Morphine-6beta-glucuronide and morphine-3-glucuronide, opioid receptor agonists with different potencies.

Using heterologous expression in Xenopus laevis oocytes, we compared the potencies of morphine, morphine-6beta-glucuronide (M6G), and morphine-3-glucuronide (M3G) for cloned human mu- (hMOR), kappa- (hKOR), and delta-opioid receptors (hDOR). Each receptor subtype was individually co-expressed with heteromultimeric G-protein coupled inwardly rectifying K(+) (GIRK) channels, consisting of GIRK1 and GIRK2 subunits, and RGS4, a regulator of G-protein signaling. The two-microelectrode voltage clamp technique was used to measure the opioid receptor-activated GIRK1/GIRK2 channel responses. Compared with morphine, M6G had higher potency at the hMOR, lower potency at the hKOR, and similar potency at the hDOR, while M3G showed a 1000-fold lower and non-selective potency via opioid receptors. In contrast to naloxone, M3G did not antagonize the effects of morphine at the hMOR. We also investigated whether Trp318 and His319 provide the molecular basis for mu/delta selectivity and mu/kappa selectivity of morphinan alkaloids by mutating these residues to their corresponding residues in kappa- and delta-opioid receptors. A single-point mutation (W318L) on hMOR completely conferred delta-like potency for morphine and M6G on the mutant mu-receptor. Double mutation at Trp318 and His319 positions (Trp318Y/His319Y) only partially conferred kappa-like potency for morphine and M6G; the decrease in potency for M6G was significantly larger than for morphine. The results of our study show that both M6G and M3G are opioid receptor agonists with different potencies and that the potency of morphinan receptor ligands can be changed by selective mutations of hMOR at the Trp318 and His319 positions.

Intramuscular ceftriaxone in the treatment of childhood meningitis due to Haemophilus influenzae type F.

OBJECTIVE: To describe a case of meningitis caused by Haemophilus influenzae type f (Hif) in a child. CASE SUMMARY: A 2.5-year-old white girl (18 kg) was hospitalized because of acute ataxia. The cerebrospinal fluid culture grew H. influenzae, which was later identified as type f. Therapy was limited by the inability to gain intravenous access. Treatment was initiated with dexamethasone 8 mg (0.44 mg/kg) intramuscularly, one dose on the day prior to initiation of ceftriaxone therapy, and intramuscular ceftriaxone 2 g (111 mg/kg/dose) once a day. After the first day, dexamethasone was administered at 3 mg (0.17 mg/kg/d) orally four times per day for four days. Within two days, the patient became afebrile and improved significantly. The remaining treatments were given during daily hospital visits on an outpatient basis. No complications occurred during the follow-up visits. DISCUSSION: The clinical presentation and therapeutic management of Hif meningitis is similar to that of H. influenzae type b (Hib) meningitis. Factors that may predispose a child to infections caused by Hif include upper respiratory tract infections, day care attendance, Down syndrome, and immunodeficiency. Hif meningitis usually is treated with a third-generation cephalosporin (frequently ceftriaxone). Although most often administered intravenously, intramuscular ceftriaxone can provide a satisfactory clinical outcome in a child with adequate peripheral perfusion but limited intravenous access. The majority of reported cases of Hif meningitis resolve with appropriate antibiotic therapy; however, long-term neurologic sequelae occasionally occur. CONCLUSIONS: Hif occasionally causes pediatric meningitis. In a patient with good perfusion and difficult intravenous access, daily intramuscular administration of ceftriaxone can be an effective treatment option. In this case, Hif meningitis occurred abruptly and resolved within 48 hours of initiation of ceftriaxone and dexamethasone without long-term sequelae. The risks of giving dexamethasone appear to be minimal, although efficacy for preventing Hif complications remains to be proven.

Effects of morphine glucuronides on the function of opioid receptors in human SK-N-SH cells.

Morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) are active metabolites of morphine. The effects of M3G and M6G on the opioid receptor transduction system has not yet been fully elucidated. Formation of cAMP after treatment with various doses of morphine, M3G, and M6G was studied. M6G and morphine, but not M3G, showed a dose dependent inhibition of cAMP accumulation. Naloxone blocked the inhibitory effect of M6G, M3G, and morphine. Pretreatment with M3G did not change the effects of morphine and M6G. The G-protein inhibitor PTX, prevented morphine, M3G, and M6G effects on cAMP. M3G and M6G vary in their ability to interact with the opioid receptor effector system. Inhibition of cAMP evoked by activation of opioid receptors and inhibitory G-proteins may play a role in the actions of M6G and M3G.

Luteinizing hormone-releasing hormone (LHRH) attenuates morphine-induced inhibition of cyclic AMP (cAMP) in opioid-responsive SK-N-SH cells.

SK-N-SH cells were used to assess the effects of luteinizing hormone-releasing hormone (LHRH) on opioid receptor-mediated changes in cyclic AMP (cAMP). Prostaglandin E1 (PGE1, 1 microM) caused a dramatic increase in cAMP levels. Treatment with 10 microM morphine (MOR) significantly inhibited the stimulatory effect of PGE1, LHRH (0.8 microM) caused an increase in the basal level of intracellular cAMP and potentiated the stimulatory effect of PGE1 on cAMP accumulation. In cells pretreated with LHRH the inhibitory effect of MOR on cAMP accumulation was significantly attenuated. An LHRH antagonist had no effect on cAMP. The involvement of pertussis toxin (PTX)-sensitive G proteins in the actions of LHRH was studied. PTX increased the stimulatory effect of PGE1 on cAMP and attenuated the inhibitory effect of MOR. However, PTX pretreatment prevented the effects of LHRH on the intracellular actions of PGE1 but exerted an additive effect with LHRH in blocking the MOR-induced decrease in cAMP levels. We conclude that LHRH attenuates the inhibitory, opioid receptor-mediated effect of MOR on intracellular cAMP accumulation in SK-N-SH cells, and that the G protein-independent mechanism may be involved in LHRH-induced attenuation of the inhibitory effect of MOR on neuronal cAMP.

Luteinizing hormone-releasing hormone in undifferentiated and differentiated SK-N-SH human neuroblastoma cells.

The presence of luteinizing hormone-releasing hormone (LHRH) in SK-N-SH human neuroblastoma cells was investigated by immunocytochemistry and enzyme-linked immunosorbent assays of whole cell extracts and culture medium. In addition, ribonuclease protection assays were utilized to quantitate LHRH messenger RNA. The expression of LHRH mRNA and LHRH protein level was correlated with neuronal differentiation induced by retinoic acid (RA). In differentiated SK-N-SH cells the LHRH mRNA level as well as the amount of LHRH in cell extracts and cell medium were significantly lower than in differentiated cells. These results suggest that RA affects the expression of LHRH mRNA and the level of LHRH protein in SK-N-SH cells. These data show that altering the growth state of the human neuroblastoma SK-N-SH cells toward more neuronal phenotype results in a significant decrease in expression of LHRH mRNA and the protein. The ability of RA to induce changes in LHRH at the mRNA and at the peptide levels will allow further study of RA regulation of LHRH at the neuronal level.

Pharmacokinetic characterization and tissue distribution of the new glucocorticoid soft drug loteprednol etabonate in rats and dogs.

Loteprednol etabonate, a new glucocorticoid soft drug with a characteristic chloromethyl ester function in the 17 beta-position, is currently in the early phases of clinical development. As the basis for human trials, this study describes a new reversed-phase high-performance liquid chromatographic method for the determination of levels of drug in plasma and urine samples and assesses the pharmacokinetic properties of loteprednol etabonate in dogs and rats. Intravenous administration of loteprednol etabonate (5 mg/kg) to dogs revealed a terminal half-life of 2.8 h, a volume of distribution of 3.7 L/kg, and a total body clearance of 0.9 L/h/kg. Intact loteprednol etabonate was not detectable in the urine. After oral administration of the drug (5 mg/kg) to dogs, only metabolites, but no intact drug, were found in the plasma, an indication for a high first-pass effect. A pronounced binding of the drug to plasma protein (> 90%) and a high erythrocyte-buffer partition coefficient of 7.8 were determined in vitro. Preliminary information about tissue distribution and possible metabolic pathways were obtained in rats after oral administration of a 14C-labeled loteprednol etabonate suspension (5 mg/kg). pH-selective extraction into ethyl acetate revealed three distinguishable fractions: (1) a neutral lipophilic fraction, presumably intact drug, (2) an acidic, lipophilic fraction, and (3) a hydrophilic nonextractable fraction. Levels of intact drug and metabolites were highest in liver and kidney, whereas significantly lower levels were found in other investigated organs (lung, brain, heart).(ABSTRACT TRUNCATED AT 250 WORDS)

