trachealis muscle from five Basenji-greyhound (BG) and four greyhound dogs to determine whether the inverse relationship between in vivo and in vitro airway responsiveness could be due to altered cyclic nucleotide metabolism. Basal cAMP levels were not significantly different (PNS) in muscle from BG (11.6 +/- 0.53 pmol/mg protein) and greyhound dogs (10.30 +/- 1.60 pmol/mg protein). The cAMP responses to stimulation with ISO were enhanced in BG compared with greyhound dogs. The low Michaelis constant (1) for Km-cAMP PDE activity (Km = 0.63 microM) was significantly less (P less than 0.005) in BG dogs (1.54 +/- 0.28 pmol.min-1.mg protein-1) than greyhounds (11.76 +/- 2.48). Endogenously active PK activity was significantly greater (P less than 0.005) in BG (54.74 +/- 5.39 pmol.min-1.mg protein-1) than in greyhound dogs (15.50 +/0 2.20). Increases in PK activity with 5 microM cAMP added were not significantly different between BG (14.79 +/- 6.00) and greyhound dogs (7.04 +/- 2.14). Approximately 90% of both endogenous PK activity and cAMP-activated PK activity in BG and greyhound dogs was inhibited by a cAMP-dependent PK inhibitor (PKI'). These data suggest that decreased cyclic nucleotide degradation due to decreased cyclic nucleotide PDE activity with increased PK could account for the in vitro hyporesponsiveness of airway smooth muscle in BG dogs as a protective adaptive mechanism.     

Effect of beta-N-oxalylamino-l-alanine on cerebellar cGMP level in vivo

Beta-N-oxalylamino-l-alanine (BOAA), a non-protein amino acid present in the seeds of Lathyrus Sativus (LS), is one of several neuroactive glutamate analogs reported to stimulate excitatory receptors and, in high concentrations, cause neuronal degeneration. In the present study, the in vivo acute effects of synthetic BOAA and LS seed extract were investigated on rat cerebellar cyclic GMP following intraperitoneal (10–100 mg/kg) or oral (100 mg/kg) administration of subconvulsive doses of toxin. Furthermore, the BOAA content in LS seeds and in the cerebellum of injected rats was determined by high performance liquid chromatograph analysis. A dose- and time-dependent increase of cerebellar cyclic guanosine monophosphate (cGMP) level was observed after intraperitoneal administration of synthetic BOAA or LS extract. The neurotoxin evoked a maximum stimulation 90 min after injection within the dose range of 50–75 mg/kg, elevating cGMP from basal levels of 5.3±0.5 pmol/mg protein to 15±1.3 pmol/mg protein. Similarly, the oral intake of LS-extracted neurotoxin resulted in the elevation of cGMP content. Kynurenic acid (300 mg/kg i.p.), a non specific excitatory amino acid antagonist, was effective in blocking LS BOAA-elicited cGMP enhancement. The data suggest that in the cerebellum acute administration of low concentrations of BOAA exert in vivo activation of glutamate receptors involved in the regulation of cGMP level.     

Compartmentalization of cyclic AMP elevation in neurons ofAplysia californica

We have measured by radioimmunoassay the amount of total, free, and bound forms of cyclic AMP (cAMP) within the abdominal ganglion and in five identified cell bodies of neurons from Aplysia californica. In the abdominal ganglion the unbound (free) cAMP levels comprised approximately 25-30% of the total cAMP content under the unstimulated condition, i.e., bathed in high-magnesium saline. Under pharmacological conditions that blocked endogenous phosphodiesterase and activated adenylate cyclase, ganglionic free cAMP levels were elevated more than fourfold, while bound cAMP levels more than doubled. Freeze-substitution techniques were employed to facilitate isolation of individual cell bodies either before or after pharmacological manipulation of cAMP levels. The basal, free cAMP content of cells R2, LP1, R15, L11, and L2-L6 was in the range of 10-40 pmol/mg of cell protein, which accounted for approximately one-half of the total cAMP content per cell body. Determinations of individual cell volumes indicated that the basal, free cAMP concentrations ranged from 1 to 6 microM. Under the same pharmacological conditions that elevated ganglionic cAMP in levels, no changes were measured in either the free or the bound forms of cAMP in isolated cell bodies. Our results indicate that the cAMP elevation was compartmentalized within the neuropilar region of the ganglion, most likely within the processes of the nerve cells. Previous results demonstrated that cAMP injections into the same Aplysia neurons studied here induced a cAMP-activated sodium current, INa (cAMP). In this report we discuss the possibility that pharmacological elevation of cAMP within neuronal processes may reach concentrations similar to those produced by cAMP injections into somata.     

