Laura Castelletti

Dopamine Uptake is Differentially Regulated in Rat Striatum and Nucleus Accumbens

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.

Potassium channels involved in the transduction mechanism of dopamine D2 receptors in rat lactotrophs.

1. Radioactive rubidium (86Rb+) efflux was used to measure potassium (K+) permeability in a study designed to asses both the presence and the sensitivity to ions and drugs of the K+ channels in the plasma membrane of rat lactotrophs. 2. Rb+ efflux from Rb+‐pre‐loaded lactotrophs into nominally calcium‐free solution containing 5 mM‐K+ was linear from 1 to 60 s, with a calculated rate of about 0.1%/s. Raising K+ concentrations to depolarize the cells stimulated the Rb+ efflux (0.2%/s), which was already significant after 1 s of exposure of the cell to 100 mM‐K+. This component of Rb+ efflux has been designated component V (sensitive to voltage and Ca2+ independent). 3. Addition of Ca2+ to 5 mM‐K+ solution had no effect on resting Rb+ efflux (0.1%/s), but did further stimulate Rb+ efflux into K+‐rich solutions. This component, which has been designated component C, was completely inhibited by 0.5 mM‐cadmium. These data fit the view that the increase in intracellular Ca2+ concentration during depolarization opens certain (Ca2+‐activated) K+ channels. 4. K+ efflux was differently affected by K+ channel blockers. Tetraethylammonium (TEA) inhibited both V and C components while 4‐aminopyridine (4‐AP) inhibited the component V without modifying the C component of Rb+ efflux. 5. Dopamine appears to affect both types of Rb+ efflux components. Dopamine increased the efflux of Rb+ in a nominally Ca2+‐free medium containing 5 mM‐K+ (component V). This effect was statistically significant 15 s after exposure of the cells to 10 nM‐dopamine. Increasing the concentrations of K+ to gradually depolarize the cells enhanced the rate of increase of Rb+ efflux induced by dopamine, being evident in the initial 2‐5 s of incubations. Dopamine also increased Rb+ efflux in a 5 mM‐K+ solution containing 1 mM‐Ca2+ (component C). This effect was rapid (2‐5 s) and inhibited by 0.5 mM‐cadmium. The combined action of dopamine on both component C and V caused the cells to be less sensitive to depolarizing concentrations of K+. The increase in Rb+ efflux and the enhancement of prolactin release induced by high concentrations of K+ were, indeed, prevented by exposure of the cells to 10 nM‐dopamine. 6. The effects of dopamine on either component V or component C were pharmacologically characterized as D2 receptor mediated, being mimicked by selective D2 receptor agonists (quinpirole and RU 24213) and stereospecifically blocked by the D2 receptor antagonist sulpiride.(ABSTRACT TRUNCATED AT 400 WORDS)

identification and Characterization of Postsynaptic D1- and D2-dopamine Receptors in the Cardiovascular System

Measuring adenylate cyclase activity (AC) as a biochemical index of dopamine (DA) receptor function, we obtained evidence for the presence in rabbit vasculature of both D1 receptors associated with stimulation of AC and of D2 receptors coupled with AC in an inhibitory way. The cAMP generating system in rabbit mesenteric artery was stimulated by DA and several DA agonists, an effect antagonized by the D1-receptor blocker SCH 23390 and by other neuroleptic drugs. When activation of D1 sites was impeded by SCH 23390, DA, (-)apomorphine, and (-)NPA inhibited cAMP formation. In addition, selective D2 agonists inhibited basal AC activity even when there was no D1-receptor blockade. The relative order of potency of various neuroleptics in antagonizing bromocriptine-induced inhibition of AC confirmed the D2 nature of these binding sites. Inhibition of AC activity elicited by bromocriptine remained unchanged after chemical sympathectomy, suggesting that vascular D2 receptors inhibiting AC activity are located postsynaptically in the arterial wall.

Identification of neurotensin receptors associated with calcium channels and prolactin release in rat pituitary.

