Krishnamurthy P. Gudehithlu

Preproenkephalin mRNA and methionine-enkephalin content are increased in mouse striatum after treatment with nicotine

Abstract: A single dose of nicotine increased methionine‐enkephalin (Met‐Enk) immunoreactivity in the striatum of mice in a time‐dependent manner. Met‐Enk content reached a maximum by ∼1 h after nicotine and returned to control values by 6 h. The response to nicotine was blocked by pretreating animals with the nicotinic receptor antagonist mecamylamine. In contrast, pretreating mice with the muscarinic receptor antagonist atropine or the dopamine receptor antagonist haloperidol did not block the response. A single dose of nicotine also increased mRNA for the precursor peptide preproenkephalin (PPE). The increase of PPE mRNA preceded that of Met‐Enk and reached a maximum by ∼30 min after nicotine. PPE mRNA levels returned to near normal by ∼3 h and increased again by 6 h after nicotine. Daily administration of nicotine for 14 days increased Met‐Enk content and PPE mRNA in the striatum of mice as well. Taken together, our results suggest that nicotinic receptors modulate Met‐Enk content and PPE mRNA in the mouse striatum.

β-endorphin and methionine-enkephalin levels in discrete brain regions, spinal cord, pituitary gland and plasma of morphine tolerant-dependent and abstinent rats

The effect of morphine tolerance-dependence, protracted and naloxone-precipitated abstinence on the levels of beta-endorphin and methionine-enkephalin in discrete brain regions, spinal cord, pituitary gland and plasma was determined in the male Sprague-Dawley rats. Among the brain regions examined, the levels of beta-endorphin in descending order were: hypothalamus, amygdala, midbrain, hippocampus corpus striatum, pons and medulla and cortex. The levels of beta-endorphin in midbrain, hypothalamus, and pituitary of morphine tolerant-dependent rats were decreased significantly. During protracted withdrawal beta-endorphin levels were decreased in amygdala, spinal cord and pituitary. During naloxone-precipitated abstinence beta-endorphin levels were increased in corpus striatum, midbrain and cortex. In addition, in naloxone-precipitated abstinence beta-endorphin levels were decreased in pituitary gland and hippocampus but increased in plasma. The levels of methionine-enkephalin in brain regions in decreasing order were: corpus striatum, pons and medulla, amygdala, hypothalamus, midbrain, hippocampus and cortex. The levels of methionine-enkephalin in pons and medulla, amygdala, hippocampus and pituitary gland were decreased in morphine tolerant-dependent rats. During protracted abstinence from morphine, methionine-enkephalin levels in spinal cord, amygdala, pons and medulla, midbrain, cortex, corpus striatum and pituitary gland were decreased. The levels of methionine-enkephalin in hypothalamus and corpus striatum of naloxone-precipitated abstinent rats were increased but were decreased in amygdala and pituitary gland. These results suggest that during morphine tolerance-dependence and during protracted abstinence beta-endorphin and methionine-enkephalin levels in discrete brain regions and pituitary gland are decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

Down-regulation of N-methyl-d-aspartate (NMDA) receptors of brain regions and spinal cord of rats treated chronically with morphine