cAMP accumulation in opioid-sensitive SH-SY5Y neuroblastoma cells is modified by estradiol and progesterone.

We have recently demonstrated that acute and chronic treatments with estradiol and progesterone induce changes in the responsiveness of endogenous opioid systems to painful stimulation. In the present study the neuroblastoma SH-SY5Y subclone known to contain predominantly mu opioid receptors was used as a model to characterize the gonadal steroid effect on this opioid receptor system. The function of opioid receptors was assessed by measuring prostaglandin E1 (PGE1)-induced cyclic AMP accumulation after various treatments with estradiol and progesterone. Differentiated SH-SY5Y cells respond to PGE1 with a dramatic increase in cAMP level. Morphine (MOR) inhibits by about 75% the stimulatory effect of PGE1 on cAMP. Pretreatment with 5 nM of estradiol for 6 days resulted in a significant increase of PGE1-stimulated cAMP accumulation. Exposure of cells for 48 h to estradiol in doses of 5 nM or 50 nM did not affect cell sensitivity to the PGE1 effect on cAMP. Moreover, neither dose of estradiol changed the inhibitory effect of morphine on PGE1-induced cAMP response. There was a significant increase in PGE1-stimulated cAMP accumulation after treatment with 100 nM progesterone for 1 h or 15 min and a marked elevation of cAMP levels was also measured after 15 min treatment with 10 nM progesterone. Exposure to either dose of progesterone for 8 h, 48 h or 6 days did not affect basal or PGE1-induced cAMP in neuroblastoma cells. Progesterone-treated groups responded to MOR with 56-67% inhibition of PGE1-stimulated cAMP accumulation. The potency of MOR-induced inhibition was comparable to the MOR effect in cells not treated with the steroid.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of estradiol and progesterone on the sensitivity to pain and on morphine-induced antinociception in female rats.

Long-term ovariectomized (OVX) rats were exposed to 2- or 14-day replacement with pellets made of cholesterol (CHOL), estradiol (E2), progesterone (P4), or a combination of E2 and P4. Following the treatment with steroids the antinociceptive effect of morphine (5 mg/kg,sc) was measured by a hot-plate method. Pellets of E2 (0.5 and 5%) caused dose- and time-dependent reductions of morphine-induced antinociception as compared with OVX rats treated with CHOL pellets. Moreover, OVX rats pretreated with E2 pellets had decreased basic sensitivity to nociceptive stimulus (hyperalgesia). Treatment for 2 and 14 days with 75% P4 pellets produced significant reduction of MOR antinociception. The low dose of P4 (10% pellet) did not change the effect of MOR on Day 2 but significantly increased the antinociceptive effect of MOR on Day 14. Replacement of OVX rats with one 0.5% E2 pellet plus one 10% P4 pellet resulted in marked inhibition of the antinociceptive effect of MOR on Day 2 as well as on Day 14. Central injection 30 min before MOR of either LHRH antagonist or the antiserum against LHRH into OVX rats pretreated for 14 days with both steroids had no effect on the degree of the antinociception. The results suggest that the effects which ovarian steroids exert on opioid systems vary according to the dose, the duration of treatment, and the type of steroid administered.

A modulatory role for luteinizing hormone-releasing hormone in nociceptive responses of female rats.

