Steroid hormones play an important role in the regulation of cyclic changes in the uterus and preparation of intrauterine environment for the egg fertilization, embryo implantation and maintenance of pregnancy. Their secretion by porcine uterus has been demonstrated. The present study aimed to establish the effect of opioid receptors (μ, δ and κ) activation by selective agonists (DAMGO, DPLPE and U 50.488, respectively) on in vitro secretion of steroid hormones (during 6-h and 24-h incubations) by the endometrial explants of gilts on days 2 - 3, 10 - 11, 12 - 3, 15 - 16, 18 - 20 of the estrous cycle and 10 - 11, 12 - 13, 15 - 16 of pregnancy. The agonists at certain of tested concentrations (10-9, 10-8 and 10-7 M) affected secretion of steroid hormones. Progesterone secretion was increased by μ-opioid receptor agonist on days 18 - 20 (6 h) and by δ-agonist on days 2 - 3 and 18 - 20 (24 h) of the cycle. During pregnancy (days 15 - 16), κ-agonist increased it (6 h), but μ-opioid agonist decreased (24 h). Androstenedione secretion was decreased during shorter incubation; by μ- and δ-receptor agonists on days 2 - 3, by all agonists on days 12 - 13, and by κ-receptor agonist on days 18 - 20 of the cycle. However, it was increased during longer incubation with agonists of κ- and μ-opioid receptors on days 10 - 11 and 18 - 20 of the cycle, respectively. Estradiol secretion was elevated by κ- and μ-agonists (6 h) on days 2 - 3 and 15 - 16 of the cycle, respectively, as well as following 24-h incubation with μ-agonist on days 15 - 16, and μ- and κ-agonists on days 18 - 20 of the cycle. During pregnancy, its secretion was increased (24 h) ondays 15 - 16 by μ- and κ-opioid agonists. Cortisol secretion did not significantly change (versus control) in response to applied treatments. These results indicate a potential involvement of EOPs in the modulation of endometrial steroidogenesis in the pig during the estrous cycle and pregnancy.

To investigate the channels and neurotransmitters in the ventrolateral medulla (VLM) oblongata that are responsible for the maintenance of sympathetic tone and cardio-respiratory reflex regulation. Microinjections of excitant amino acid (glutamate, 100 nl, 100 mM), calcium channel blockers, agonists and antagonists were made throughout the VLM in anaesthetized rats. Arterial blood pressure, sympathetic nerve activity and phrenic nerve discharge was recorded. Responses to activation of the baroreceptor, somatosympathetic, and chemoreceptor reflexes were recorded. Injection sites were marked with albumin-colloidal gold or methylene blue. At the end of recording, brains were removed, fixed, sectioned and stained for gold spots, and counterstained to localize injection sites. Bilateral microinjection of calcium channel antagonists into the rostral VLM (RVLM) had little effect on arterial blood pressure with the exception of Ni++ (an agent that blocks low voltage T-type calcium channels). Ni++ caused a profound fall in arterial blood pressure and sympathetic nerve activity, but did not affect reflex function. By contrast the high voltage channel (P, Q, L, N) blockers increased blood pressure and sympathetic activity following injection into the caudal VLM (CVLM). The agents applied were 8-OHDPAT; 5HT1a agonist, DAMGO; mu opioid agonist, DPDPE; delta opioid agonist and sar9-substance P; substance P agonist. In addition, the effect of changing the inhaled concentration of carbon dioxide was tested. In all cases except injection DAMGO, there was an attenuation of the somato-sympathetic reflex. DAMGO attenuated the baroreceptor reflex. None of these agents affected the chemoreceptor reflex. The data suggest that tonic maintenance of sympathetic activity is mediated, at least in part, by a Ni++ sensitive conductance in RVLM and that the neurochemical heterogeneity found within the inputs to sympathoexcitatory and inhibitory neurons in the RVLM has functional significance, with different neurotransmitters affecting cardiorespiratory reflexes differentially. Further functional studies are required to determine the precise connectivity between different neuronal populations that are subserving different functions in the RVLM.

Opioid receptor pharmacology in vivo has predicted a greater number of receptor subtypes than explained by the profiles of the three cloned opioid receptors, and the functional dependence of the receptors on each other shown in gene-deleted animal models remains unexplained. One mechanism for such findings is the generation of novel signaling complexes by receptor hetero-oligomerization, which we previously showed results in significantly different pharmacology for mu and delta receptor hetero-oligomers compared with the individual receptors. In the present study, we show that deltorphin-II is a fully functional agonist of the mu-delta heteromer, which induced desensitization and inhibited adenylyl cyclase through a pertussis toxin-insensitive G protein. Activation of the mu-delta receptor heteromer resulted in preferential activation of Galpha(z), illustrated by incorporation of GTPgamma(35)S, whereas activation of the individually expressed mu and delta receptors preferentially activated Galpha(i). The unique pharmacology of the mu-delta heteromer was dependent on the reciprocal involvement of the distal carboxyl tails of both receptors, so that truncation of the distal mu receptor carboxyl tail modified the delta-selective ligand-binding pocket, and truncation of the delta receptor distal carboxyl tail modified the mu-selective binding pocket. The distal carboxyl tails of both receptors also had a significant role in receptor interaction, as evidenced by the reduced ability to co-immunoprecipitate when the carboxyl tails were truncated. The interaction between mu and delta receptors occurred constitutively when the receptors were co-expressed, but did not occur when receptor expression was temporally separated, indicating that the hetero-oligomers were generated by a co-translational mechanism.

Hypothalamic proopiomelanocortin (POMC) neurons are critical for controlling homeostatic functions in mammals. We used a transgenic mouse model in which the POMC neurons were labeled with enhanced-green fluorescent protein (EGFP) to perform visualized, whole-cell patch recordings from pre-pubertal female hypothalamic slices. The mouse POMC-EGFP neurons expressed the same endogenous conductance (IA and Ih) that has been described for guinea pig POMC neurons. In addition, the selective opioid receptor agonist DAMGO induced an outward current (maximum of 12.8 ± 1.2 pA), which reversed at EK+, in the majority (85%) of POMC neurons with an EC50 of 102 nM. This response was blocked by the opioid receptor antagonist naloxone with a Ki of 3.1 nM. In addition, the GABAB agonist baclofen (40 ?M) caused an outward current (21.6 ± 4.0 pA) that reversed at E K+ in these same neurons. The KATP channel opener diazoxide also induced an outward K+ current (maximum of 18.7 ± 2.2 pA) in the majority (92%) of POMC neurons with an EC50 of 61 ?M. The response to diazoxide was blocked by the sulfonylurea tolbutamide, indicating that the POMC neurons express both Kir6.2 and SUR1 channel subunits, which was verified using single cell RT-PCR. This pharmacological and molecular profile suggested that POMC neurons might be sensitive to metabolic inhibition, and indeed, we found that their firing rate varied with changes in glucose concentrations. Therefore, it appears that POMC neurons may function as an integrator of metabolic cues and synaptic input for controlling homeostasis in the mammal.

Lack of tolerance in peripheral opioid analgesia in mice