Inhibition of catecholamine release by tolbutamide and other sulfonylureas.

Tolbutamide and other sulfonylureas inhibited spontaneous and nicotine-induced release of catecholamines from the perfused cat adrenal gland and nicotine-induced release of [3-H]norepinephrine from isolated guinea pig hearts. Of the sulfonylureas tested, the order to potency of this inhibitory effect paralled the hypoglycemic action. These results raise the possibility that the inhibition of the sympathoadrenal system may contribute in part to the hypoglycemic action of sulfonylureas.

Catecholamine uptake, accumulation, and release in acute porphyria.

Hypertension and tachycardia are well known features of acute porphyria and have been shown to be related to increased circulating catecholamines. The mechanism by which circulating catecholamines are increased was studied using the isolated perfused rat heart and human platelets as a model of adrenergic neuronal function. It was found that neither delta-aminolevulinate (ALA) nor porphobilinogen (PBG) blocked uptake or caused release in the isolated perfused rat heart. Platelets from six patients with acute prophyria, three in remission and three latent, with matching normal controls were studied with regard to their uptake of [(3)H]norepinephrine in the presence of ALA or PBG. It was found that ALA and PBG significantly reduced uptake and accumulation of [(3)H]-norepinephrine in patients with acute porphyria; however, no similar reduction in uptake and accumulation was observed in the platelets of normal controls. Therefore, it appears that there is a latent defect in the catecholamine uptake and (or) accumulation of platelets of patients with acute prophyria which only manifests itself in the presence of ALA or PBG. If platelet uptake serves as a model of adrenergic neuron uptake, this suggests that elevated circulating catecholamine levels during acute attacks of acute porphyria are caused at least partially by blockade of re-uptake into the sympathetic neurons.

A novel mechanism prevents the development of hypertension during chronic cold stress.

1 Chronic cold exposure of rats (7 days in a cold room at 4 degrees C) attenuated the sympathetic nerve stimulation (NS)-induced overflow of noradrenaline (NE) (measured by high-performance liquid chromatography, coupled to electrochemical detection) appearing in the perfusate/superfusate of the perfused mesenteric arterial bed as well as the increase in the perfusion pressure. 2 The same type of cold exposure resulted in an increase in tyrosine hydroxylase (TH) gene expression measured in the superior cervical ganglion and NE content measured in the mesenteric artery obtained from cold-exposed rats. 3 Addition of sodium nitroprusside, a nitric oxide (NO) donor, to the buffer perfusing the mesenteric arterial bed obtained from rats maintained at room temperature also resulted in an attenuation of the NS-induced overflow of NE and increase in perfusion pressure. 4 N(c)-nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor, placed in the drinking water prevented the attenuation of the pre- and post-junctional responses to NS of the mesenteric arterial bed obtained from cold-exposed rats. 5 L-NAME treatment also increased the cold-induced elevation of blood pressure seen in whole animals. 6 The present results are consistent with the idea that cold exposure leads to a concomitant increase in sympathetic nerve activity and production of NO. We hypothesize that the increase in production and release of NO results in a decrease in the biologically active form of NE despite increased synthesis and release of the catecholamine. 7 It is concluded that the above-mentioned interactions serve as a protective mechanism offsetting the increased release and action of NE from sympathetic nerves and thus preventing the development of hypertension.

Effects of differentiation on neuropeptide-Y receptors and responses in rat pheochromocytoma cells.

