Pier Franco Spano

The functional status of dopaminergic nerve terminals has been studied with a method that allows the simultaneous determination of the specific activities of dopamine (DM), tyrosine (Tyr), 3-methoxytyramine (3-MT) and 3,4-dihydroxyphenylacetic acid (DOPAC), after administration of [3H]tyrosine ([3H]Tyr).Combined fluorimetric, mass fragmentographic and radiometric techniques have been used. [3H]Tyrosine was given intraventricularly to unanaesthetized rats and the animals were killed by exposure for 4 s to high energy microwave radiations.The specific activities of 3-MT and DOPAC measured 5 and 20 min after administration of [3H]Tyr, i. e. at time intervals in which the specific activity of DM is rising, are much higher than those their physiological precursor, suggesting that they are generated by more than one DM compartment.This hypothesis seems to be supported by the finding that in animals killed by decapitation instead of microwave radiations the post mortem accumulation of 3-MT occurs to a much smaller extent for the radioactive fraction than for the endogenous one, indicating that 3-MT formed after death may be mainly derived from DM coming from a compartment where this monoamine has been poorly labeled by the radioactive precursor.

Ibopamine has hemodynamic and neurohumoral effects potentially useful for the treatment of congestive heart failure (CHF), but its mechanism of action is not completely clear. To evaluate the role of dopaminergic receptor stimulation in the hemodynamic and neurohumoral activity of ibopamine, we compared the effects of ibopamine, 100 mg orally (p.o.) with those of the dopamine 2, 4, and 6 micrograms/kg/min intravenously (i.v.) and of the DA2 agonist dihydroergotoxine 6 micrograms/kg i.v. in 13 patients with chronic CHF [left ventricular ejection fraction (LVEF) < or = 35%]. All patients underwent right heart Swan-Ganz catheterization with determination of hemodynamic parameters at baseline, after 30 min of infusion of each dose of dopamine (DA) and < or = 6 h after ibopamine and dihydroergotoxine administration. Blood samples for the assessment of plasma renin activity (PRA), aldosterone, norepinephrine (NE), and epinephrine (Epi) were also obtained. Ibopamine induced a peak 21% increase of cardiac index (CI) with a 23 and 25% increase in stroke volume (SV) and stroke work indexes (SWI), respectively, and an 18% reduction in systemic vascular resistance (SVR). Similar changes were observed after DA infused at the doses of 2 and 4 micrograms/kg/min, whereas with the dose of 6 micrograms/kg/min heart rate (HR) increased by 23% and SV index (SVI) did not change further. Dihydroergotoxine administration induced only a significant 9% decrease in mean arterial pressure (MAP), with a 13% reduction in SVR.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: The present study demonstrates that 3,4-dihydroxyphenylethylamine (DA, dopamine) prevents neurotensin (NT) stimulation of both prolactin (PRL) release and calcium influx by interacting with specific receptors that are functionally linked to calcium channels. As shown by the studies with dispersed cells from rat anterior pituitary, the pharmacology of the control of PRL release and calcium influx, both induced by NT, was found to be typical of a DAergic process. This was demonstrated (1) by the order of potency of agonists in inhibiting PRL release and calcium influx (DA > epinephrine > norepinephrine isoproterenol); (2) by the high affinity of antagonists such as haloperidol and fluphenazine for this process; and (3) by the high degree of stereoselectivity of sulphide. Specific D2 receptor agonists, such as bromocriptine and lisuride, and the specific D2 receptor antagonist (-)-sulpiride were found to be highly potent on the DA receptors negatively coupled with calcium channels and PRL release. DA was found to lack the capacity to change the influx of calcium induced by either the sodium channel activator veratridine or high extracellular potassium levels, thus indicating a specific action of this amine on calcium channels sensitive to NT. In a range of concentrations that are effective in inhibiting either the calcium influx or the PRL release, both induced by NT, DA did not alter the cyclic AMP generating system. DA (from 1.0 nM to 50 nM) did not affect adenylate cyclase activity in rat pituitary gland homogenates and did not modify intracellular cyclic AMP levels in pituitary cells. The putative correlation between DA suppressing activity of the NT effects and the adenylate cyclase system was verified in pertussis toxin-treated pituitary cells. At the time that DA receptors negatively coupled to inhibition of adenylate cyclase were not operative, DA was still fully effective in preventing NT stimulation of PRL release. The lack of effect of low concentrations of DA on both basal adenylate cyclase activity and cyclic AMP content in pituitary cells, together with their capacity to inhibit the NT effects in pertussis toxin-treated cells, suggest that DA may act independently of cyclic AMP in the control of PRL secretion. These observations lead to the hypothesis of the existence of two inhibitory mechanisms for DA in mammotrophs.