1. The effects of morphine tolerance and abstinence on the characteristics of N-methyl-D-aspartate (NMDA) receptors, labeled with [3H]MK-801, were determined in the brain regions and spinal cord of the rat. 2. Male Sprague-Dawley rats were rendered tolerant to and physically dependent on morphine by subcutaneous implantation of six morphine pellets during a 7-day period. In tolerant (non-abstinent) rats, the pellets were left intact at the time of sacrificing, whereas in the abstinent rats the pellets were removed 16 hr prior to sacrificing. 3. The binding of [3H]MK-801, an NMDA receptor antagonist, to membranes prepared from spinal cord and brain regions (cortex, striatum, amygdala, hippocampus, hypothalamus, midbrain and pons-medulla) was determined using 5 nM concentration of the ligand in the presence of 30 microM glycine and 50 microM of glutamate. 4. In non-abstinent morphine tolerant rats, the binding of [3H]MK-801 was decreased by 40 and 33% in the midbrain and spinal cord, respectively, in comparison with their placebo controls. In morphine abstinent rats, the binding of [3H]MK-801 was decreased by 42, 29 and 50% in hypothalamus, midbrain and spinal cord, respectively, in comparison with their placebo controls. The binding of [3H]MK-801 to other brain regions and spinal cord of morphine tolerant and abstinent rats did not differ from their respective placebo controls. 5. Thus, these studies demonstrate, for the first time, that in the presence of glutamate and glycine, NMDA receptors of selected brain regions and spinal cord are down-regulated in rats treated chronically with morphine.

Antineoplastic properties of Maharishi-4 against DMBA-induced mammary tumors in rats

Maharishi-4 (M-4), an ayurvedic food supplement, was tested for anticarcinogenic and anticancer properties against 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumors in rats. The 6% M-4-supplemented diet protected DMBA-induced carcinogenesis by reducing both tumor incidence and multiplicity during initiation and promotion phases. The control animals who developed tumors when supplemented with M-4 diet for four weeks showed tumor regression in 60% of cases. There was no significant difference in the food intake or weight gain in rats who were on M-4-supplemented diet compared to control group. Possible mechanisms of action of M-4 are discussed.

c-fos and NGFI-A mRNA of rat retina: evidence for light-induced augmentation and a role for cholinergic and glutamate receptors.

When rats are exposed to room light from the dark, there is a transient increase of mRNA for the immediate-early genes c-fos and NGFI-A in the retina. Augmentation of c-fos and NGFI-A mRNA by light is apparently associated with activation of cholinergic nicotinic and muscarinic receptors as it can be suppressed by the nicotinic antagonist mecamylamine and the muscarinic antagonist atropine. Moreover, the light-induced increase of c-fos mRNA in retina appears to be associated with activation of glutamate receptors also as the noncompetitive inhibitor of N-methyl-D-aspartate receptors dizocilpine (MK-801) partially suppressed the increase of the c-fos message. Light-induced NGFI-A mRNA augmentation is apparently modulated by the same receptors. We were unable to detect light-induced changes of c-jun mRNA.

Preproenkephalin mRNA and methionine-enkephalin increase in mouse striatum after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment.

Abstract: Dopaminergic neurons that project to the striatum from the substantia nigra are thought to modulate methionine‐enkephalin (Met‐Enk) metabolism in the striatum. We administered a dose of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) that produces a moderate depletion of dopamine in striatum, about 50%, without overt motor deficits, and found that Met‐Enk‐like immunoreactivity and preproenkephalin mRNA content increased in the tissue. Pretreatment with the monoamine oxidase B inhibitor deprenyl or the dopamine transport blocker nomifensine prevented these changes, suggesting that the changes were related to the partial loss of dopaminergic neurons rather than to MPTP. Moreover, administering GM1 ganglioside, which partially restores the MPTP‐induced dopaminergic deficit, partially corrected the Met‐Enk changes in the striatum as well. These findings are consistent with the hypothesis that dopaminergic input to the striatum, in part, modulates Met‐Enk metabolism. Moreover, they show that moderate nigrostriatal lesions are sufficient to elevate Met‐Enk and preproenkephalin mRNA contents and that restoration of dopaminergic function, as in our studies with GM1 ganglioside, restores the content of Met‐Enk.