We have shown that responsiveness to noxious stimuli change after gonadal steroid treatment and during the estrous cycle. In the present study, we evaluate the role of LHRH in modulating nociceptive responses in female rats. In ovariectomized (OVX) rats, an LHRH agonist ([ Des-Gly10] LHRH ethyl amide; 1 ng/rat/microliters), given intraventricularly (icv) at either 90, 60, or 30 min before a hot-plate test caused a time-dependent, significant increase in sensitivity to the noxious thermal stimulus (hyperalgesia) vs. saline-treated controls. Further, the LHRH agonist (1 ng/rat/microliters; icv) attenuated morphine (5 mg/kg, sc)-induced antinociception. The injection of an LHRH antagonist, [D-Phe2,Pro3,D-Phe6] LHRH, to OVX rats in doses of 0.1, 1, or 10 ng/rat 30 min prior to morphine, enhanced and prolonged morphine-induced antinociception in a dose-dependent manner. Moreover, the hyperalgesia observed in OVX rats treated with naloxone (1 mg/kg, sc) was reversed by preinjection of either the LHRH antagonist (0.1 ng/rat, icv) or LHRH antiserum. OVX rats primed with estradiol benzoate (EB) and progesterone (P) were less sensitive to the antinociceptive effect of morphine than OVX rats. When EBP-treated rats received the LHRH antagonist prior to morphine, a twofold increase in morphine-induced antinociception was observed. A similar effect was observed in EBP-treated rats after the injection of LHRH antiserum. In conclusion, LHRH may interact with central opioid systems causing an increased sensitivity to nociceptive stimulation (hyperalgesia) and reduction of the antinociceptive effect of morphine.

Dose-dependent effects of chronic treatment with estradiol or progesterone on LH secretion in ovariectomized rats.

Ovariectomized (OVX) Sprague-Dawley rats bearing atrial cannulae were implanted subcutaneously with fused pellets containing estradiol (E2) or progesterone (P4). Variable doses of E2 (0.1, 0.5, 1 and 5%) or P4 (10, 50, 75, 100%) were achieved by varying the ratio of the hormone to cholesterol (CHOL) in the pellet. Control groups were treated with CHOL containing pellets. Blood samples were collected in the morning and afternoon the day before and 1, 2, 5, 8, 11 and 14 days after pellet implantation. The concentrations of E2, P4 and LH were measured by RIA. Throughout the sampling period, plasma concentrations of both steroids were proportional to pellet composition. On days 1 and 2, high concentrations of E2 and P4 in plasma were obtained, but between days 5 and 14 stable levels at E2 and P4 were observed. The effectiveness of chronic replacement with E2 and P4 on the negative feedback on LH secretion was assessed from morning samples and positive feedback on LH from afternoon samples. E2 implants suppressed the morning LH levels in plasma in a time and dose-dependent manner. The afternoon concentrations of LH were significantly elevated on each sampling day, except for day 1. P4 pellets had no effect on morning-afternoon difference in LH level, but low doses suppressed LH shortly after the implantation and high doses suppressed LH level after the 8th day of implantation. These results indicate that fused pellets of E2 and P4 are effective in chronically maintaining plasma E2 and P4 at levels observed during various normal and pathological reproductive states. Further, these studies indicate that E2 can stimulate afternoon hypersecretion of LH for at least 14 days in ovariectomized rats.

On the role of brain mineralocorticoid (type I) and glucocorticoid (type II) receptors in neuroendocrine regulation.

