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To determine whether opioid receptors in sensory neurons are regulated by chronic exposure to opioids, we assessed the binding of various opioid ligands to membranes derived from isolated rat dorsal root ganglia neurons grown in culture. Equilibrium binding of [3H]diprenorphine onto membranes from cells grown for 13-15 days revealed a saturable binding site with a Kd value of 0.3 +/- 0.2 nM and an approximate Bmax value of 1300 +/- 200 fmol/mg of protein. [3H]Diprenorphine binding increased 3-fold from 1-15 days in culture. The mu receptors represent approximately 70 +/- 11% of the [3H]diprenorphine binding sites, as indicated by saturation binding of [3H]DAMGO. The kappa and delta receptors represent approximately 10 +/- 3% and approximately 5 +/- 2% of the [3H]diprenorphine binding sites, respectively. Preexposure of neurons to 10 microM naloxone for 48 hr up-regulated the receptors by 40%, whereas incubation with 100 nM to 10 microM DAMGO for 48 hr resulted in a significant decrease in the Bmax value of opioid receptors, with a maximum reduction of 70%. The identification of a high level of opioid receptors expressed in isolated sensory neurons and their modulation by opioids demonstrates that cultured sensory neurons are an excellent model with which to study opioid receptor regulation.

Endothelin (ET), a recently discovered vasoconstrictor peptide, is widely distributed in different tissues including brain and pituitary. Although evidence regarding the role of ET in neuroendocrine processes is still fragmentary, it appears that the release of several anterior pituitary (AP) hormones can be modulated by peptides of the ET family. In the present study, we compared the effects of ET-1 and ET-3 on the release of PRL from AP cells cultured in serum-free (SFM) or serum-containing (SCM) medium. AP obtained from adult male rats were enzymatically dispersed, and the cells were plated in either SFM or SCM. After 4 days, cells were incubated with ET-1 or ET-3 for designated periods of time, and PRL levels in the incubation media were measured by RIA. When AP cells maintained in SCM were exposed to ET for 20 min, both peptides inhibited PRL release, with ET-1 being a more potent inhibitor than ET-3. In contrast, a biphasic response was observed in cultures grown in SFM: low concentrations of ET peptides inhibited the secretion of PRL, while high concentrations caused a significant stimulation. Further characterization of the effects of ET-1 revealed that the stimulatory phase was relatively short (15-30 min) and was followed by inhibition of PRL release. The addition of either horse serum or fetal bovine serum to SFM on the day of plating resulted in a dose-dependent reduction in the ET-induced stimulation of lactotrophs. These findings indicate that the presence of serum in culture medium alters the expression of cell properties underlying inhibitory and stimulatory responses to ET in terms of PRL secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

The synthesis and release of PRL are regulated by a variety of factors that originate in the hypothalamus, peripheral tissues, or posterior pituitary (PP). We recently reported that coculture of anterior pituitary (AP) and PP cells induced an increase in both PRL cell content and the responsiveness of lactotrophs to TRH. The aim of the present study was to determine whether the augmented response to TRH is due to increased lactotroph sensitivity to this particular secretagogue or to enhancement of the releasable pool of PRL. Cells obtained from anterior pituitaries of adult male rats were plated either alone or together with PP cells at the same total density. Cells cultures were maintained in serum-free medium for 4 days and then incubated for 20 min with the designated substances. Angiotensin-II and TRH evoked a significantly larger release of PRL in AP + PP cocultures than in AP cells cultured alone; the greatest difference between the culture types was observed at the highest concentrations of both secretagogues. The stimulation of PRL release by KCl, the calcium ionophore A23187, and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate was higher in the presence of PP cells than in cultures of AP cells alone, although the magnitude of this effect was lower than that seen with PRL secretagogues. The concomitant application of A23187 and 12-O-tetradecanoylphorbol-13-acetate resulted in an increased response in both types of culture and a greater relative effect of PP cells on the evoked PRL release. In contrast to other secretagogues, oxytocin (OT) elicited a smaller response in AP + PP cocultures than in AP cultures. OT was present in significant amounts in medium from cocultures, apparently after being released from the severed neuronal terminals. When AP cultures were pretreated for 4 days with comparable concentrations of OT, the acute OT-evoked PRL release was greatly diminished. These findings suggest that coculture with PP cells increases the releasable pool of PRL in lactotrophs. The stored PRL is accessible for release by secretagogues known to act via the Ca2+ second messenger system, involving both Ca2+/calmodulin and protein kinase-C pathways. The diminished response of cocultures to OT is probably due to desensitization of lactotrophs by the residual amounts of this peptide present in the disrupted nerve endings.