Undifferentiated rat pheochromocytoma PC12 cells resemble immature adrenal chromaffin cells, express neuropeptide-Y (NPY) receptors of the Y1 subtype, and synthesize catecholamines as well as NPY. In the present study, we examined how phenotypic alteration of PC12 cells by nerve growth factor (NGF) or glucocorticoid affected cellular responsiveness to NPY and related agonists, especially with regard to modulation of catecholamine overflow. Unlike undifferentiated PC12 cells, cells differentiated to a sympathetic neuronal phenotype with NGF were responsive to the Y2 receptor-selective agonist, NPY 13-36. NPY 13-36 1) inhibited binding of [125I]NPY 1-36, 2) inhibited accumulation of evoked cAMP, and 3) inhibited evoked catecholamine overflow. NGF-differentiated cells were also responsive to the Y1 receptor-selective agonist [Leu31,Pro34]NPY (LP-NPY). Like NPY-(13-36), LP-NPY inhibited binding of [125I]NPY-(1-36); however, LP-NPY and NPY-(13-36) exerted their effects through heterogeneous receptors, as LP-NPY enhanced while NPY 13-36 inhibited evoked catecholamine overflow in NGF-differentiated cells, despite the fact that both agonists inhibited the evoked cAMP. In contrast to NGF-differentiated cells, cells differentiated to a mature chromaffin phenotype with dexamethasone were unresponsive to NPY-(13-36), nor did the Y2 agonist inhibit binding of [125I]NPY-(1-36). Dexamethasone-differentiated PC12 cells were, however, responsive to LP-NPY, as this agonist enhanced evoked catecholamine overflow and inhibited binding of [125I]NPY-(1-36). Peptide-YY also enhanced catecholamine overflow, but only significantly at 100 nM. The data suggest differential expression of NPY receptor subtypes on neuronal and endocrine cells where catecholamine overflow is a key feature. These studies further demonstrate inhibitory or excitatory modulation of catecholamine transmission by NPY via distinct receptor subtypes in homogeneous sympathoadrenomedullary models resembling sympathetic neurons and chromaffin cells.

Maternal diabetes-induced hyperglycemia and acute intracerebral hyperinsulinism suppress fetal brain neuropeptide Y concentrations.

We examined the effect of streptozotocin-induced maternal diabetes of 6-day duration and 4- to 24-h intracerebroventricular and systemic hyperinsulinism on fetal brain neuropeptide Y (NPY) synthesis and concentrations. Maternal diabetes (n = 6) leading to fetal hyperglycemia (5-fold increase; P < 0.05) and normoinsulinemia caused a 40% decline (P < 0.05) in fetal brain NPY messenger RNA (mRNA) and a 50% decline (P < 0.05) in NPY radioimmunoassayable levels compared to levels in streptozotocin-treated nondiabetic (n = 7) and vehicle-treated control (n = 8) animals. In contrast, systemic hyperinsulinemia (n = 7) of 5- to 100-fold increase (P < 0.05) over the respective control (n = 7) with normoglycemia caused an insignificant (20-30%) decrease in fetal brain NPY mRNA and protein concentrations. However, fetal intracerebroventricular hyperinsulinism (n = 7) with no change in fetal glucose concentrations caused a 50-60% decline (P < 0.05) in only the NPY peptide levels, with no change in the corresponding mRNA amounts. We conclude that fetal hyperglycemia of 6-day duration and intracerebroventricular hyperinsulinism of 4-24 h suppress fetal brain NPY concentrations, the former by a pretranslational and the latter by either a translational/posttranslational mechanism or depletion of intracellular secretory stores. We speculate that fetal hyperglycemia and intracerebroventricular hyperinsulinism additively can inhibit various intrauterine and immediate postnatal NPY-mediated biological functions.

Noradrenergic transmission in the isolated portal vein of the spontaneously hypertensive rat.

The effect of electrical field stimulation (1, 2, 5, 10 Hz for a total of 480 pulses at 15-minute intervals) on the release of 3H-norepinephrine from the superfused portal vein of spontaneously hypertensive rats (SHR) or Wistar-Kyoto rats (WKY) of various ages was studied. The ages of the animals were (in weeks) 5-6 (prehypertensive), 8-10 (young hypertensives), 16-18 (older hypertensives), and 28 (mature hypertensives). There was no difference in the release of 3H-norepinephrine or developed tension of the portal vein to any frequency of field stimulation of SHR or WKY at 5-6 weeks of age. However, there was a significantly greater release of 3H-norepinephrine and developed tension of veins of SHR in response to low (1 or 2 Hz) but not high frequencies (5 or 10 Hz) at 8-10, 16-18, and 28 weeks of age. Vessels from hypertensive animals also developed greater resting tension and spontaneous activity, which was reduced to that of WKY in the presence of an alpha-adrenergic antagonist. The alpha 2 selective adrenergic antagonist yohimbine produced the same degree of enhancement of release of 3H-norepinephrine to field stimulation of veins obtained from both SHR and WKY at 5-6, 8-10 and 16-18 weeks of age. However, the facilitory effect of yohimbine was significantly attenuated in portal veins obtained from SHR at 28 weeks of age compared to age-matched WKY.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of aging on catecholamine levels, accumulation, and release in F-344 rats.

