Dopamine, dopamine D2 receptor short isoform, transforming growth factor (TGF)-beta1, and TGF-beta type II receptor interact to inhibit the growth of pituitary lactotropes.

The neurotransmitter dopamine is known to inhibit prolactin secretion and the proliferation of lactotropes in the pituitary gland. In this study, we determined whether dopamine and TGFbeta1 interact to regulate lactotropic cell proliferation. We found that dopamine and the dopamine agonist bromocriptine stimulated TGFbeta1 secretion and TGFbeta1 mRNA expression but inhibited lactotropic cell proliferation both in vivo and in vitro. The dopamine's inhibitory action on lactotropic cell proliferation was blocked by a TGFbeta1-neutralizing antibody. We also found that PR1 cells, which express low amounts of the dopamine D2 receptor, demonstrated reduced expression of TGFbeta1 type II receptor and TGFbeta1 mRNA levels and had undetectable levels of TGFbeta1 protein. These cells showed a reduced TGFbeta1 growth-inhibitory response. Constitutive expression of the D2 receptor short isoform, but not the D2 receptor long isoform, induced TGFbeta1 and TGFbeta1 type II receptor gene expression and recovered dopamine- and TGFbeta1-induced growth inhibition in PR1 cells. The constitutive expression of D2 receptor short isoform also reduced the tumor cell growth rate. These data suggest that a TGFbeta1 system may mediate, in part, the growth-inhibitory action of dopamine on lactotropes.

Effect of voltage-dependent calcium channel blockers on ethanol-induced beta-endorphin release from hypothalamic neurons in primary cultures.

The voltage-dependent calcium channel (VDCC) has been shown to mediate calcium entry into neurons that regulates neurotransmission in many neuronal cells. Four major types of VDCCs (three high-voltage-activated L-, N-, and P-types and one low-voltage-activated T-type) have been identified in neurons. Involvement of the VDCC in ethanol-stimulated beta-endorphin (beta-EP) release from hypothalamic neurons has not been studied. In the present study, the role of VDCC on basal and ethanol-induced beta-EP release was determined by using rat fetal hypothalamic cells in primary cultures. Treatments with a 50 mM dose of ethanol for 3 hr increased immunoreactive beta-EP (IR-beta-EP) release from hypothalamic cells maintained in cultures for 9 days. Ethanol-induced IR-beta-EP release was inhibited by a P/Q-type channel blocker omega-agatoxin TK (0.1-1 microM), an N-type channel blocker omega-conotoxin (0.1-1 microM), an L-type blocker nifedipine (1-10 microM), and a T-type blocker flunarizine (1-10 microM). The minimal effective doses of these blockers that blocked the ethanol response produced no significant effects on basal release of IR-beta-EP; neither did these doses of the blockers produce any significant effects on cell viability. These results suggest that ethanol-stimulated IR-beta-EP release is regulated by extracellular calcium involving P-, N-, L- and T-type channels.

Effects of ethanol on alpha-adrenergic and beta-adrenergic agonist-stimulated beta-endorphin release and cAMP production in hypothalamic cells in primary cultures.

We have previously shown that low concentrations of ethanol rapidly stimulate beta-endorphin (beta-EP) release from hypothalamic neurons in primary cultures and that chronic exposures to these concentrations of ethanol desensitize beta-EP neurons to ethanol challenges. We have also shown that chronic ethanol desensitizes dibutyryl cAMP-, adenosine-, and prostaglandin E1-stimulated beta-EP release and the cAMP content in hypothalamic neurons. In this study, we determined the effects of ethanol (50 mM) on beta-adrenergic agonist (isoproterenol) or alpha-adrenergic agonist (l-phenylephrine)-induced beta-EP release and cellular contents of cAMP to identify whether ethanol causes heterologous desensitization of the adenylate cyclase system in this neuronal cell population. Both isoproterenol and l-phenylephrine increased beta-EP levels in culture media and elevated the cAMP content in cell extracts in a concentration (0.1 and 10 microM)-dependent fashion between 3 to 6 hr. A 50 mM dose of ethanol increased beta-EP and cAMP levels at 3 hr, but it did not elevate beta-EP and cAMP levels after 48 hr of exposure. Acute exposure (3 hr) of these cells to ethanol moderately enhanced the isoproterenol-stimulated and l-phenylephrine-stimulated levels of media beta-EP and intracellular levels of cAMP. However, chronic exposure (48 hr) to ethanol reduced the magnitude of both alpha- and beta-adrenergic receptor agonist-stimulated beta-EP release and cAMP production. These results confirm our previous findings that the ethanol action on beta-EP secretion is mediated by the cAMP system and further suggest that chronic ethanol causes heterologous desensitization of the adenylate cyclase system in the beta-EP neuronal cell population.

