Inhibition of nitric oxide synthase enhances cocaine's developmental toxicity: vascular and CNS effects.

Ischemia and/or reperfusion injury from free radicals may cause cocaine's toxicity, including its effect upon neurobehavioral development. We previously used salicylate to measure hydroxyl free radicals in chick embryos exposed to cocaine. The combination was more toxic than cocaine alone. We postulated that salicylate enhanced the vasoconstriction and toxicity via inhibition of compensatory processes (eg by inhibition of the synthesis of vasodilatory prostanoids and/or nitric oxide). A nontoxic dose of N(G)-nitro-L-arginine methyl ester (L-NAME) was used to inhibit nitric oxide synthase to test this hypothesis. In one experiment, cocaine was injected every 1.5 h (total dose =67.5 mg/kg egg) on day 15 of development, 1 h after injection of L-NAME (200 mg/kg egg) to determine viability and hatchability, which are measures of toxicity. Another experiment measured diameters of blood vessels after L-NAME injection, followed by NaCl or cocaine infusion (0.23 mg/egg/min; total dose=67.5 mg/kg egg) at 15 and 5 min afterwards. Lastly, brains of embryos pretreated with L-NAME before cocaine injections were analyzed for nitric oxide synthase activity. Cocaine decreased viability and hatchability. L-NAME enhanced cocaine's effect upon both parameters. Blood vessel diameters were decreased by cocaine after 15 min of infusion. L-NAME+cocaine caused a decrease in vessel diameter as soon as 5 min into the infusion and was greater with time, compared with other groups. Enzyme activity in brains was decreased only in the L-NAME+cocaine group. Thus, inhibition of nitric oxide synthesis interferes with the embryos' capacity to mount a compensatory vasodilatory response.

HPA axis dysregulation following prenatal opiate exposure and postnatal withdrawal.

We examined the effects of prenatal exposure to the long acting opiate l-alpha-acetylmethadol (LAAM) followed by postnatal withdrawal on hypothalamic-pituitary-adrenal (HPA) axis reactivity in neonatal and adult rats and anxiety-like behavior in adult rats. Female rats were treated with LAAM (0, 0.2, or 1.0 mg/kg/day) via oral gavage for 28 days prior to and continuing throughout pregnancy. Pups were fostered at birth to nontreated, lactating dams and underwent opiate withdrawal. On postnatal day (PND) 18, prenatal opiate-exposed male and female rat pups displayed a decreased corticosterone response 2 h after the application of an immunological stressor and 15 min following a social stressor compared to controls. In contrast, in adulthood, prenatal opiate-treated rats showed a heightened corticosterone response compared to prenatal water-treated controls at 3 h, but not 8 h, following an immunological stressor. Males prenatally treated with 1.0 mg/kg LAAM displayed elevated startle responding compared to the other prenatally treated male groups, but there was no effect of prenatal treatment in females. There were no effects of prenatal treatment in the open field test in either sex. These results suggest that prenatal opiate exposure followed by postnatal withdrawal dysregulated the HPA axis response to stressors in the neonate and adult and differentially affected adult anxiety-like behavior in males and females.

Suppressed fever and hypersensitivity responses in chicks prenatally exposed to opiates.

We have established procedures to reliably induce opiate dependence in the chick embryo via in ovo injection, early in embryonic development, of the long-acting and potent opiate N-desmethyl-l-alpha-noracetylmethadol (NLAAM). Prior studies found that there is continual exposure to NLAAM throughout embryogenesis and shortly after hatching there are signs of spontaneous withdrawal. In the present study, we used three doses of NLAAM (2.5, 5, and 10 mg/kg egg weight) to determine if prenatal opiate exposure followed by postnatal withdrawal interfered with appropriate neural-endocrine-immune interactions in the young chick. To ensure that effects were not a consequence of inappropriately large doses, we first examined acute and chronic toxicity and additional characteristics of postnatal opiate withdrawal. We then measured the corticosterone and fever responses to LPS stimulation during the withdrawal period. After the conclusion of opiate withdrawal, we assessed the hypersensitivity response to phytohemagglutinin (PHA). The fever response to LPS and the hypersensitivity response to PHA were suppressed by prenatal opiate exposure and postnatal withdrawal. The corticosterone response to LPS was not affected, but there were exaggerated corticosterone responses to saline injection in chicks exposed in ovo to NLAAM. It was unlikely that the effects of prenatal NLAAM were the result of toxicity, as little chronic toxicity was seen with the lower two doses of NLAAM, doses that yielded significant suppressions of neural-endocrine-immune responses. However, effects found in the chicks treated with 10 mg NLAAM/kg may have been partly related to the greater toxicity and/or protracted postnatal withdrawal in this group.