Neuronal function in selected brain areas has been evaluated in Fischer 344 rats aged 2-4 months, 11-14 months and 21-26 months. In vitro release of 3H-norepinephrine from hypothalamus and occipital cortex and 3H-dopamine from striatum has been evaluated using potassium, amphetamine, and field-stimulation. In vitro uptake of 3H-catecholamines has been evaluated in the same tissues. Catecholamine levels were measured in six brain areas: hypothalamus, striatum, cortex, cerebellum, brainstem and midbrain. Significant age-related decreases of NE levels, uptake, and release to high frequency stimulation were seen in the hypothalamus. The decreases in 3H-NE uptake and NE levels in the hypothalamus were apparent at 12 months, whereas the decrease in 3H-NE release after high frequency stimulation was seen in the senile rats.

Pharmacological characterization of the release of neuropeptide Y-like immunoreactivity from the rat hypothalamus.

Radioimmunoassay for NPY and detection by HPLC-EC were used to detect the concurrent release of norepinephrine and NPY from slices of the rat hypothalamus and to examine the modulation by adrenoceptors of the release of this neuropeptide. Basal and potassium-evoked (56 mM K+) release of both compounds were easily measured, with evoked release occurring in a calcium-dependent manner. The effect of the alpha 2-adrenoceptor agonist clonidine, the antagonists prazosin (alpha 1 selective) and yohimbine (alpha 2 selective) and the beta-adrenoceptor antagonist propranolol were all shown to modulate the evoked release of NPY. The alpha 2 agonist clonidine decreased evoked release of NPY, while the alpha 2 antagonist yohimbine increased the potassium-evoked release. Prazosin decreased both the basal and potassium-evoked release of NPY. Propranolol had the most profound effect on release of NPY, causing a significant decrease in basal release and a large decrease in the potassium-evoked release of NPY from slices of hypothalamus.

Effects of social isolation on brain catecholamines and forced swimming in rats: prevention by antidepressant treatment.

Post-weaning rats were housed alone or in groups for a period of 4 or 8 weeks. A portion of the animals received tricyclic antidepressant treatment, desipramine 20 mg/kg/day, during this period. Animals were then tested behaviorally by forced swimming. Isolation was associated with significantly longer durations of immobility during forced swimming. This was blocked by desipramine treatment. Desipramine treatment did not have a significant effect on the swimming durations of group-housed rats. Hindbrain and midbrain levels of catecholamines were subsequently measured and turnover rates estimated by administration of alpha-methyl-p-tyrosine or saline. Isolated rats had increased levels and decreased turnover of catecholamines. The increase in norepinephrine but not dopamine levels was blocked by desipramine, while antidepressant effects on turnover could not be tested with this method. Reduced social stimulation thus appears to be associated with reduced catecholamine release which may result in the accumulation of these transmitters in the central nervous system. Treatment with desipramine appeared essentially to compensate for reduced social stimulation, blocking isolation-induced noradrenergic neurochemical changes, while having few significant effects on control animals. This study may be helpful in furthering our understanding of how the interaction of organisms with their environment influence catecholamine systems and how antidepressants may act to restore function.

Neuropeptide Y receptors involved in calcium channel regulation in PC12 cells.

Our laboratory has previously used NGF-differentiated PC12 cells as a sympathetic neuronal model to investigate the effects of NPY on catecholamine synthesis and release. We have additionally used these cells to demonstrate the NPY-induced inhibition of Ca2+ channels which was suggested by those studies. In the present work, multiple NPY, PYY, and PP analogs are utilized to further define the receptor subtypes involved in this Ca2+ channel modulation. We find that in PC12 cells NPY and PP modulate Ca2+ channels through Y1, Y2, Y3, and Y4 receptors. In addition, we show that these receptors are differentially coupled to N, L, and non-N, non-L Ca2+ channel subtypes. The results of the present study in combination with our previous investigations demonstrate an intriguing and complex role for NPY and PP in the modulation of sympathetic neurotransmission.