Acute ethanol treatment produces a significant decrease of specific 3H-GABA binding in cerebellum while no changes were detectable in other brain areas. Scatchard analysis shows a decrease in receptor affinity but not in the number of GABA binding sites. On the other hand, chronic administration of ethanol selectively increases specific 3H-GABA binding in the striatum. Kinetic analysis of these data shows that ethanol chronic administration produces a significant increase in the number of GABA binding sites. These data may be useful for the understanding of clinical pictures following acute and chronic ethanol intoxication.

There is now evidence for the presence of a dopaminergic inhibitory modulation of aldosterone production that is mediated by D-2 receptors in the adrenal cortex. In this study we evaluated the effects of dopamine and the dopaminergic agonists ibopamine and dihydroergotoxine on aldosterone secretion and plasma renin activity in 13 patients with chronic heart failure. Two groups of patients were noted: one responding to dopaminergic drugs with a decrease in plasma aldosterone and the other without dopamine agonist-related aldosterone suppression. No effect on plasma renin activity was found after each drug administration. A correlation was found between the response to dopamine agonists and basal plasma aldosterone levels. These data are of therapeutic value in showing the detrimental effect of the activation of the renin-angiotensin-aldosterone system occurring in heart failure: a drug reducing this activation appears promising for both its acute and long-term effects.

Abstract: Brain microvessels were prepared from rat cerebral cortex. The purity was confirmed by phase-contrast microscopy and by the measurement of an enzymatic marker, γ-glutamyltranspeptidase. The microvessel preparation was subjected to radioreceptor assay using a 125I-labelled β-adrenergic antagonist, hydroxybenzylpindolol (IHYP). The binding was linear with protein concentration up to at least 80 μg per tube. It was saturated at 200 PM IHYP concentration. The KD, value calculated by Scatchard analysis was 69.4 ± 9.9 PM. The maximum binding (Bmax) was 107 ± 4 fmol/mg protein. The binding reached equilibrium within 30 min and was dissociated by addition of (−)-propranolol. The inhibitory effects of isomers of propranolol and iso-proterenol on this binding showed that (−)-isomers were two orders of magnitude more potent than the (+)-isomers. Other neurotransmitters did not affect IHYP binding. The characteristics of the binding, saturability, high affinity, reversibility and stereospecificity, suggest that IHYP is bound to β-adrenergic receptor sites located on brain microvessels.

Several studies suggested that dopamine may be one of the inhibitory modulators of aldosterone secretion. Metoclopramide, a selective antagonist for dopamine D-2 receptors, increases both basal plasma aldosterone levels and the aldosterone response to angiotensin II (Ang II) in rats and humans kept on a high sodium intake, these effects being blocked by dopamine infusion. Dopamine, which has no significant effects on Ang II-induced aldosterone secretion in sodium-replete subjects, inhibits the hormonal response to Ang II infusion in sodium-depleted normal subjects, suggesting that the sodium balance state may be an important factor in the dopaminergic mechanisms controlling aldosterone secretion. The effect of dopamine on the hormone production is mediated by D-2 receptors in the adrenal cortex as shown by in vitro studies with isolated adrenal glomerulosa cells. Clinical studies have shown that dihydroergotoxine, a selective D-2 agonist, suppresses the aldosterone secretion induced by sodium depletion in hypertensive patients, an effect blocked by sulpiride. This mechanism could be of relevant therapeutic interest in its contribution to the natriuretic effects of dopaminergic agonists, which have clinical applications in the treatment of hypertension and congestive heart failure.