Elevations of intracellular cAMP result in a change in cell shape that resembles dome formation in cultured rat glomerular epithelial cells

Summary Cultured glomerular epithelial cells form a continuous monolayer of polyhedral-shaped cells. PGE₂ (1 μg/ml) in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine (MIX) markedly raises intracellular and medium cyclic AMP (cAMP) levels at 20 min (intracellular: MIX alone, 112 ± 6.6 pmol cAMP/mg protein, MIX plus PGE₂, 2252±63 pmol cAMP/mg protein; medium: MIX, 20.6±2.1 pmol cAMP/mg protein; MIX plus PGE₂, 117±3.8 pmol cAMP/mg protein). By 2 h, when cellular and medium cAMP levels were still elevated, the cells underwent a change in shape that was similar to dome formation (15 to 20% of the monolayer changing shape). Derivatives of cAMP [i. e. dibutyryl and 8-(4-chlorophenylthio)-cAMP], when added to the incubation medium also caused shape change in glomerular epithelial cells at 2 h; cAMP itself did not. The formation of domes has been used as a morphological indicator of the vertorial transport of salt and water in other cultured epithelial cells. This work was supported by grant AM 29787 from the National Institutes of Health, Bethesda, MD.     

Concentration of components of the adenylate system in heavy and light fractions of the sarcoplasmic reticulum of skeletal muscles and sensitivity of these fractions to the effects of imidazole-containing compounds and caffeine]

Fragments of sarcoplasmic reticulum from rabbit sceletal muscles sedimented within the range from 2000 g to 8000 g (heavy fraction) and 8000 g to 40000 g (light fraction) and washed with 0.6 M KCl, were practically free of adenylatecyclase activity. Phosphodiesterase cAMP was not found in the light fraction, while its activity in the heavy fraction was 500 pmol of cAMP/min per mg of protein. Both fractions contain bound cAMP (1-2 pmol/mg of protein) and specific sites of cAMP binding, the binding constant being approximately 10(6)M-1. The number of binding sites is 60 pmol/mg of protein for the heavy and 30 pmol/mg of protein for the light fractions. The level of phosphodiesterase activity in the heavy fraction correlates with its sensitivity to imidazole, anserine and caffeine. Imidazole and anserine increase in 1.5-1.8 times the value of Ca2+/ATP in the heavy fraction and produce no effect on Ca2+ transport by the light fraction. Caffeine decreases almost twice the Ca2+/ATP value in the heavy fraction and has practically no effect on Ca2+ absorption by enzymes of the light reticulum fraction. Imidazole and anserine activate membrane-bound phosphodiesterase, while caffeine inhibits it. It is suggested that structural rearrangements of membrane-bound phosphodiesterase under the effect of caffeine, imidazole and anserine are responsible for changes in the efficiency of Ca2+ transport by fragments of the heavy reticulum fractions.     

Distribution and persistence of kainic acid in brain.

The distribution of ³H-kainic acid in rat brain was studied as a function of time after injections of 5 nmoles into the neostriatum, substantia nigra or cerebellum. More than half of the injected material had disappeared from the injection site and the brain by 1/2 hour post injection. Under the conditions used very small amounts of radioactivity (corresponding to less than 7 pmol/ mg of tissue) were found in areas other than the injection site, suggesting that the histological damage reported in the hippocampus and pyriform cortex after striatal injections may be due to a secondary process not dependent on the presence of toxic concentrations of kainic acid in those areas. No radioactivity was found in the TCA-insoluble material nor did it appear that there was rapid metabolism of the bulk of the kainic acid.     