Neuropeptide Y gene expression in immortalized rat hippocampal and pheochromocytoma-12 cell lines.

Employing clonal cell lines derived from rat embryonic hippocampal cells, we detected neuropeptide Y (NPY) mRNA in three progenitor subcloned cell lines. These cell lines upon differentiation express markers indicative of commitment to either neuronal (H19-7; NF +, GFAP -), glial (H19-5; GFAP +, NF -), or bipotential (H583-5; NF +, GFAP + ) lineages. Induction of differentiation was associated with the persistence of the NPY mRNA, however, in the differentiated H19-7 cells a 20-fold increase in NPY mRNA levels was observed (P<0.05). NPY immunoreactivity was observed only in cells with a differentiated neuronal phenotype. The cellular radioimmunoassayable NPY peptide levels increased twelve-fold without a change in extracellular NPY peptide levels by multi-factorially induced neuronal or glial cell differentiation. The differentiated H19-5 cells expressed lower levels of NPY that could not be immunocytochemically detected. The peripheral sympathetic PC-12 neuronal cells examined in the undifferentiated and nerve growth factor-driven differentiated states expressed NPY only upon differentiation. We conclude that NPY is expressed by the cultured undifferentiated and differentiated rat hippocampal clonal cell lines, while the peripheral sympathetic PC-12 neuronal cell line only expresses the NPY gene upon differentiation. These immortalized embryonic neural cell line(s) will provide a hippocampal cell line(s) to conduct future in-vitro investigations targeted at determining the cellular and molecular mechanisms governing NPY gene expression.

Modulation of terminal excitability of mesolimbic dopaminergic neurons by D-amphetamine and haloperidol.

Electrophysiological techniques were used to study the changes in the terminal excitability of mesolimbic DA and non-DA neurons following the infusion of D-amphetamine (D-AMP) and haloperidol (HAL) into the nucleus accumbens (NAc) of rats. The amount of current needed to evoke antidromic spikes by electrical stimulation of the NAc was used as an index of the excitability of axon terminals of these neurons. The excitability of DA neurons was decreased by D-AMP and increased by HAL. In addition, the effect produced by D-AMP was reversed by HAL. By contrast, these drugs either induced an opposite effect or were ineffective in inducing changes on the excitability of nerve terminals of mesolimbic non-DA neurons. Infusion of the vehicle or saline produced no effect. D-AMP and HAL were still effective in modulating the excitability of mesolimbic DA nerve terminals after the destruction of NAc neurons by ibotenic acid. The results suggest that the effects seen after D-AMP and HAL are mediated primarily by DA autoreceptors. It is likely that the increase in the current needed for evoking antidromic spikes after infusion of D-AMP into the terminal region is the consequence of DA autoreceptor-mediated hyperpolarization of terminal membranes. On the other hand, HAL could exert its actions by blocking autoreceptor-mediated hyperpolarization.

Effect of prolactin on dopamine synthesis in medial basal hypothalamus: evidence for a short loop feedback.

Several procedures were utilized to study the effects of prolactin on dopamine synthesis in the medial basal hypothalamus of the rat. Elevation of serum prolactin was produced by the administration of trifluoperazine (5 mg/kg, i.p.) and resulted in a significant increase in the conversion of [3',5'-3H]tyrosine to dopamine when measured in slices of medial basal hypothalamus and striatum. Hypophysectomy abolished this effect of trifluoperazine in the medial basal hypothalamus but not in the striatum. In addition, the synthesis of dopamine was significantly elevated in slices of medial basal hypothalamus obtained from rats bearing pituitary tumor implants that secreted microgram quantities of prolactin. In contrast, the in vitro synthesis of dopamine in the striatum of such rats was increased by the secretory products in one tumor line but decreased in another compared to that observed in control animals. It is suggested that the ability of prolactin to accelerate the synthesis of dopamine in the medial basal hypothalamus might constitute a short loop feedback system that finely regulates prolactin secretion.

Failure of subchronic lisuride to modify A10 dopamine autoreceptors' sensitivity.