Effects of ethanol on basal and prostaglandin E1-induced increases in beta-endorphin release and intracellular cAMP levels in hypothalamic cells.

Our recent studies determining the effect of cAMP-elevating agents forskolin and dibutyryl cAMP on ethanol-induced immunoreactive beta-endorphin (IR-beta-EP) release from hypothalamic cells in primary cultures suggested the possibility that both stimulatory and adaptive secretory responses of beta-EP neurons after ethanol exposure may involve the cAMP system. To determine further the role of cAMP, the effects of prostaglandin E1 (PGE1) on basal and ethanol-regulated IR-beta-EP secretion and cAMP productions were determined in primary cultures of hypothalamic cells. The results presented in this study show that a 50 mM dose of ethanol, which is within the EC50 dose of ethanol required to elevate IR-beta-EP release from hypothalamic cells, increased cellular levels of cAMP and elevated IR-beta-EP release simultaneously from the cultured neurons for a period of 6 hr. The cAMP and IR-beta-EP secretory responses developed desensitization to ethanol challenge after 24 hr of constant ethanol incubation. The cAMP-elevating agent PGE1 increased the cellular content of cAMP and IR-beta-EP release in a dose-dependent manner. The EC50 dose of PGE1 for elevation of IR-beta-EP and cAMP was approximately 0.5 microM. As with ethanol, chronic treatment with PGE1 desensitized the cAMP and IR-beta-EP responses of hypothalamic neurons to PGE1. Acute exposure to ethanol increased the PGE1-stimulated levels of cAMP and IR-beta-EP, whereas chronic exposure to ethanol resulted in diminished cAMP responses to PGE1. These data provide evidence that the cAMP system may be involved in controlling hypothalamic beta-EP secretion, as well in regulating the stimulatory and adaptive responses of beta-EP neurons to ethanol.

The role of cAMP in ethanol-regulated beta-endorphin release from hypothalamic neurons.

We have previously shown that ethanol acutely stimulates immunoreactive beta-endorphin (IR-beta-EP) release from hypothalamic neurons, whereas chronic administration of ethanol desensitizes these neurons. In the study reported herein, the role of the intracellular cAMP system in the ethanol-regulated IR-beta-EP release from hypothalamic cells in primary cultures was investigated. Acute treatment with ethanol or with the cAMP analog, dibutyryl cAMP, revealed that both agents stimulate the release of IR-beta-EP from the hypothalamic cells. Combined treatment of ethanol and the cAMP analog produced a synergistic effect on IR-beta-EP release. Treatment with ethanol and a cAMP-elevating agent, forskolin, increased cAMP levels in cultured hypothalamic cells. However, prior exposure to ethanol reduced the cAMP-elevating responses of these neurons to ethanol and forskolin. These results indicate that the stimulatory and adaptive responses of IR-beta-EP neurons to ethanol may involve the cAMP system.

The secretory response of hypothalamic beta-endorphin neurons to acute and chronic nicotine treatments and following nicotine withdrawal.

In the present study, we determined the effect of acute and chronic nicotine treatments on the secretion of immunoreactive beta-endorphin (IR-beta-EP) and cell viability of cultured hypothalamic neurons. Also, we determined the secretory response of IR-beta-EP following withdrawal from a long-term nicotine treatment. Fetal hypothalamic cells were dissociated and maintained in cultures for 9 days and were treated with various doses of nicotine (1, 6, 12 and 18 microM) for 6 h (acute treatment) or treated with nicotine at 12 h intervals for 96 h (chronic treatment). Determination of IR-beta-EP concentrations in the media revealed that 6-18 microM doses of nicotine increased IR-beta-EP secretion from these cultures for a period of 24 h; after this period, the cultured cells did not respond to these doses of nicotine. The desensitization of beta-EP neurons 24 h after treatment with nicotine did not appear to be related to the loss of viable cells. Determination of withdrawal response after 72 h of constant nicotine (6 microM) treatments revealed that the hypothalamic neurons secrete elevated amounts of IR-beta-EP for a period of 72 h after nicotine withdrawal. These results suggest that: 1) acute treatment with nicotine stimulates hypothalamic IR-beta-EP release; 2) chronic nicotine treatment desensitizes beta-EP-secreting neurons and, 3) beta-EP neurons in primary culture show withdrawal response to nicotine.