Response of hepatic carbohydrate and cyclic AMP metabolism to cadmium treatment in rats.

Daily intraperitoneal injection of cadmium chloride (0.25 or 1 mg/kg) for 21 or 45 days into rats significantly stimulated the activities of hepatic pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose-1, 6-diphosphatase, and glucose-6-phosphatase, increased the concentrations of glucose and urea in the blood, and decreased the levels of glycogen in the liver. Whereas chronic cadmium treatment failed to alter adenosine-3',5'-monophosphate phosphodiesterase (phosphodiesterase) activity, the endogenous levels of cyclic AMP (cAMP) and the activity of basal- and fluoride-stimulated forms of hepatic adenylate cyclase (AC) were markedly increased in cadmium-injected animals. Treatment with the higher dose (1.0 mg/kg) of cadmium chloride for 45 days produced greater metabolic alterations in hepatic tissue than those seen with the lower dose (0.25 mg/kg) given for a shorter period of time (21 days). Discontinuation of cadmium administration for 14 days in rats previously injected with cadmium chloride (1 mg/kg per day) for 21 days, failed to reverse the observed changes in hepatic cAMP or carbohydrate metabolism. A similar persistence of metabolic alterations was noted in rats treated with cadmium (1 mg/kg per day) for 45 days and subsequently maintained without additional treatment for 28 days. Administration of an acute dose of cadmium chloride (60 mg/kg) decreased hepatic phosphodiesterase activity and glycogen content 1 h after the injection. In addition, acute cadmium exposure increased blood glucose, serum urea, and hepatic cAMP levels, and produced an augmentation of basal- and fluoride-activated AC. However, the activities of various hepatic gluconeogenic enzymes remained unaffected in animals given an acute dose of cadmium chloride (60 mg/kg). Data provide evidence that suggests that the gluconeogenic potential of liver is markedly enhanced following chronic exposure to cadmium and that the cadmium-induced changes in carbohydrate metabolism may be associated with an enhanced synthesis of cAMP. In addition, the present study shows that the cadmium-induced metabolic alterations persist even after the cessation of cadmium treatment for a period of 28 days.     

Evoked dopamine overflow is augmented in the striatum of calcitriol treated rats

Calcitriol, the active metabolite of vitamin D, has been shown to have significant effects on the brain. These actions include reducing the severity of some central nervous system lesions, possibly by upregulating trophic factors such as glial cell line-derived neurotrophic factor (GDNF). GDNF has substantial effects on the nigrostriatal dopamine (DA) system of young adult, aged and lesioned animals. Thus, the administration of calcitriol may lead to significant effects on nigrostriatal DA neuron functioning. The present experiments were designed to examine the ability of calcitriol to alter striatal DA release, and striatal and nigral tissue levels of DA. Male Fischer-344 rats were administered vehicle or calcitriol (0.3, 1.0, or 3.0 μg/kg, s.c.) once daily for eight consecutive days. Three weeks later in vivo microdialysis experiments were conducted to measure basal and stimulus evoked overflow of DA from the striatum. Basal levels of extracellular DA were not significantly affected by the calcitriol treatments. However, the 1.0 and 3.0 μg/kg doses of calcitriol led to increases in both potassium and amphetamine evoked overflow of striatal DA. Although post-mortem tissue levels of striatal DA were not altered by the calcitriol injections, nigral tissue levels of DA and its main metabolites were increased by both the 1.0 and 3.0 μg/kg doses of calcitriol. In a separate group of animals GDNF levels were augmented in the striatum and substantia nigra after eight consecutive daily injections of calcitriol. These results suggest that systemically administered calcitriol can upregulate dopaminergic release processes in the striatum and DA levels in the substantia nigra. Increases in the levels of endogenous GDNF following calcitriol treatment may in part be responsible for these changes. The ability of calcitriol to lead to augmented DA release in the striatum suggests that calcitriol may be beneficial in disease processes involving dopaminergic dysfunction.     