The concept that prolonged treatment with dopamine (DA) mimetics results in a subsensitivity of DA autoreceptors generally is accepted. However, the present study indicates that the administration of a rather specific DA-D2 agonist, lisuride hydrogen maleate (LIS), for one week (200 micrograms/kg/daily) failed to modify the sensitivity of DA autoreceptors of A10 neurons. Indeed, by using extracellular single unit recording in chloral hydrate-anesthetized rats, we observed that neither intravenous apomorphine nor microiontophoresis of DA changed their firing rate-depressant potency when it was estimated at 1 or 3 days after the last LIS injection. A possible interpretation of the results is that the subchronic stimulation of DA-D2 receptors activates an unknown compensatory mechanism which avoids the changes in their sensitivity. Alternatively, the possibility that LIS may also possess antagonistic properties for DA receptors, which might balance the D2 receptors activation, is discussed.

Nicotinic effect of acetylcholine on the release of newly synthesized (3H)dopamine in rat striatal slices and cat caudate nucleus.

The effect of acetylcholine (ACh), carbachol and nicotinic blocking agents on the release of newly synthesized [3H]dopamine ([3H]DA) was studied in vitro on rat striatal slices and in vivo on the cat caudate nucleus. In the latter case, the animals were anaesthetized with halothane; in some experimetns an 'encéphale isolé' preparation was used to eliminate anaesthesia. Rat striatal slices placed in a superfusion chamber were continuously superfused with L-[3,5-3H]tyrosine. A cup placed on the ventricular surface of the cat caudate nucleus similarly allowed a continuous superfusion of the structure with the 3H amino acid. In both cases the quantities of [3H]DA contained in serial superfusate fractions were estimated; the drugs were always added in superfusing medium. In vitro ACh (10(-5) M) and carbachol (10(-5) M) enhanced the release of [3H]DA (90%). Similar results were obtained in vivo in anaesthetized cats. The effect of ACh (10(-5) M) was more pronounced (125%) in presence of eserine (10(-4) M) than with ACh alone (65%). ACh was also effective in unanaesthetized cats. The ACh effect on [3H]DA release was reproducible within the same experiment both in vitro and in vivo. This allowed to test the effect of anticholinergic agents on the ACh induced release of [3H]DA. In vivo hexamethonium (10(-4) M, 10(-5) M) partially blocked the release of [3H]DA induced by ACh (10(-5) M) alone; the effect was not seen when ACh was added in the presence of eserine (10(-4) M). Both in vivo and in vitro the prior introduction of mecamylamine into the superfusing medium antagonized the stimulating effect of ACh (10(-5) M) on [3H]DA release. The effects of this nicotinic blocking agent were seen with various concentrations (10(-6); (10(-5) 10(-4) in the in vitro experiments.

Catecholamine monoamine oxidase a metabolite in adrenergic neurons is cytotoxic in vivo.

3,4-Dihydroxyphenylglycolaldehyde is the monoamine oxidase-A metabolite of two catecholamine neurotransmitters, epinephrine and norepinephrine. This aldehyde metabolite and its synthesizing enzymes increase in cell bodies of catecholamine neurons in Alzheimer's disease. To test the hypothesis that 3,4-dihydroxyphenylglycolaldehyde, but not epinephrine or its major metabolite 4-hydroxy-3-methoxyphenylglycol, is a neurotoxin, we injected 3,4-dihydroxyphenylglycolaldehyde onto adrenergic neurons in the rostral ventrolateral medulla. Injections of epinephrine or 4-hydroxy-3-methoxyphenylglycol were made into the same area of controls. A dose response and time study were performed. Adrenergic neurons were identified by their content of the epinephrine synthesizing enzyme, phenylethanolamine N-methyltransferase, immunohistochemically. Apoptosis was evaluated by in situ terminal deoxynucleotidyl-transferase mediated dUTP nick end label staining. Injection of 3,4-dihydroxyphenylglycolaldehyde in amounts as low as 50 ng results in loss of adrenergic neurons and apoptosis after 18 h. The degree of neurotoxicity is dose and time dependent. Doses of 3,4-dihydroxyphenylglycolaldehyde 10-fold higher produce necrosis. Neither epinephrine nor 4-hydroxy-3-methoxyphenylglycol are toxic. A 2.5 microg injection of 3,4-dihydroxyphenylglycolaldehyde is toxic to cortical neurons but not glia. Active uptake of the catecholamine-derived aldehyde into differentiated PC-12 cells is demonstrated. Implications of these findings for catecholamine neuron death in neurodegenerative diseases are discussed.