Administrations of the glucocorticoid receptor antagonist (anti-glucocorticoid, RU38486) and the mineralocorticoid antagonist (anti-mineralocorticoid, RU28318) followed by frequent, sequential blood sampling were employed to investigate the possible role the brain mineralocorticoid receptor (MR, type I) and glucocorticoid receptor (GR, type II) have in the regulation of basal and stress-induced adrenocortical secretion in the rat. The anti-mineralocorticoid and anti-glucocorticoid were administered subcutaneously (s.c.) at doses of 2.5 mg and 1.0 mg/100 g body weight, respectively. Both antagonists were also given intracerebroventricularly (i.c.v.) at a dose of 100 ng/rat. Under basal non-stressed conditions (at the diurnal trough in the morning), injections of either saline, anti-glucocorticoid (s.c. or i.c.v.) or anti-mineralocorticoid (s.c.) did not have effect on the plasma corticosterone level. The anti-mineralocorticoid given intracerebroventricularly, however, caused an elevation of plasma corticosterone up to 60 min after the injection. Exposure of the rats to a novel environment resulted in a large increase in the plasma corticosterone level, which was slightly reduced in the rats treated with the anti-glucocorticoid. In vehicle-treated rats, the level returned to basal values at 90 min, while in the anti-glucocorticoid- and anti-mineralocorticoid-treated groups, it remained elevated for prolonged periods. The present study thus shows that (1) the anti-glucocorticoid RU38486 via the brain GR has no effect on the basal plasma corticosterone level in the morning but interferes with a glucocorticoid negative feedback following stress and (2) the anti-mineralocorticoid RU28318 via the brain MR elevates the basal plasma corticosterone level and enhances adrenocortical secretion following stress.(ABSTRACT TRUNCATED AT 250 WORDS)

Long-lasting glucocorticoid suppression of opioid-induced antinociception.

The antinociceptive effect of morphine (5 mg/kg body weight i.p.) in rats subjected to various experimental manipulations of the pituitary-adrenocortical system was studied. The absence of adrenal steroids increased the sensitivity to morphine. The following findings suggest that glucocorticosteroids have a long-lasting influence on opioid-induced antinociception, even when the steroids have been removed by adrenalectomy. First, when rats were adrenalectomized in the morning under basal conditions of pituitary-adrenocortical activity (plasma corticosterone level less than 1 microgram %), the subsequent hypersensitivity to morphine-induced antinociception following adrenalectomy either in the morning or in the evening persisted for at least 2 weeks. Second, exposure to a novel environmental (stress of a new cage) or administration of corticosterone (10 mg/kg body weight s.c.) prior to morning adrenalectomy decreased the sensitivity to morphine measured 1 week later. Third, RU 38486, a glucocorticoid antagonist, injected in the lateral cerebral ventricle prior to the evening adrenalectomy increased subsequent morphine antinociception. In attempts to understand the long-term effect on morphine antinociception, the opioid receptor sites were quantified by an in vivo procedure. Quantitative autoradiography of binding sites labeled after intravenous administration of a tracer dose of [3H]-diprenorphine showed a decrease in retention of the labeled opioid in cortical and midbrain regions of rats adrenalectomized in the evening when compared with rats operated in the morning.(ABSTRACT TRUNCATED AT 250 WORDS)

Vasopressin and adrenalectomy-induced sensitivity to morphine.

Arginine vasopressin, vasopressin antiserum and a specific vasopressin pressor antagonist were injected intracerebroventricularly into adrenalectomized rats before morphine-induced antinociception was tested. In these experiments we have exploited previous findings which showed that the antinociceptive effect of opioids was potentiated after adrenalectomy; rats that were adrenalectomized in the morning under basal resting conditions of the pituitary-adrenal system displayed significantly higher response latencies after morphine administration than rats adrenalectomized in the evening. These effects were measured 7 days after adrenalectomy. The same conditions were used in this study. Both, the vasopressin antiserum and the vasopressin antagonist abolished the morning adrenalectomy-induced hypersensitivity to centrally injected morphine and were not effective when administered to rats that had been adrenalectomized in the evening. The reverse was observed after intraventricular administration of vasopressin. The peptide significantly raised the sensitivity to morphine-induced antinociception of rats that had been adrenalectomized in the evening whereas it did not affect antinociception in animals that had been adrenalectomized in the morning. Vasopressin levels determined by radioimmunoassay in the cerebrospinal fluid were significantly higher in adrenalectomized animals. We propose that vasopressin is a critical neuropeptide factor involved in the adrenalectomy-induced hypersensitivity to morphine antinociception.

Corticosteroid effects on morphine-induced antinociception as a function of two types of corticosteroid receptors in brain.