Cyclic Guanosine Monophosphate in Primary Cultures of Glial Cells

Cyclic GMP was found in primary cultures of glial cells obtained by dissociation of newborn mouse brain hemispheres. Its basal level (0.52 pmoles/mg cell protein) was as high as that found in adult mouse brain cortex but 10 times lower than in cerebellum. When glia were grown in the presence of dBcAMP, astrocytes changed their morphology; cGMP level increased and reached about 8 to 10 times the basal value. This increase was dose dependant with cAMP and was enhanced by the presence of 5mM Theophylline. Two hypothesis are discussed, either a direct action oc cAMP on glial cGMP metabolism or an indirect one on the protein activator of cGMP phosphodiesterase.     

An In Vivo Study of Dopamine Release and Metabolism in Rat Brain Regions Using Intracerebral Dialysis

Intracerebral dialysis was used with a specifically designed HPLC with electrochemical detection assay to monitor extracellular levels of endogenous 3,4-dihydroxyphenylethylamine (dopamine, DA) and its major metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), in brain regions of the halothane-anesthetized rat. Significant amounts of DA, DOPAC, and HVA were detected in control perfusates collected from striatum and n. accumbens whereas the medial prefrontal cortex showed lower monoamine levels. The ratio of DA in perfusate to DA in whole tissue suggests that in f. cortex, compared to n. accumbens and striatum, there is a greater amount of DA in the extracellular space relative to the intraneuronal DA content. The DOPAC/HVA ratio in control perfusates varied between regions in accordance with whole tissue measurements. This ratio was highest in n. accumbens and lowest in f. cortex. The monoamine oxidase inhibitor pargyline (100 mg/kg i.p.) caused an exponential decline in DOPAC, but not of HVA, in regional perfusates, an effect that was associated with an increase in DA. The data indicated a higher turnover of extracellular DOPAC in n. accumbens than in striatum and the lowest DOPAC turnover in f. cortex. The rate of decline in extracellular DA metabolite levels was slow compared to whole tissue measurements. In the perfusates there was no statistical correlation between basal amounts of DA in the perfusates and DOPAC and HVA levels or DOPAC turnover for any of the areas, indicating that measurement of DA metabolism in the brain under basal conditions does not provide a good index of DA release. In summary, this study shows clear regional differences in basal DA release and metabolite levels, metabolite patterns, and DOPAC turnover rates in rat brain in vivo.     

Effects of ethanol on gastric mucosal adenosine 3', 5' monophosphate (cAMP)

The effects of ethanol on the gastric mucosal adenosine 3′, 5′-monophosphate (cAMP) system were evaluated. The activity of adenylate cyclase (AC), phosphodiesterase (PDE), and tissue content of cAMP were determined in the presence of ethanol. NaF stimulated AC in rat gastric mucosa was inhibited in vitro and in vivo by 20% ethanol. Basal AC activity was so low (0.05 ± 0.10 pmoles cAMP formed/min/mg protein) that consistent results without NaF could not be obtained. The PDE activity (172 ± 11 pmoles cAMP consumed/min/mg protein) was approximately 350 fold greater than the basal AC activity. All levels of ethanol tested (2.0–20.0%) significantly inhibited (p<0.05) PDE in vitro. Gastric mucosal levels of cAMP are not measurably altered by ethanol in vivo (5–20%).     

CYCLIC NUCLEOTIDE METABOLISM IN ELECTROPLAX OF ELECTROPHORUS ELECTRICUS

Abstract— In order to describe the regulation of cyclic nucleotide metabolism in a cholinergic tissue, the properties of cyclic nucleotide phosphodiesterase were determined in electroplax of Electrophorus electricus and compared to those of mammalian brain. Electroplax phosphodiesterase was Mg²⁺ -dependent. localized in the soluble fraction and displayed normal linear Lineweaver-Burk kinetics ( K _m: cyclic AMP. 1.4 μ m ; cyclic GMP, 0.54 μ m ). No low affinity (i.e. high K _m) activity was detected. These results were correlated with comparatively low tissue levels of cyclic AMP (67 pmol/g) and cyclic GMP (3.2 pmol/g). Attempts were made to detect calcium-dependent phosphodiesterase because of the presence of large amounts of the calcium-dependent regulator protein (CDR) in electroplax, as this protein has been shown to activate brain phosphodiesterase. Assay with EGTA under a variety of conditions revealed that no calcium-dependent activity could be detected. Preparation of CDR-deficient phosphodiesterase also failed to produce calcium-dependent activity. Assay of phosphodiesterase in other cholinergic tissues revealed calcium-dependent activity in Electrophorus muscle and rat diaphragm but not in Torpedo electroplax. The results suggest that calcium-dependent activity is not a significant portion of phosphodiesterase in electroplax and indicate alternate roles for CDR in electric tissue.     