Allosteric modulation by leucine-enkephalin of [3H]naloxone binding in rat brain.

The interaction of morphine and leucine-enkephalin with the binding site labeled by [3H]naloxone in the presence of sodium was compared. The effect of fixed concentrations of morphine and leucine enkephalin on the saturation binding of [3H]naloxone demonstrated that whereas morphine was a competitive inhibitor, leucine enkephalin caused a dose-dependent masking of binding sites. From these data we conclude that the enkephalin receptor is allosterically coupled to the morphine receptor.

Neuropeptide Y inhibits depolarization-stimulated catecholamine synthesis in rat pheochromocytoma cells.

In PC12 rat pheochromocytoma cells differentiated with nerve growth factor (NGF), neuropeptide Y inhibited depolarization-stimulated catecholamine synthesis as determined by in situ measurement of 3,4-dihydroxyphenylalanine (DOPA) production in the presence of the decarboxylase inhibitor m-hydroxybenzylhydrazine (NSD-1015). The inhibition by neuropeptide Y was concentration-dependent and was prevented by pretreatment with pertussis toxin, suggesting the involvement of a GTP-binding protein of the Gi or Go subtype. The neuropeptide Y analog [Leu31,Pro34]neuropeptide Y also caused inhibition of DOPA production, but was less potent than neuropeptide Y itself, while peptide YY and neuropeptide Y-(13-36) had no significant effect. This pattern is most consistent with the involvement of the neuropeptide Y Y3 receptor subtype. In PC12 cells differentiated with dexamethasone, neuropeptide Y also caused a concentration-dependent inhibition of DOPA production, while peptide YY was again without effect. Neuropeptide Y had no effect on DOPA production in undifferentiated PC12 cells. These results indicate that neuropeptide Y can modulate catecholamine synthesis in addition to its modulatory effects on catecholamine release.

Effects of adrenalectomy and adrenal steroids on norepinephrine synthesis and monamine oxidase activity.

Rat heart norepinephrine (NE) tunover was increased 6--10 days after bilateral adrenalectomy. This increase was prevented by administration of deoxycorticosterone acetate (DOCA) but not by either hydrocortisone or corticosterone. Blood pressure decreased following adrenalectomy. This decrease was prevented by DOCA, hydrocortisone and corticosterone. Monoamine oxidase (MAO) activity increased in the heart but not in the liver following adrenalectomy. DOCA prevented the increase in heart MAO activity whereas hydrocortisone and corticosterone were ineffective. In intact animals, heart and liver MAO activity were not changed by 5 days of cold exposure, a procedure which increases NE turnover. It is suggested that the increase in heart NE turnover may be related to the increase in MAO activity seen after adrenalectomy.

The effect of opioid drugs on the release of dopamine and 5-hydroxytryptamine from rat striatum following activation of nicotinic-cholinergic receptors.

The effect of (Met5)enkephalin, (D-Ala2,D-Met5)enkephalin, (Leu5)enkephalin, (D-Ala2,D-Met5)enkephalin and morphine on the release of [3H]dopamine, endogenous dopamine and [3H]5-hydroxytryptamine produced by the nicotinic-cholinergic agonist, dimethylphenyl piperazinium iodide (DMPP), was examined in rat striatal slices. The DMPP-induced release of [3H]dopamine and endogenous dopamine was reduced by the presence of (Met5)enkephalin, (D-Ala2,D-Met5)enkephalin (1-10 microM) or morphine (10 microM) but not by (Leu5)enkephalin or (D-Ala2,D-Leu5)enkephalin. The DMPP-induced release of [3H]5-hydroxytryptamine was reduced by (Leu5)enkephalin, (D-Ala2,D-Leu5)enkephalin, (Met5)enkephalin, (D-Ala2,D-Leu5)enkephalin (1-10 microM), and morphine (10 microM). All three opioids failed to alter the release of [3H]dopamine induced by field stimulation or potassium depolarization (30 microM). The inhibitory effects of opioid peptides and morphine demonstrated in the present study appear to be due to an initial interaction with nicotinic-cholinergic receptors in the striatum.