Abstract: Active uptake of 3,4-dihydroxyphenylethylamine (dopamine) is sodium- and temperature-dependent, strongly inhibited by benztropine and nomifensine, and present in corpus striatum and nucleus accumbens. In rat striatum dopamine uptake is related to a receptor that is specifically labelled by [3H]cocaine in the presence of Na+ and is located on dopaminergic terminals. The dopamine uptake is differentially affected in the two areas by single or repeated injections of cocaine. Cocaine inhibits dopamine uptake in slices of corpus striatum. Moreover Na+-dependent [3H]cocaine binding is not detectable in nucleus accumbens. Nomifensine inhibits [3H]dopamine uptake by interacting with low- and high-affinity sites in corpus striatum, but shows only low affinity for dopamine uptake in nucleus accumbens. The present data indicate that different mechanisms are involved in the regulation of dopamine uptake in corpus striatum and nucleus accumbens.

The effects of ethanol have been studied in three strains of mice (DBA 2J, albino, and C57 BL/6J) having different populations of opiate receptors. Acute ethanol treatment induces a significant increase in striatal dopamine metabolism only in the mouse strains (C57 and albino) that are rich in enkephalinergic receptors upon nigrostriatal dopaminergic fibers. After chronic ethanol, the same strains develop tolerance to striatal dihydroxyphenylacetic acid increase, while the striatal dopaminergic recognition sites become supersensitive. DBA mice, which have lower numbers of enkephalinergic receptors and higher levels of enkephalins in the striatum, fail to show changes in central dopaminergic function after acute or chronic ethanol treatment. Our results indicate the importance of an interaction between ethanol and opiate receptors in determining the neurochemical and behavioral effects of ethanol.

Reverse Transcriptase-Polymerase Chain Reaction technique was used to determine the levels of the mRNAs encoding different receptors belonging to the D2 family in various brain areas of 6-, 12-, 18-, and 24-month-old rats. We found a progressive, age-dependent reduction in the mRNA levels of D2 short and D2 long receptors in corpus striatum, substantia nigra, and frontal cortex. D2 short and D2 long receptor mRNA levels were unchanged in hippocampus and olfactory tubercle, while they increased in pituitary. D3 receptor mRNA levels were reduced in olfactory tubercle and unchanged in striatum. D4 receptor mRNA levels were unmodified in pituitary gland. These data suggest that the molecular mechanisms responsible for regulating the pattern of expression of the various dopamine receptors are differentially regulated by aging.

Several studies suggested that dopamine may be one of the inhibitory modulators of aldosterone secretion. Metoclopramide, a selective antagonist for dopamine D-2 receptors, increases both basal plasma aldosterone levels and the aldosterone response to angiotensin II (Ang II) in rats and humans kept on a high sodium intake, these effects being blocked by dopamine infusion. Dopamine, which has no significant effects on Ang II-induced aldosterone secretion in sodium-replete subjects, inhibits the hormonal response to Ang II infusion in sodium-depleted normal subjects, suggesting that the sodium balance state may be an important factor in the dopaminergic mechanisms controlling aldosterone secretion. The effect of dopamine on the hormone production is mediated by D-2 receptors in the adrenal cortex as shown by in vitro studies with isolated adrenal glomerulosa cells. Clinical studies have shown that dihydroergotoxine, a selective D-2 agonist, suppresses the aldosterone secretion induced by sodium depletion in hypertensive patients, an effect blocked by sulpiride. This mechanism could be of relevant therapeutic interest in its contribution to the natriuretic effects of dopaminergic agonists, which have clinical applications in the treatment of hypertension and congestive heart failure.

