Catecholamine biosynthesis in brains of rats treated with morphine.

In the brains of rats without tolerance to morphine, the accumulation of [(14)C]dopamine formed from [(14)C]tyrosine injected intracisternally is increased, reaching a maximum in the hypothalamus and striatum 1 hour after administration of morphine. In tolerant rats, the rate of incorporation of carbon-14 into dopamine and into norepinephrine in these areas is more than twice that in animals that have received only one injection of morphine.

Effects of narcotic analgesic drugs on the cyclic adenosine 3',5'-monophosphate-adenylate cyclase system in rat brain.

1. The concentrations of cyclic adenosine 3',5'-monophosphate (cyclic AMP), measured in discrete brain areas removed from rats killed by microwave irradiation, rose transiently in most areas after the administration of morphine. The most pronounced changes, however, were found 2 h after doses of either 10 or 60 mg/kg morphine when cyclic AMP levels declined significantly in the hypothalamus, medulla and cerebellum. In morphine-tolerant rat brains there were no decreases in cyclic AMP levels. 2. Basal adenylate cyclase activity in crude nerve-ending fractions from discrete areas of rat brain was unaffected by the addition of active analgesic agonists, antagonists or inactive isomers to the assay medium in vitro, except for a nonspecific inhibition at drug concentrations of 1 mM. 3. The acute administration of morphine or levorphanol, but not dextrorphan produced transient increases in basal cyclase activity of crude nerve-ending preparations from midbrain and striatum. In morphine-tolerant rats, these changes in basal adenylate cyclase activity were no longer seen.

The effects of etorphine on the carboxylmethylation of synaptosomal proteins of rat striatum.

The stimulation of protein carboxylmethyl transferase (PCMT) activity in rat striatal synaptosomes by the dopamine agonist, apomorphine, and a PCMT substrate, calmodulin, was measured in normal and opioid-treated rats to see if inactivation of calmodulin by methylation is a factor in opioid action. Total carboxyl methyl acceptors were measured in preparations from alkaline homogenates, while those already occupied in vivo were measured in acidic homogenates, since the carboxylmethyl group is stable in acid. The administration of etorphine acutely increased the number of already occupied acceptors while chronic morphine treatment decreased this number. Apomorphine stimulation of PCMT activity was significant only when tested for direction of change from control values. Calmodulin was a substrate for PCMT in all preparations.

A method for dissection of discrete regions of rat brain following microwave irradiation.

A simple microdissection technique for obtaining discrete areas from rat brains exposed to microwave irradiation is described and illustrated. Using the atlas of Pellegrino et al. [10] as a guide, stereotaxically defined areas were removed from coronal sections prepared with sectioning stages constructed from microscope slides. The dissection of sixteen discrete regions is shown in photographic and schematic form. This technique may prove useful for examining neurochemical processes in discrete areas of the rat central nervous system and may aid in establishing the distribution of pharmacological and toxicological agents at a neuroanatomical level.

The effect of opioids on striatal calmodulin levels.

Phosphorylation of proteins in synaptic membranes of rat striatum by endogenous kinases was quantified by scanning the optical density on radioautograms made from gels after electrophoresis of assay samples. Neither the decreased phosphorylation found in samples from morphine-tolerant rats could be raised to control levels, nor could samples from untreated rats be raised to those found after acute opioid administration by the addition of Ca++ and calmodulin to the assay. However, the addition of opioids in vitro to the assay was able to inhibit the stimulation of protein kinase activity by Ca++ and calmodulin. Calmodulin seemed to be transported from membranes to cytosol of striatal synaptosomes from morphine tolerant rats. This phenomenon has been related to striatal dopamine receptor supersensitivity (5, 10), a condition suggested by many kinds of evidence in tolerant animals. After acute administration of etorphine or methionine-enkephalin, the levels of calmodulin were increased in both compartments of striatal synaptosomes. The increased membranal levels may be related to its ease of extraction from the membranes and thus to a physical change in calmodulin.

Is a calmodulin-opiopeptide interaction related to the mechanism of opioid action?