Vasoconstriction caused by cocaine is enhanced by sodium salicylate: is inducible nitric oxide synthase mRNA related?

We have previously found that sodium salicylate (NaSal), injected into chicken eggs at nontoxic doses used for quantifying hydroxyl free radicals in hearts and brains of embryos, caused or exacerbated hemorrhages and dramatically reduced hatchability when combined with cocaine (Coc). It has also been reported that inducible nitric oxide synthase (iNOS) gene expression is altered in brain in response to vascular damage and inflammation. In this study we measured diameters of membrane-bound blood vessels (BV) before and after pretreatment with saline (NaCl) or NaSal (100 mg/kg egg), followed by infusion of either NaCl or Coc HCl (total of 67.5 mg/kg egg) during 15 min. Brains and hearts of the embryos were then analyzed for iNOS messenger RNA (mRNA) concentrations. Coc caused vasoconstriction that was significant 5 min postinfusion (5 min PI) of the entire dose (ie after 67.5 mg/kg egg). Significant vasoconstriction was evident within 5 min in the group injected with NaSal followed by infusion with Coc (ie after 22.5 mg Coc/kg egg). Expression of iNOS mRNA was significantly increased only in the brains of the group exposed to NaSal plus Coc, and the increase was inversely related to BV diameter. These data are discussed in relation to effects of salicylate upon prostanoid synthesis and/or nitric oxide synthesis via iNOS inhibition and their possible relationship to Coc-associated cerebral vascular and/or cardiovascular events in abusing humans.

Prenatal opiate withdrawal activates the chick embryo hypothalamic pituitary-adrenal axis and dilates vitelline blood vessels via serotonin(2) receptors.

Opiate withdrawal during pregnancy may occur because of voluntary or forced detoxification, or from rapid cycling associated with exposure to short-acting "street" opiates. Thus, animal modeling of prenatal withdrawal and development of potential therapeutic interventions is important. Direct developmental effects of opiates and/or withdrawal can be studied using a chick model. In ovo administration of the long-acting opiate N-desmethyl-l-alpha-noracetylmethadol (NLAAM) induces opiate dependence in the chick embryo. We examined activation of the hypothalamic-pituitary-adrenal (HPA) axis (assessed via serum corticosterone) and hemodynamic changes (assessed as changes in apparent diameter of vitelline (extraembryonic) blood vessels) after chronic NLAAM exposure and naloxone (Nx)-precipitated withdrawal during late stages of embryogenesis. Nx-precipitated withdrawal increased corticosterone 2- to 4.5-fold and diameters of vitelline blood vessels by 15 to 45%. NLAAM exposure itself did not effect these measures. In a second set of experiments, isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor, was injected into eggs with embryos. IBMX similarly increased corticosterone and vitelline vessel diameter, with a similar time course and response magnitude. Previous studies found that serotonin(2) (5-HT(2)) receptors were involved in other withdrawal manifestations, so we determined whether they were likewise involved. Pretreatment with the 5-HT(2) antagonist ritanserin completely blocked HPA axis activation and vasodilation associated with both Nx-precipitated withdrawal and IBMX administration. This indicates that 5-HT(2) receptors, directly or indirectly, mediate these withdrawal manifestations in the chick embryo.