Short-term exposure of primary cultures of cerebellar granule cells from neonatal rat brain to high concentrations of glutamate results in neuronal degeneration. We found that glutamate, before causing neuronal degeneration, induced a significant increase of Tau protein immunostaining. Time-course experiments revealed the increase ot Tau immunoreactivity to be maximal 2 h after the glutamate pulse. To investigate the possible role of newly synthesized Tau protein in the neurotoxic process activated by glutamate, cerebellar granule cells were preincubated with a specific Tau antisense oligonucleotide. This treatment resulted in (i) an inhibition of the glutamate-induced increase of Tau immunoreactivity and (ii) a decrease in the sensitivity of the neurones to neurotoxic concentrations of glutamate. These data indicate that new synthesis of the cytoskeleton-associated Tau protein is a crucial step in the cascade of events promoted by glutamate and leading to neurodegeneration.

GH3 cells are a clonal strain from a rat pituitary tumor that synthesizes and secretes both PRL and GH. The peculiarity of these cells is that they do not express receptors for dopamine; thus the hormone release is insensitive to the inhibitory effect of dopamine and D2 receptor agonists. Exposure of GH3 cells to epidermal growth factor for 4 consecutive days markedly altered the cell morphology, from a spherical appearance to an elongated flattened shape, and increased the cell size. These morphological changes were accompanied by the functional expression of D2 dopamine receptors as shown by the presence of a specific, saturable, and stereoselective high affinity binding for [3H]spiroperidol in epidermal growth factor-treated cells and by the fact that the selective D2 agonist quinpirole recovered the property to inhibit PRL secretion in the cell cultures exposed to the neurotrophic factor. The effect of EGF on the functional expression of D2 receptors was dose dependent (EC50 = 8 pM) and reversible. These data suggest that EGF elicits major effects on the expression of specific genes leading to the differentiation of GH3 cells into lactotroph-like cells endowed with dopamine D2 receptors.

Abstract: Acute ethanol treatment in rats elicits a selective increase in dihydroxyphenylacetic acid (DOPAC) content in striatum. In contrast, striatal DOPAC concentration does not differ from normal values after chronic ethanol treatment. Chronic administration of ethanol however causes a selective increase of specific [3H]spiroperidol binding and met-enkephalin content in the striatum. Kinetic analysis of [3H]spiroperidol binding data shows that after chronic ethanol treatment there is a significant increase in the affinity constant rather than in the number of binding sites for the ligand. Our results support the hypothesis that dopaminergic mechanisms at both pre- and postsynaptic level may be involved in the mediation of some of the central effects observed after ethanol consumption.

Various ergot alkaloids and derivatives were investigated for their interaction with dopaminergic receptors at the level of the rat corpus striatum and nucleus accumbens. Dihydro(DH)-ergotoxine, DH-ergocornine, DH-ergocryptine, DH-ergocristine, 2-Br-alpha-ergocryptine, ergotamine and DH-ergotamine were shown to inhibit, at micromolar concentrations, the dopamine-stimulated adenylate cyclase activity of rat striatal and nucleus accumbens homogenates. Interestingly, the inhibitory effect of the ergot drugs was higher in the nucleus accumbens than in the striatum. Moreover, the ergot drugs were more active in displacing 3H-haloperidol than 3H-dopamine from striatal membranes. The results, which are in apparent contradiction with previously obtained behavioral, pharmacological and clinical data, are discussed in the light of the possible presence in the central nervous system of distinct dopaminergic receptors with different conformations.

There is now evidence for the presence of a dopaminergic inhibitory modulation of aldosterone production that is mediated by D-2 receptors in the adrenal cortex. In this study we evaluated the effects of dopamine and the dopaminergic agonists ibopamine and dihydroergotoxine on aldosterone secretion and plasma renin activity in 13 patients with chronic heart failure. Two groups of patients were noted: one responding to dopaminergic drugs with a decrease in plasma aldosterone and the other without dopamine agonist-related aldosterone suppression. No effect on plasma renin activity was found after each drug administration. A correlation was found between the response to dopamine agonists and basal plasma aldosterone levels. These data are of therapeutic value in showing the detrimental effect of the activation of the renin-angiotensin-aldosterone system occurring in heart failure: a drug reducing this activation appears promising for both its acute and long-term effects.