Abstract: Neurotensin (NT) is now reasonably well established as a neurotransmitter or neuromodulator candidate in the CNS. In the present study, we characterized the NT receptors in dispersed cells from the anterior lobe of rat pituitary and investigated the involvement of both cyclic AMP and calcium in the release of prolactin (PRL) induced by NT receptor stimulation. The [3H]NT binding to membranes from anterior pituitary dispersed cells was found saturable and stereospecific. Scatchard analysis of the data gave a straight line indicating a Bmax value of 121 ± 11 fmol/mg protein and a KD value of 1.4 ± 0.2 nM. The calculated IC50 values for [3H]NT binding were 5.8 nM for NT, 7.8 nM for L‐Phe‐NT, and 3,000 nM for the pharmacologically inactive form D‐Phe‐NT. NT, up to a concentration of 1 μM, did not affect the cyclic AMP generating system in homogenates of anterior pituitary from male or lactating female rats. The same pattern of results was obtained for cyclic AMP formation in intact cells. NT and its analogs stereospecifically enhanced the influx of calcium into dispersed cells from rat anterior pituitary. The effect was time‐ and dose‐dependent. It appeared to be associated with neurotransmitter‐operated calcium channels since: (1) preincubation of the cells with tetrodotoxin did not affect the increase in calcium influx induced by NT; (2) concentrations of verapamil that counteract the influx of calcium induced by potassium lacked the capacity to modify the influx of calcium induced by NT; and (3) NT lost its capacity to release PRL in the absence of extracellular calcium. The significant correlation of the effective concentrations of NT and its analogs for binding and biological activities indicates that occupancy of NT receptors in anterior pituitary modifies calcium channel permeability of the cell membranes, resulting in an accumulation of intracellular calcium that participates in the cascade of events that are ultimately expressed as an enhanced release of PRL.

Striatal adenylate cyclase-inhibiting dopamine D2 receptors are not affected by the aging process.

Radioreceptor binding studies with various labelled ligands and positron emission tomography have revealed a decline in D2 receptor concentration with age in both animal and human caudate nucleus. In this study we found that during senescence the functional characteristics of D2 receptors that inhibit adenylate cyclase (AC) are unchanged, by measuring the extent of inhibition of AC activity by dopamine mimetic drugs as a direct indicator of D2 receptor function.

Epidermal Growth Factor Promotes Uncoupling from Adenylyl Cyclase of the Rat D2S Receptor Expressed in GH4C1 Cells

Abstract: In anterior pituitary cells or when transfected into host cell lines, the D2 dopamine receptor inhibits adenylyl cyclase and activates potassium channels. The GH‐3 pituitary tumor cell line, which lacks functional D2 receptors, responds to epidermal growth factor (EGF) by expressing a D2 receptor that, paradoxically, couples to potassium channel activation but poorly inhibits adenylyl cyclase; this was correlated with a pronounced increase in α subunit of the G protein G13. In this study we have investigated the effects of EGF on the transduction mechanisms of D2 receptors in GH4C1 cells transfected and permanently overexpressing the rat short D2 receptor. Activation of D2 receptors in these cells resulted in both inhibition of adenylyl cyclase and opening of potassium channels and inhibition of prolactin release by both cyclic AMP‐dependent and independent mechanisms. Exposure of the transfected GH4C1 cells to EGF caused a dramatic decrease in the coupling efficiency of the D2 receptor to inhibit cyclic AMP‐dependent responses, leaving its activity toward potassium channels unchanged. The EGF treatment led to the concomitant increase in the membrane content of G13 protein. These results suggest that the transmembrane signaling specificity of G protein‐coupled receptors can be modulated by the relative amounts of different G proteins at the cell membrane.

Age related changes of enkephalin in rat spinal cord.

Met-enkephalin immunoreactive material content was found to be decreased in the cervical and thoracic segments of the spinal cord from rats aged 25 months as compared to young, 3-month-old, rats. No age-related variations were detectable at the lumbar level. Bio-Gel P 30 column chromatography of thoracic segment extracts indicates that the composition of the immunoreactive material is similar in the two age-groups investigated. At the thoracic level opiate receptor binding was also measured. Opiate receptor number is increased in the thoracic segments of the spinal cord from older rats. These age-related changes in immunoreactive Met-enkephalin content and opiate receptor number at spinal levels may contribute to determine an altered pain sensitivity during aging.

Decreased content of met-enkephalin-like peptides in superior cervical and coeliac ganglia of aged rats.

Enkephalin like peptides seem to have an important regulatory role at ganglia level. The aim of the present study is to investigate whether the content of enkephalin-like peptides in sympathetic ganglia is affected by the aging process. The results show that the enkephalin like peptides content is low in superior cervical and coeliac ganglia of aged rats (25 months). The age-related decrease of enkephalin content in these structures may be of importance in determining an altered sympathetic control during aging.

Intracellular Ionic Changes Induced by Dopamine Receptor Stimulation in Mammotrophs: Evidence for a K+ Channel-linked Dopamine D-2 Receptor

In mammalian CNS and peripheral tissues two categories of dopamine (DA) receptors have been identified on the basis of biochemical and pharmacological criteria. These two receptors were designated as D-1 and D-2 (Spano et al, 1978; Kebabian and Calne, 1979; Memo et al, 1987).