Culture-expanded human dermal stem cells exhibit donor to donor differences in cAMP generation

Stem cell techniques have facilitated a number of potential uses for such cells in cell therapy and drug development. Studies of the cAMP/protein kinase A (PKA) pathway are widely employed to investigate the effects of a large variety of substances. We assayed the cAMP pathway in human skin-derived mesenchymal stem cells (S-MSC) to evaluate donor to donor variations in response to pharmacological manipulations in vitro. Immunophenotyping of S-MSC revealed that, in general, 95% of S-MSCs were positive for CD90, CD73 and CD105 and negative for the expression of haemopoetic markers CD14, CD45 and human leukocyte antigen-DR (HLA-DR). Nevertheless, fluctuations occurred in basal cAMP levels from 5 pmol/mg to 18 pmol/mg. Total cAMP response element binding protein (CREB) concentrations ranged from 0.8 ng/ml to 1 ng/ml, whereas the proportions of phospho-CREB versus total CREB differed between the cell lines. Basic fibroblast growth factor (FGF-2) and epidermal growth factor (EGF) stimulated cAMP generation, whereas leukaemia inhibiting factor reduced some of their effects. Forskolin (0.05 and 1 mM) acted in synergy with FGF-2 and EGF; however, it caused pronounced donor to donor differences in the increase of cAMP and phospho-CREB levels. Additionally, dibutyryl-cAMP caused significant donor to donor variations in cell proliferation, possibly indicating a change of cell differentiation status. We speculate that similar donor diversity might be observed after cell stimulation with various G_s-protein-coupled receptor ligands. Heterogeneity of donor cell responses to stimulation of the cAMP pathway indicates the need for wide safety margins for S-MSC use in drug screening; nevertheless, knowledge of this heterogeneity might be useful for the design of donor-specific cell therapy.     

Regulation of Nervous System-Specific S-100 Protein and Enolase Levels in Adipose Tissue by Catecholamines

The effect of catecholamines on the levels of S-100 protein and nervous system-specific enolase (NSE) in epididymal adipose tissue of Wistar rats in vivo was examined by sensitive enzyme immunoassay methods. Soluble S-100 protein levels in the adipose tissue of 9-12-week-old rats (1.46 +/- 0.19 microgram/mg protein) were decreased to less than 50% of those of controls by serial injection (for 4-7 days) of epinephrine (0.1 mg/day) or norepinephrine (0.15 mg) with, however, little effect on the levels of membrane-bound (pentanol-extractable) S-100 protein. A significant decrease in the soluble S-100 protein levels was observed at 2 h after a single injection of epinephrine (1.04 +/- 0.13 microgram/mg protein). On the other hand, levels of NSE subunit (gamma subunit or 14-3-2 protein) in adipose tissue (0.51 +/- 0.03 gamma gamma-equivalent pmol/mg protein) were increased to 170% of control by serial injection (for 7 days) of epinephrine or norepinephrine with little change of the level of enolase alpha subunit on a mg protein basis. Isoproterenol had no apparent effect on the levels of soluble S-100 protein and NSE subunit. These results suggest that the levels of S-100 protein and NSE in adipose tissue are regulated by catecholamines.     