The effect of several opioids: methadone, etorphine, beta-endorphin and D-ala2met enkephalin on Ca++/calmodulin stimulation of enzyme activities either in pure solution (cyclic nucleotide phosphodiesterase) or in striatal membranes (protein kinases in synaptic membranes) were compared to see if a direct opioid/calmodulin interaction could eliminate the stimulation of enzyme activity as part of the mechanism by which opioids alter ion flow and neurotransmitter release. In other experiments, in which endogenous phosphorylation of proteins in striatal synaptic membranes was altered by opioid treatments, the possibility of restoring protein kinase activity to normal levels in the membrane preparation by supplementation with calmodulin at optimal Ca++ concentration was examined. Some opioids (methadone and D-ala2met enkephalin) did not inhibit calmodulin-stimulated phosphodiesterase, which suggests that they were not able to bind to calmodulin. In addition, it was not possible to restore decreases in protein kinase activity to normal levels by adding calmodulin to the assay in the presence of optimal Ca++. We conclude that a direct binding of opioids to calmodulin is not a general mechanism of opioid action, although the binding may participate in the action of some neuropeptides, including beta-endorphin.

Effects of morphine and haloperidol on the electrical activity of rat nigrostriatal neurons.

The action of opiates on electrical activity in nigrostriatal neurons was compared with the actions of known dopamine receptor agonists and antagonists in order to clarify the effects of opiates on postsynaptic dopamine receptors in the striatum. The systemic administration of either morphine or haloperidol increased the rate of spontaneous firing of neurons in substantia nigra. The injection of either drug directly into the caudate nucleus of the striatum also increased the firing rates of nigral neurons. The administration of the opiate antagonist, naloxone, blocked or reversed the action of systemically or locally applied morphine on firing rates, but not those of haloperidol. The dopamine receptor agonists, dopa and apomorphine, decreased the firing rate in nigral neurons and also reversed the stimulation of firing rates by morphine. The agonists were only partially successful in reversing the effects of haloperidol. The differences in the ability of naloxone and dopa to reverse the stimulated firing rates produced by morphine or haloperidol support the hypothesis that opiates do not act directly on the postsynaptic dopamine receptor in the striatum.

The effect of acute opioid administration on the phosphorylation of rat striatal synaptic membrane proteins.

The acute administration of morphine, methadone, etorphine, methionine-enkephalin, leucine-enkephalin and some related pharmacologically active peptides to rats produces biphasic effects on the degree of phosphorylation of striatal synaptic plasma membrane (SPM) proteins by endogenous protein kinases in the membranes. At 1 to 5 min after opioid administration, the phosphorylation of several SPM proteins is enhanced, followed at 10 to 20 min by a severe reduction in the rate of phosphorylation of the same proteins. Multiphasic responses follow the initial biphasic response after the injection of some opioids. Naloxone is able to block the agonist effects. The injection of saline or an inactive narcotic isomer has no effect on the phosphorylation of striatal SPM proteins. The administration of other psychoactive drugs such as clonidine or haloperidol also produces changes in phosphorylation, although the patterns of change are distinctly different from those produced by opioids. The proteins affected by opioid administration resemble those susceptible to stimulation of phosphorylation in the presence of Ca++/calmodulin. The role of Ca++/calmodulin-regulated reactions in the mechanisms of action of opioids has been evaluated by the addition of these modulators to the assays of preparation from opioid-treated rats. Neither modulators, added separately or together to the assay, was able to restore a depressed phosphorylation to normal levels, although each modulator was effective in stimulating the phosphorylation of many SPM proteins.

The effect of morphine tolerance on the incorporation of 3H-leucine into proteins of rat synaptic membranes.

The rate of incorporation of 3H-leucine into proteins of the synaptic junctional and nonjunctional membranes and synaptic vesicles of rat brain has been examined in control and morphine-tolerant rats. There are no discernible differences between control and tolerant animals in amount of protein as measured by densitometric tracings of dye-stained proteins separated by acrylamide gel electrophoresis from the three membrane fractions of whole brain areas. However, there are differences in the turnover of membrane protein: three vesicle protein bands and one junctional-membrane protein band are significantly more highly labeled, and one junctional-membrane protein is significantly less highly labeled by 3H-leucine in samples from tolerant rats. Of these, the two junctional-membrane proteins can be tentatively identified as components of the post synaptic densities, and one of the vesicle proteins as tubulin.

Effects of delta and mu opiopeptides on the turnover and release of dopamine in rat striatum.