Differential binding of [3H]MK-801 to brain regions and spinal cord of mice treated chronically with morphine

1. The effects of morphine tolerance and abstinence on the binding of [3H]MK-801, a noncompetitive antagonist of the N-methyl-D-aspartate (NMDA) receptors were determined in brain regions and spinal cord of the mouse. 2. Male Swiss-Webster mice were rendered tolerant to and physically dependent on morphine by subcutaneous implantation of a pellet containing 75 mg of morphine base for 3 days. Placebo pellet-implanted mice served as controls. In tolerant (nonabstinent) mice, the pellets were left intact at the time of sacrificing whereas, in the abstinent mice, the pellets were removed 6 hr prior to sacrificing. 3. The binding of [3H]MK-801 to membranes prepared from spinal cord and brain regions (cortex, pons-medulla, hypothalamus, hippocampus, amygdala, striatum, and midbrain) was determined by using a 5 nM concentration of the ligand in the presence of 30 microM glycine and 50 microM of glutamate. 4. In nonabstinent morphine-tolerant mice, the binding of [3H]MK-801 was decreased in pons-medulla and hypothalamus, but was increased in the spinal cord in comparison to that in placebo controls. The reduction in binding in pons-medulla was due to a decrease in the Bmax value; the Kd value remained unchanged. The binding of [3H]MK-801 was increased in the hippocampus of morphine-abstinent mice. 5. These studies demonstrate that NMDA receptors of brain regions and spinal cord are differentially affected in morphine-tolerant and abstinent mice.

c-fos mRNA in mouse brain after MPTP treatment

The neurotoxin, MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) induces a transient increase of mRNA for the immediate-early gene c-fos in the mouse brain. The c-fos mRNA level is MPTP dose-dependent and is evident in all brain regions tested including striatum, hypothalamus, cortex, hippocampus, cerebellum and midbrain. There are regional differences in the time-course for the rise of c-fos mRNA. Pretreatment with deprenyl, a selective monoamine oxidase B inhibitor, pargyline, a nonselective monoamine oxidase inhibitor, or mazindol, a dopamine uptake transport inhibitor, does not prevent the c-fos mRNA increase, suggesting that the elevation is due to the action of MPTP and not its neurotoxic metabolite MPP+.

Nicotine-induced changes of brain β-endorphin

A consensus has emerged that endogenous opioid peptides and their receptors play an important role in the psychoactive properties of nicotine. Although behavioral studies have shown that β-endorphin contributes to the rewarding and emotional effects of nicotine, whether the drug alters the function of brain endorphinergic neurons is not fully explored. These studies investigated the effect of acute, 1mg/kg, sc, and chronic, daily injection of 1mg/kg, sc, for 14 days, administration of free base nicotine on brain β-endorphin and its precursor proopiomelanocortin (POMC). Acute and chronic treatment with nicotine decreased β-endorphin content in hypothalamus, the principal site of β-endorphin producing neurons in the brain, and in the endorphinergic terminal fields in striatum and hippocampus. The acute effect of nicotine on β-endorphin was reversed by the nicotinic antagonist mecamylamine and the dopamine antagonist haloperidol, indicating pharmacological specificity and involvement of dopamine D2-like receptors. Similar observations were made in prefrontal cortex. POMC mRNA in hypothalamus and prefrontal cortex was unchanged following acute nicotine, but it decreased moderately with chronic treatment. The nicotine treatments had no effect on pituitary and plasma β-endorphin. Taken together, these results could be interpreted to indicate that nicotine alters the synthesis and release of β-endorphin in the limbic brain in vivo. Altered endorphinergic function may contribute to the behavioral effects of acute and chronic nicotine treatment and play a role in nicotine addiction.

Expression of cloned aromatic L-amino acid decarboxylase in Xenopus laevis oocytes

Sense mRNA coding for bovine adrenal medulla aromatic L-amino acid decarboxylase (AADC) was expressed following microinjection into Xenopus laevis oocytes. The expressed enzyme activity was stereoselective for L-5-hydroxytryptophan and L-DOPA and blocked by NSD-1015 an inhibitor of AADC. Heating the expressed enzyme at 55 degrees C resulted in a parallel loss of activity towards both substrates. Our findings are consistent with the prevailing notion that a single enzyme is able to decarboxylate both substrates in vivo.