The antinociceptive effect of parenterally and intracerebroventricularly injected morphine and beta-endorphin in adrenalectomized rats and in adrenalectomized rats treated with adrenal steroids was examined employing the hot-plate method. (1) Adrenalectomy sensitized the rats to an analgesic effect of morphine and beta-endorphin. (2) Replacement therapy (chronic and acute) with corticosterone, dexamethasone or RU 28362 (glucocorticoid receptor agonist) effectively reversed the increase in the sensitivity to the analgesic effect of peripherally injected morphine (5 mg/kg i.p.) induced by adrenalectomy to the level of sham-operated animals. Glucocorticosteroids administered to non-adrenalectomized rats did not change the sensitivity to morphine. (3) Corticosterone had a biphasic, dose-dependent effect; the most significant attenuation of the hypersensitivity to morphine-induced antinociception in adrenalectomized rats was achieved after 0.01 mg and after 10 mg (per kg b.w.). Doses of corticosterone of 0.005 mg/kg and in a range of 0.05-0.30 mg/kg were ineffective. (4) Corticosterone in a dose of 0.01 mg/kg (s.c.) had suppressant effects on the adrenalectomy-induced increase in the sensitivity to antinociception induced by morphine when given prior to morphine (60, 30 and 5 min) as well as after the injection of morphine (before the first and the second testing on the hot-plate, 15 and 5 min, respectively). (5) Intracerebroventricularly (i.c.v.) injected morphine and beta-endorphin also displayed the hypersensitivity to the analgesic effect in adrenalectomized rats which in both cases could be suppressed by 0.01 mg/kg of corticosterone given subcutaneously 5 min prior to administration of the opiate.(ABSTRACT TRUNCATED AT 250 WORDS)

Function and plasticity of brain corticosteroid receptor systems: action of neuropeptides.

Two types of corticosteroid receptors may be distinguished in rat brain. Type 1 resembles the kidney mineralocorticoid receptor and Type 2 is similar to the liver glucocorticoid receptor (GR). Type 1 receptor system displays two functional expressions, i.e. Type 1 corticosterone (CORT)-preferring sites (CR) and Type 1 mineralocorticoid receptors (MR). MR occurs in circumventricular organs and mediates behaviors such as salt appetite. CR has its principal localization in neurons of the hippocampus, and mediates tonic influences of CORT on hippocampus-associated functions. CR responds with stringent specificity to CORT. Differentiation between CR and MR is due to a different accessibility of the receptor by CORT and ALDO, which seems dependent on the presence of extravascular corticosteroid binding globulin (CBG). GR has a wide distribution in brain, occurs in neurons and glial cells and has its principal localization in such regions as the paraventricular nucleus and the n. tractus solitarii (site of CRF synthesis and of blood pressure regulation, respectively). GR mediates the feedback action of CORT on stress-activated brain processes. GR is subject to autoregulation by CORT. Chronic stress, senescence, and chronic CORT administration reduce receptor number, while GR capacity is increased after adrenalectomy. Reduced GR receptor number results in a less-efficient feedback action. The CORT signal via CR probably can only be altered via changes in CR density evoked rather by neural factors than by autoregulation. CR density is reduced at senescence, but is increased to receptor number of young control animals after chronic treatment with a behaviorally potent ACTH-(4-9) analogue, Org 2766. CR plasticity is prominent for the hippocampus, which is a structure involved in cognition, emotional state and subtle regulation of pituitary-adrenal function.

Interaction of morphine and steroid hormones in the postirradiation disease in rats.

Male Wistar rats were irradiated with a single 600 R dose of X rays and then treated for 3 or 6 days with intraperitoneal injections of steroid hormones. In both irradiated and non-irradiated rats the morphine analgesia was potentiated after 3, and even more after 6 days of pretreatment with testosterone and hydrocortisone, while the effect of morphine was weaker after pretreatment with estradiol and desoxycortone. The results correlate with an increase or decrease of the morphine level in the brain. The results cannot be explained by changes in the activity of beta-glucuronidase.