Opioids and clonidine modulate cytokine production and opioid receptor expression in neonatal immune cells.

OBJECTIVE: Opioids and clonidine, used in for sedation, analgesia and control of opioid withdrawal in neonates, directly or indirectly activate opioid receptors (OPRs) expressed in immune cells. Therefore, our objective is to study how clinically relevant concentrations of different opioids and clonidine change cytokine levels in cultured whole blood from preterm and full-term infants. STUDY DESIGN: Using blood from preterm (≤ 30 weeks gestational age (GA), n=7) and full-term ( ≥ 37 weeks GA, n=19) infants, we investigated the changes in cytokine profile (IL-1ß, IL-6, IL-8, IL-10, IL-12p70 and TNF-α), cyclic adenosine monophosphate (cAMP) levels and µ-, δ- and κ- opioid receptor (OPR) gene and protein expression, following in-vitro exposure to morphine, methadone, fentanyl or clonidine at increasing concentrations ranging from 0 to 1 mM. RESULT: Following lipopolysaccharide activation, IL-10 levels were 146-fold greater in cultured blood from full-term than from preterm infants. Morphine and methadone, but not fentanyl, at >10(-5) M decreased all tested cytokines except IL-8. In contrast, clonidine at <10(-9) M increased IL-6, while at >10(-5) M increased IL-1ß and decreased TNF-α levels. All cytokine changes followed the same patterns in preterm and full-term infant cultured blood and matched increases in cAMP levels. All three µ-, δ- and κ-OPR genes were expressed in mononuclear cells (MNC) from preterm and full-term infants. Morphine, methadone and clonidine, but not fentanyl, at >10(-5)M decreased the expression of µ-OPR, but not δ- or κ-OPRs. CONCLUSION: Generalized cytokine suppression along with downregulation of µ-OPR expression observed in neonatal MNC exposed to morphine and methadone at clinically relevant concentrations contrast with the modest effects observed with fentanyl and clonidine. Therefore, we speculate that fentanyl and clonidine may be safer therapeutic choices for sedation and control of opioid withdrawal and pain in neonates.

Cognitive function and cholinergic neurochemistry in weanling rats exposed to chlorpyrifos.

Previous studies have shown that moderate to high levels of chlorpyrifos (CPF) alter cognitive function in adult and immature rats. In the present study, we tested the hypothesis that lower-level exposure to CPF before or immediately after weaning causes deficits in cognitive function. A total of 78 Long-Evans rats were injected subcutaneously with 0, 0.3 or 7.0 mg/kg CPF every 4 days before or after weaning and were tested with the Morris swim task from postnatal day 24 through 28. Exposure to CPF before weaning did not cause signs of overt cholinergic intoxication or impaired growth nor did the exposures cause significant inhibition of regional brain cholinesterase (ChE) activity or reduction in muscarinic receptors 24 h after the last injection. However, spatial learning was impaired after 5 days of training in the group of weanling rats administered 7.0 mg/kg CPF. Rats administered 0.3 or 7.0 mg/kg CPF after weaning were also impaired in the task, without significant changes in brain ChE activity. These data indicate that low-level exposure to CPF caused deficits in cognitive function in weanling rats, and these effects did not appear to be mediated by the inhibition of brain ChE. It is suggested that the alteration of cognitive function in juvenile rats is an important functional correlate of the cellular and molecular effects of CPF in the immature brain. The mechanisms for CPF-induced cognitive dysfunction are unknown.

Acute morphine dependence in mice selectively-bred for high and low analgesia.

Acute morphine dependence was compared in mice selectively-bred for high (HA) and low (LA) swim stress-induced analgesia and high (HAR) and low (LAR) levorphanol analgesia by counting the number of naloxone-precipitated jumps. Whereas LAR mice displayed greater acute morphine dependence than HAR mice, HA and LA mice did not differ. No genotypic differences were observed in non-dependent mice, discounting possible differences in basal naloxone sensitivity and/or opioid peptide levels. Thus, the two selection projects, while both producing lines exhibiting highly divergent sensitivity to morphine analgesia, have not had analogous effects on all opioid measures, supporting the notion of independent genetic mediation of opioid analgesia and dependence. Further, these data suggest that analgesic sensitivity may not predict sensitivity to morphine dependence.

The effects of LY293558, an AMPA receptor antagonist, on acute and chronic morphine dependence.