Brain changes in rats induced by prenatal injection of methylazoxymethanol

Various doses (0, 1, 5, 10, 15, 20, or 25 mg/kg) of methylazoxymethanol acetate (MAM), a potent alkylating agent, were injected singly into pregnant rats intraperitoneally on day 15 of gestation. Relationships between brain weights and neurochemical changes in the cerebral hemispheres (CHs; cerebral cortex and subjacent white matter, hippocampus, amygdala) and remainder of the brain (BGDM; basal ganglia, diencephalon, and mesencephalon) were examined at 60 days of age in offspring; varying degrees of microencephaly were observed. Dose-dependent reductions in the weights of CH and BGDM were observed. Reductions in total DNA content positively correlated with decreases in brain weights also observed. Dose-dependent elevations of noradrenaline (NA) and dopamine (DA) were observed in CH at MAM levels 10 mg/kg and above; dose-dependent elevations of 5-hydroxytryptamine (5-HT) were observed at 15 mg/kg and above; and in BGDM at 20 mg/kg and above dose-dependent elevations for NA and 5-HT were observed; dose-dependent elevations at 15 mg/kg and above were observed for DA. Monoamine concentrations were negatively correlated with brain weights or total DNA contents. NA and DA concentrations increased to the extent of approximately 1.3 times of control at a time when an 18% loss of CH weight was noted in animals treated with 10 mg/kg MAM. It is suggested that the above variables might be appropriately sensitive neurochemical markers for detecting minor developmental anomalies in the brain.     

Effect of brain serotonin on the arterial pressure and plasma renin in normal and hypertensive rats]

The effects of changes in brain serotonin content after injections of p-chlorophenylalanine (p-CPA), L-5-hydroxytryptophan (L-5HTP) and 5-6-dihydroxytryptamine (5-6DHT) on the mean arterial pressure (MAP), plasma renin activity (PRA) and peripheral levels of atrial natriuretic peptide (ANP) have been studied in normal and hypertensive (2K:1C model) male Wistar rats. The p-CPA (250 mg/kg) and L-5HTP (200 mg/kg) were injected i.p., while 5-6 DHT (15 micrograms/animal in 10 mu/animal vehicle) was injected into lateral brain ventricles. The effects were studied 24 h after the p-CPA injection, 2 h after L-5HTP and 10 or 20 days after 5-6DHT administration. The fall in brain serotonin produced by p-CPA and 5-6DHT did not modify the MAP values in the normal and hypertensive rat model, whereas the increase induced after L-5HTP injection only caused a slight decrease in arterial pressure in normotensive animals. The ARP experimented remarkable rises in the normal and hypertensive rats, these values increasing after L-5HTP and falling after p-CPA and 5-6 DHT injections. Similar changes are detected in the normal group after administration of these substances related to serotoninergic brain activity. The ANP levels rose after renal artery constriction, and they are not affected by the above mentioned substances. Only p-CPA and 5-6DHT reduced a low decrease in the ANP levels 10 days after their administration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Impaired parathyroid hormone action on urinary phosphorus excretion in isolated perfused kidney of streptozotocin-induced diabetic rats.

To study the effects of diabetes on the renal actions of parathyroid hormone (PTH), we observed urinary excretion of cyclic adenosine monophosphate (cAMP) and phosphorus in isolated perfused rat kidney. Diabetic rats were kept for 7 days after an intraperitoneal injection of 70 mg/kg streptozotocin (STZ). STZ-induced diabetic rats were treated with a daily injection of 20 U/kg lente-type insulin for 7 days. Plasma albumin, calcium, phosphorus, and PTH levels were not different among normal control, diabetic and insulin-treated diabetic groups. In the control rat kidney, the addition of PTH increased urinary cAMP excretion from 8 +/- 3 to 190 +/- 49 pmol/5 min and urinary phosphorus excretion from 11.3 +/- 4.4 to 33.6 +/- 10.8 microg/5 min. In the STZ-diabetic rat kidney, basal urinary cAMP was impaired, and PTH altered neither urinary cAMP nor phosphorus excretion (from below 0.7 to below 0.7 pmol/5 min, and from 15.5 +/-4.5 to 13.6 +/- 8.1 microg/5 min, respectively). Insulin treatment completely recovered the PTH actions. These results show that insulinopenic diabetes induces PTH resistance in the kidney.     

Differential response to gonadotropins and prostaglandin E2 in ovarian tissue during prenatal and postnatal development in cattle.