Effects of dopamine D1 and D2 receptor agonists and antagonists on basal and ethanol-modulated beta-endorphin secretion from hypothalamic neurons in primary cultures.

In the present study, we determined the effects of dopamine receptor agonists and antagonists on basal and ethanol-modulated beta-endorphin (beta-EP) secretion from hypothalamic neurons in primary cultures. Treatment with various concentrations of dopamine D1 agonist SKF 38393 and D1 antagonist SCH 23390 did not affect basal IR-beta-EP release. However, dopamine D2 receptor agonist LY 141865 reduced basal immunoreactive (IR)-beta-EP release in a concentration dependent manner. D2 receptor antagonist, sulpiride, on the other hand, stimulated basal IR-beta-EP release and blocked LY 141865-induced inhibition of IR-beta-EP release in a concentration dependent manner. When the actions of these DA receptor agents on ethanol-modulated IR-beta-EP release were studied, both D1 and D2 receptor agents failed to affect ethanol-modulated IR-beta-EP release. These data suggest that the endogenous secretion of beta-EP from hypothalamic neurons is under the influence of an inhibitory dopaminergic system involving the D2 receptor. Furthermore, ethanol's effects on beta-EP secretion are not mediated by dopamine.

Effects of chronic alcohol on immunoreactive beta-endorphin secretion from hypothalamic neurons in primary cultures: evidence for alcohol tolerance, withdrawal, and sensitization responses.

Endogenous opioid peptides are known to be involved in the alcohol tolerance and dependence following alcohol abuse. However, the cellular mechanisms involved in the ethanol tolerance and dependence are not well established. We have previously shown that low concentrations of ethanol stimulate immunoreactive beta-endorphin (IR-beta-EP) release from the cultured hypothalamic neurons and that chronic ethanol exposure desensitizes these neurons to ethanol challenges. In this study, we determined the IR-beta-EP response to increasing doses of ethanol during the desensitizing phase of moderate ethanol doses to test whether the cultured IR-beta-EP-secreting neurons develop tolerance to ethanol following constant exposure. We also determined IR-beta-EP responses following withdrawal from chronic ethanol challenge and compared the IR-beta-EP secretory response to various doses of ethanol in ethanol-naive and ethanol-preexposed cultures. The IR-beta-EP responses to increasing doses of ethanol (50-150 mM) were markedly reduced in the cultures preexposed to a 50 mM dose of ethanol when compared with those that were naive to ethanol. The ethanol-exposed cultures showed hypersecretion of IR-beta-EP after removal from 48 hr of constant ethanol, as compared with ethanol-naive cultures. When ethanol-preexposed cultures were challenged with various doses of ethanol 4 days after ethanol withdrawal, the cultures showed higher IR-beta-EP secretory responses than did the ethanol-naive cultures. These data suggest that IR-beta-EP secretory neurons in primary cultures develop tolerance to chronic ethanol, show withdrawal response after removal of chronic ethanol exposure, and develop sensitization following repeated ethanol challenges.

Cyclic AMP and ethanol interact to control apoptosis and differentiation in hypothalamic beta-endorphin neurons.

In this study we have determined the role of cyclic AMP on the function and differentiation of beta-endorphin (beta-EP) neurons in rat fetal hypothalamic cell cultures. Addition of Bt2cAMP or the cAMP elevating agent, forskolin, in cultures, dose and time dependently increased beta-endorphin secretion. The increased beta-EP secretion after Bt2cAMP or forskolin treatment was associated with proopiomelanocortin gene expression, enhanced neurite growth, and increased neuronal viability. Determination of internucleosomal cleavage of DNA by agarose gel electrophoresis revealed that apoptosis occurred in hypothalamic neurons during the first 6-8 days in culture. Addition of Bt2cAMP during this developmental period inhibited DNA degradation in hypothalamic neurons. Furthermore, incubation with various doses of ethanol, which is known to reduce intracellular levels of Bt2cAMP, increased DNA degradation in these cells. Ethanol-induced DNA degradation was blocked by concomitant incubation with Bt2cAMP. Histochemical identification of apoptotic cells following ethanol and Bt2cAMP treatments further revealed that apoptosis occurred in beta-EP neurons during the developmental period, and that ethanol increased and Bt2cAMP reduced apoptotic beta-EP cell numbers. These results suggest that ethanol neurotoxicity on beta-EP neurons during early neuronal differentiation involves an apoptotic process and that the cAMP signaling system plays an important role in controlling apoptosis and differentiation of the beta-EP neuronal system.