In rodents, noncompetitive and competitive NMDA receptor antagonists have been shown to attenuate and, in some cases, reverse tolerance to the analgesic effects of morphine. However, the ability of these same excitatory amino acid (EAA) receptor antagonists to modulate morphine dependence is controversial, and very little is known about the role of AMPA receptors in morphine dependence. LY293558, a novel, systemically active, competitive AMPA receptor antagonist and the NMDA receptor antagonists, MK-801 and/or LY235959, were evaluated in tolerant or dependent CD-1 mice. In mice rendered tolerant by morphine injection or pellet implantation, continuous s.c. infusion of LY293558 (60 mg/kg per 24 h) or MK-801 (1 mg/kg per 24 h) attenuated the development of tolerance. Neither LY293558 nor MK-801 produced analgesia or altered the ED50 value of morphine. Continuous s.c. infusion of LY293558 (60 mg/kg per 24 h), MK-801 (1 mg/kg per 24 h) or LY235959 (12 mg/kg per 24 h) attenuated the development of acute (3 h) morphine dependence (i.e., decreased naloxone-precipitated withdrawal jumping). In contrast, continuous s.c. infusion of LY293558 (60 mg/kg per 24 h) or LY235959 (12 mg/kg per 24 h) did not significantly attenuate the development of chronic dependence produced by morphine pellet implantation. These data indicate that the development of morphine tolerance is more sensitive to modulation by EAA receptor antagonists than is the development of morphine dependence as assessed by naloxone-precipitated withdrawal jumping.

An antisense oligodeoxynucleotide to the delta opioid receptor (DOR-1) inhibits morphine tolerance and acute dependence in mice.

Pharmacological data from several laboratories support a modulatory role for the delta opioid receptor in morphine analgesia, tolerance, and physical dependence. We examined the role of the delta opioid receptor in these processes using an in vivo antisense strategy in mice. Intracerebroventricular administration of a 20mer antisense or a mismatch control oligodeoxynucleotide (ODN) targeting the mRNA of the cloned delta opioid receptor (DOR-1) for 3 days did not affect baseline nociceptive thresholds or morphine analgesia compared to untreated or saline-treated mice. However, dose-response studies indicate that the induction of morphine tolerance following 3 days of chronic morphine administration was blocked in antisense but not mismatch ODN or saline-treated mice. Antisense ODN treatment also blocked the development of acute morphine dependence, whereas similar protection was not afforded to mice treated with saline or mismatch ODN. This study demonstrates the relevance of the cloned DOR-1 in morphine tolerance and dependence and provides new evidence for a modulatory role of the delta opioid receptor using this novel approach.

Cardiac noradrenergic mechanisms mediate GABA-enhanced ouabain cardiotoxicity.

Peripherally administered gamma-aminobutyric acid (GABA) alters cardiovascular function and has been reported to enhance ouabain-induced cardiotoxicity in vivo. Control and reserpinized rat hearts were perfused in vitro to determine if GABA directly evokes bradycardia by GABAA receptors, interacts with ouabain, and if noradrenergic mechanisms are required. Also, double-staining immunohistochemistry was employed to determine whether GABA-ergic and noradrenergic synthetic enzymes were juxtaposed within atrial tissue. The main results were as follows. GABA produced a dose-dependent bradycardia (p < 0.05) by stimulating GABAA receptors in Langendorff-perfused hearts. Reserpinized hearts were unresponsive (p < 0.05) to GABA, except at the highest dose (20 mg/ml). Ouabain-induced cardiotoxicity was enhanced (p < 0.05) by GABA in isolated control, but not reserpinized hearts. Lastly, glutamic acid decarboxylase and tyrosine hydroxylase immunoreactivities were in close proximity in atrial slices. Collectively, the results document that GABA causes bradycardia and enhances ouabain cardiotoxicity by modulating noradrenergic mechanisms in the isolated rat heart. Since the synthetic enzymes for GABA and norepinephrine were in close proximity in atrial tissue, these transmitters may interact under physiological conditions.

Response of the magnocellular system in rats to hypovolemia and cholecystokinin during pregnancy and lactation.

To define changes in the magnocellular neuroendocrine system during lactation and pregnancy, we compared plasma levels of oxytocin (OT) and vasopressin (VP) after polyethylene glycol (PEG)-induced hypovolemia and cholecystokinin (CCK) stimulation. Conscious virgin, pregnant (day 20), and lactating (day 6) Sprague-Dawley rats were injected with either PEG (70-600 mg/ml; 35 or 70 ml/kg sc), CCK (100 micrograms/ml; 4 ml/kg ip), or vehicle and decapitated 4 h (PEG) or 5 min (CCK) later. Changes in thresholds for release of hormone and the responsiveness (slopes relating [hormone] to blood volume depletion or to plasma osmolality) of the OT and VP systems were determined using an iterative nonlinear threshold regression model. After PEG, plasma osmolality increased coincident with a decrease in blood volume, with both stimuli contributing to the rise in plasma VP and OT. Compared with virgin rats, neither the threshold nor the responsiveness of the VP system was altered by the combined stimulus, whereas the oxytocinergic system of pregnant rats was more responsive to osmotic component. Lactating rats, however, had a higher threshold for VP release and an apparent elevation of the OT threshold beyond 25% volume depletion. Regardless of the reproductive state, the threshold for VP release was always lower than that for OT. Intraperitoneal CCK elevated plasma [OT] in each reproductive state, although the response in lactating animals was attenuated. In virgin and lactating rats, plasma levels of VP also increased slightly but significantly in response to CCK. During gestation when cardiovascular volume is expanded, both the VP and OT neuroendocrine systems were reset, enabling secretion of both hormones in response to hypovolemia with hypertonicity. During lactation, both neuroendocrine systems are reset such that greater changes in fluid balance are needed to stimulate hormone release. Regardless of the reproductive state, the threshold for VP release was always lower than that for OT, indicative of preferential release of VP with less than a 5% (virgin, pregnant) or a 20% (lactating) loss in blood volume.