There is extensive evidence that the posterior pituitary (PP) participates in the regulation of PRL secretion. We recently reported that a putative PRL-releasing factor is localized, and possibly produced, in the intermediate lobe of the PP. The aim of the present investigation was to determine whether cultured PP cells affect anterior pituitary (AP) function in terms of cell content and cumulative release of PRL. Anterior and posterior pituitaries from adult male rats were dispersed with trypsin and cultured either alone or together for 4 and 8 days in serum-free medium. The concentrations of PRL, GH, and LH in cell extracts and culture media were measured by RIA. Coculturing of AP and PP cells at different plating densities resulted in a 2-fold rise in PRL cell content after 4 days. The cumulative release of PRL in the cocultures was significantly increased only after 8 days. LH and GH were affected slightly, or not at all. Medium conditioned by PP cells mimicked the effects of coculture on the cumulative release of PRL, but not on the cell content. Short-term incubation with TRH induced a much larger release of PRL from AP + PP cocultures than from AP cells cultured alone. In conclusion, these data suggest that 1) PP cells stimulate the production and release of PRL in a hormone-specific manner, and 2) coculturing of AP + PP cells augments the responsiveness of lactotrophs to secretagogues such as TRH. We propose that at least two factors, one of which might be PRL-releasing factor, are involved in these effects.

The influence of chemical depolarization on the survival and differentiation of acetylcholinesterase (AChE)-containing neurons was examined in primary rat striatal cultures, maintained in different types of media (serum-free and serum-supplemented) and substrate (poly-ornithine and astrocyte monolayer). Chronic application of 5 microM veratridine resulted in a significant loss of neurites by AChE-positive cells, while a higher concentration (20 microM) reduced the number of stained cell bodies. These effects appeared to be selective with regard to AChE-positive cells, as indicated by morphological observations of the cells in the treated cultures and receptor binding measurements. Similarly, elevation of extracellular KCl levels (20-60 mM) produced a dose-dependent neurite loss by AChE-containing cells. Blockers of voltage-sensitive Ca2+ channels--verapamil (1 microM) and nifedipine (1 microM)--did not affect the veratridine-induced neurite loss, while tetrodotoxin (0.1 microM) had a partial effect. When cultures treated with 5 microM veratridine were allowed to recuperate for several days, the number of AChE-positive cells possessing neurites returned close to control values, thus indicating the reversibility of the effect of chemical depolarization. The possibility that chronic neuronal depolarization in the striatum might play a role in regulation of the neuronal processes outgrowth by AChE-containing cells is discussed.

Rats from two genetically high self stimulating lines (LC1-Hi, LC2-Hi) and from two genetically low self stimulating lines (LC1-Lo, LC2-Lo) were subjected to septal lesions at various ages. The genetically high self-stimulators exhibited the typical marked increase in emotionality following septal lesions, while the genetically low self stimulators showed an attentuated emotional response following septal lesions. In a subsequent experiment, animals from the same lines were subjected to VMH lesions. Once again, animals from the genetic high self stimulating lines showed a marked increase in emotionality following the lesions while the animals from the genetic low self-stimulating lines showed an attenuated emotional response. Hyperphagia was observed in all lines with no relation to the self stimulation genetic background. It is suggested that the septal and VMH systems modulating affective behavior are functionally related to the system modulating self stimulation, while these mechanisms seem to be basically independent of the VMH systems involved in maintenance of body weight.

It has been demonstrated that the effects of exogenous opiates like morphine could be modified by exposure of an organism to stress, but it is uncertain whether this modification is due to the action of endogenous opioid peptides released by stressful stimuli. The stress of restraint produced an antinociceptive response in mice measured by a latency to escape from a hot plate and, in addition, markedly potentiated analgesia induced by low doses of morphine. Both effects were antagonized by naloxone in a dose-dependent manner. On the other hand, restraint reduced the naloxone-precipitated jumping after single morphine injection. Morphine analgesia and a jumping response were not correlated when tested in two different strains of mice. It is suggested that the enhancement of morphine analgesia by restraint and the reduction in naloxone-induced jumping are mediated via independent mechanisms.