In cattle, growing follicles are present in fetal ovaries during the last part of gestation. This study examines the extent of changes in basal and hormone-stimulated adenylyl cyclase (AC) activity in ovaries of the bovine fetus when the first follicles begin to grow. The first growing follicles appeared in fetal ovaries around Day 180 and consisted mainly of primary and secondary follicles; few antral follicles were present before Day 220 of gestation. Basal AC activity in ovarian membranes increased simultaneously with the beginning of follicle growth in the fetus (5.8 +/- 0.9 vs. 9.3 +/- 1.3 pmol cAMP/mg protein/min at 130-180 and 180-210 days of gestation, respectively p less than 0.05). During the same time period, there was a significant increase in both the absolute (16.1 +/- 1.2 to 39.9 +/- 1.4 pmol cAMP/mg protein/min) and the relative (2.8 +/- 0.1 to 4.3 +/- 0.3 times the basal level, p less than 0.05) effects of guanosine triphosphate (GTP). After birth, basal and GTP-stimulated AC activities (pmol cAMP/mg protein/min) increased markedly in ovarian membranes of 1-wk-old calves and then decreased with age; the lowest levels were measured in mature cyclic cows. However, the relative effect of GTP (times the basal level) did not show this age-related variation. Prostaglandin E2 (PGE2) stimulation of AC in ovarian membranes from fetuses was high even on Day 120 (2.1 +/- 0.3 times the control level).(ABSTRACT TRUNCATED AT 250 WORDS)     

Amphetamine augments action potential-dependent dopaminergic signaling in the striatum in vivo

Amphetamine (AMPH) is thought to disrupt normal patterns of action potential-dependent dopaminergic signaling by depleting dopamine (DA) vesicular stores and promoting non-exocytotic DA efflux. Voltammetry in brain slices concurrently demonstrates these key drug effects, along with competitive inhibition of neuronal DA uptake. Here, we perform comparable kinetic and voltammetric analyses in vivo to determine whether AMPH acts qualitatively and quantitatively similar in the intact brain. Fast-scan cyclic voltammetry measured extracellular DA in dorsal and ventral striata of urethane-anesthetized rats. Electrically evoked recordings were analyzed to determine K(m) and V(max) for DA uptake and vesicular DA release, while background voltammetric current indexed basal DA concentration. AMPH (0.5, 3, and 10 mg/kg i.p.) robustly increased evoked DA responses in both striatal subregions. The predominant contributor to these elevated levels was competitive uptake inhibition, as exocytotic release was unchanged in the ventral striatum and only modestly decreased in the dorsal striatum. Increases in basal DA levels were not detected. These results are consistent with AMPH augmenting action potential-dependent dopaminergic signaling in vivo across a wide, behaviorally relevant dose range. Future work should be directed at possible causes for the distinct in vitro and in vivo pharmacology of AMPH.     

Effects of Ethanol In Vitro and In Vivo on the Release of Endogenous Catecholamines from Specific Regions of Rat Brain

Blocks of tissue from the hypothalamus, olfactory bulb, or striatum of rats were incubated in vitro to study the basal and potassium-stimulated release of endogenous catecholamines. When ethanol (100-250 mM) was added to these preparations in vitro no changes in release were observed. When ethanol (3.0 g X kg-1) was injected intraperitoneally in vivo, however, and 3,4-dihydroxyphenylethylamine (DA, dopamine) release was measured in vitro at various times after drug administration, significant increases in the basal release and decreases in the potassium-stimulated release were observed in striatum and olfactory bulb. In striatum, these changes showed a more rapid onset and a longer duration than in olfactory bulb. In both brain regions, DA release did not differ from controls at 4-6 h after the ethanol injection, although blood ethanol concentrations remained elevated. This may imply the tissue's acquisition of acute functional tolerance to the drug. Similar increases and decreases in the basal and the potassium-induced release of DA from striatal tissues were also found at 1 h after injection of a lower dose of ethanol (1.0 g X kg-1). In terms of behavior, this lower dose of ethanol produced only mild intoxication and ataxia, in contrast to the loss of righting reflex following the higher dose.