Two mu and two delta opiopeptides were administered intracisternally and morphine was administered systemically to rats. The level of dopamine (DA) and its catabolites, homovanillic acid, dihydroxyphenylacetic acid and 3-methoxytramine were measured by high-pressure liquid chromatography with electrical detector in rat striatum to determine: 1) whether opioids alter the release of DA from striatal neuron (which would be indicated by changes in the level of 3-methoxytramine, the extraneuronal catabolite) and 2) whether delta or mu ligands have a greater effect on DA turnover. We found that the levels of 3-methoxytramine did not rise in response to the administration of any opiopeptide or morphine. However, mu opiopeptides produced a small but significant decrease in these levels, indicating that there was no increase, but instead a slight decrease in DA release. The delta opiopeptides produced larger increases in homovanillic acid and dihydroxyphenylacetic acid than the mu ligands, indicating that delta ligands are more effective on an equidose basis in increasing the turnover of striatal DA. The opiopeptides were also tested for pharmacological activity at the same dose (3 micrograms/rat). All four peptides were effective in reducing locomotor activity and producing analgesia. One peptide, Tyr-d-Ala-Gly-N-Mephe-Met-O-ol, also produced catalepsy. There was no segregation of these two behavioral responses according to ligand specificity. Morphine acted like a delta ligand in affecting DA turnover.

The role of calmodulin in opioid-induced changes in the phosphorylation of rat striatal synaptic membrane proteins.

Chronic morphine treatment of rats decreased the level of phosphorylation of synaptic membrane proteins of the striatum assayed in vitro. Although the patterns of phosphorylated proteins separated on SDS-gel electrophoresis from morphine-tolerant rats resembled patterns produced by lowering Ca2+ levels in the assay, supplementation of the protein kinase assay with Ca2+ and its binding protein, calmodulin, did not restore full kinase activity. The addition of methadone or etorphine to the protein kinase in vitro however, was able to block the Ca2+-calmodulin stimulation of phosphorylation in both synaptic membranes and intact synaptosomes. These data suggest that opioids produce an irreversible (or slowly reversible) defect in the Ca2+-dependent protein kinase system of striatal membranes.

The effect of morphine on the endogenous phosphorylation of synaptic plasma membrane proteins of rat striatum.

The synaptic plasma membrane (SPM) fraction was prepared from rat striata and assayed for endogenous phosphorylation in vitro. After the separation of membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the incorporation of phosphate into specific membrane proteins was analyzed by gel slicing and autoradiographic techniques. Phosphate was incorporated into several SPM protein bands, two of which were regulated by cyclic adenosine 3':5' monophosphate. As a consequence of the chronic (in vivo) administration of morphine, the phosphorylation of two protein bands, designated protein II (MW 48,000-52,000) and protein III (MW 11,000-18,000), was reduced by approximately 20 and 50%, respectively; the phosphorylation of the former proteins was regulated by cyclic adenosine monophosphate whereas the phosphorylation of the latter proteins was not. The incorporation of phosphate into these same two protein bands was similar to control values when the membranes were obtained from rats undergoing naloxone-precipitated withdrawal. Morphine, in vitro, did not alter the phosphorylation of any SPM proteins in the assay, although at a high concentration morphine did antagonize the calcium-stimulated phosphorylation of one or more proteins in band II. The development of tolerance to and dependence on opiates may include changes in the phosphorylation of specific SPM proteins.

The specificity of binding of the narcotic agonist etorphine in synaptic membranes of rat brain in vivo.

When 3H-etorphine was administered to rats in a pharmacologically effective dose (0.75 mug/kg intracisternally), the labeled drug was concentrated in synaptic membrane fractions isolated from the brains of rats killed 10 min after etorphine injection. Pretreatment of the animals with the narcotic antagonists naloxone, diprenorphine or l-cyclorphan, blocked the pharmacological responses to etorphine and reduced 3H-etorphine binding in the membrane fractions. The differences between 3H-etorphine bound in synaptic membranes of rats treated with d-cyclorphan (inactive isomer) and l-cyclorphan (active antagonist) were in the same range as the reductions in etorphine binding in antagonist-treated rats, indicating that stereospecific and pharmacologically-specific binding sites in synaptic membranes in vivo were of the same magnitude: about 0.04 pmol/g brain.