Ouabain cardiotoxicity is enhanced by GABA in anesthetized rats.

Potential alteration of ouabain-induced cardiotoxicity by gamma-aminobutyric acid (GABA) in rats was tested by infusing ouabain for 10 min (0.7 mg/kg/min, i.v.) before or after continuous infusion of Ringer's solution with or without GABA (1 mg/min, i.v.). GABA evoked hypotension and bradycardia of similar magnitude under both conditions. The incidence of ouabain-induced ventricular fibrillation (VF) or cardiac arrest (CA) was similar in both groups. However, the time intervals to onset of VF and CA, in rats given ouabain before, but not after, GABA were shorter than in rats treated with Ringer's solution (p < 0.05). In experiments where baclofen (0.034 mg/min, i.v.) was infused after ouabain, hypotension and bradycardia occurred, but the incidence and times of ouabain-induced VF and CA were similar to control values. These results suggest that the enhancement in ouabain cardiotoxicity was mediated by GABAA receptors.

Osmoregulation of the magnocellular system during pregnancy and lactation.

We compared the responsiveness of both the vasopressin (VP) and oxytocin (OT) magnocellular systems to osmotic stimulation during pregnancy and lactation to determine if changes in thresholds and sensitivities were similar for both neuropeptides. Virgin, pregnant (day 20), and lactating (day 6) Sprague-Dawley rats were injected with a hypertonic NaCl solution (0.25 M-8.0 M NaCl; 15 ml/kg sc) and decapitated 2 h later. Late in gestation, the apparent osmotic threshold for both VP and OT release was lower by approximately 10 mosmol/kgH2O than that of virgin and lactating animals. The sensitivity (i.e., slope of the linear regression relating plasma osmolality and VP or OT levels) of the magnocellular system to osmoregulation, however, was unchanged in pregnant animals but was significantly attenuated (P < 0.01) for both peptides during lactation (slopes of lactating vs. virgin rats: OT, 1.7 vs. 3.4; VP, 1.1 vs. 1.9). The neural lobe content of VP decreased (P < 0.05) in pregnant rats, whereas OT stores were reduced (P < 0.05) in lactating animals. Thus, during pregnancy, the lower tonicity of plasma is perceived as normal by both VP and OT neuroendocrine systems enabling excretion of an acute sodium or water load.

Kappa opiate receptors inhibit release of oxytocin from the magnocellular system during dehydration.

Magnocellular neurons synthesize vasopressin (VP) or oxytocin (OT) and release these hormones preferentially from the neural lobe during physiological stimulation. In the rat, VP is secreted preferentially during dehydration and hemorrhage, whereas OT is released without VP by suckling, parturition, stress, and nausea. Vasopressinergic neurons also synthesize and release dynorphin-related peptides--alpha- and beta-neoendorphin, dynorphin A (1-8) or (1-17), dynorphin B--which are agonists selective for kappa opiate receptors in the neural lobe. We proposed that one mechanism for preferential secretion of neurohypophysial hormones is that a dynorphin-related peptide(s) coreleased with VP inhibits selectively OT secretion from magnocellular neurons. We tested this hypothesis in conscious adult male Sprague-Dawley rats which were stimulated by either hypertonic saline administered intraperitoneally (2.5%, 20 ml/kg) or subcutaneously (1 M, 15 ml/kg) or by 24 h of water deprivation. Two approaches were used: (1) dynorphin-related peptides (0.02-20.4 mM) were injected intracerebroventricularly 1 min before decapitating the animal, and (2) the action of endogenous opioid peptides was blocked by injecting subcutaneously or intracerebroventricularly either naloxone or a selective kappa receptor antagonist, Mr 2266 or nor-binaltorphimine. VP and OT were measured by radioimmunoassay. After 24 h of water deprivation, the elevation in plasma [OT] but not [VP] was attenuated (p less than 0.05) by alpha-neoendorphin. Dynorphin A (1-8) also inhibited the release of OT and not VP after intraperitoneal administration of hypertonic saline. Blocking the action of endogenous opioid peptides at kappa receptors with Mr 2266 given peripherally (s.c.) elevated plasma [OT] but not [VP] after stimulation with hypertonic saline administered intraperitoneally or subcutaneously.(ABSTRACT TRUNCATED AT 250 WORDS)