Alteration of alpha and muscarinic receptors in rat brain and heart following chronic nicotine treatment

Adrenergic and muscarinic binding sites in 4 brain regions (cerebral cortex, corpus striatum, hypothalamus/thalamus and brainstem) and in heart ventricles were measured in rats chronically treated with nicotine added to the drinking water in doses ranging from 6 to 8 mg/kg/day, for 4 weeks. Control rats received only tap water. The nicotine treatment led to increases in the specific binding of both [³H]prazosin and [³H]clonidine in the cerebral cortex. An increase in [³H]prazosin binding was also observed in the hypothalamus/thalamus of nicotine-treated rats. These changes were all due to an increase of about 23% in B_max. In the brainstem and heart left ventricle, respectively, an increase and a decrease in the affinity of [³H]quinuclidinyl benzilate binding were observed. There were no changes of the binding parameters for the 3 radioligands in other regions tested, and no alteration of [³H]dihydroalprenolol binding was detected in any region examined. These results indicate that chronic administration of nicotine causes an increase in the density of α₁-and α₂-binding sites in some brain regions and reciprocal changes of the affinity of muscarinic binding sites in the brain and in the heart.     

Modulation of tyrosine hydroxylase gene expression in the rat adrenal gland by exercise: effects of age.

Both aging and exercise are associated with alterations in circulating levels of catecholamines. To determine the interactions of age and exercise on tyrosine hydroxylase (TH) activity and TH mRNA, Fischer-344 female rats aged 5 months (young) and 25 months (old) were trained by treadmill running for 10 weeks. The elevation in maximum oxygen consumption in both groups was equivalent following exercise, indicating that training had occurred. In control rats, both TH activity and TH mRNA were greater in the older groups when compared with the younger animals. In young rats, exercise decreased TH activity by 25% and TH mRNA by 27%. In older rats, exercise was not associated with a decrease in TH activity and TH mRNA. Choline acetyltransferase activity (ChAT) was decreased and glutamic acid decarboxylase activity (GAD) was increased by exercise in young rats. The decrease in ChAT activity and increase in GAD activity suggest that trans-synaptic mechanisms play a role in the exercise-induced alteration of TH gene expression. Neither ChAT nor GAD was altered by exercise in older groups. Our data suggest that the previously reported diminution in catecholamines associated with exercise may be due to a decrease in TH mRNA and a resulting decrease in TH activity. There was no effect of exercise in the old rats, supporting previous observations that the plasticity of the sympathoadrenal system diminishes with age.     

Different effects of insulin and 2-deoxy- -glucose administration on tyrosine hydroxylase gene expression in the locus coeruleus and the adrenal medulla in rats

The major brain norepinephrinergic nucleus, locus coeruleus, is an important integrating element of extero- and interoceptive stimuli in organisms facing different physiological challenges. We investigated the effects of single and repeated (seven times) exposure to immobilization stress (120 min daily), insulin (5 IU/kg, i.p. daily) or 2-deoxy- -glucose (500 mg/kg, i.p. daily) administration on tyrosine hydroxylase (TH) mRNA levels, the rate-limiting enzyme in catecholamine biosynthesis, by in situ hybridization in locus coeruleus and by Northern blot analysis in the adrenal medulla of rats. Both the single and repeated immobilization caused a significant increase in TH mRNA levels in the locus coeruleus (1.5–2-fold; p < 0.05) and in the adrenal medulla (about 4-fold; p < 0.05) when compared with unstressed controls. Hypoglycemia induced by a single or repeated insulin administration led to about fourfold (p < 0.01) elevation in adrenal medullary TH mRNA levels, whereas TH mRNA in locus coeruleus remained unchanged when compared with saline-treated controls. In contrast to the effect of insulin-induced hypoglycemia, cellular glucoprivation caused by a single or repeated 2-deoxy- -glucose administration significantly elevated TH mRNA levels in both the adrenal medulla (fourfold; p < 0.01) and the locus coeruleus (twofold; p < 0.01). Our data suggest that in contrast to immobilization or cellular glucoprivation caused by 2-deoxy- -glucose administration, insulin-induced hypoglycemia is not a specific or quantitatively sufficient stimulus for induction of TH gene expression in the locus coeruleus, although all these stressors strongly activate the process in the adrenal medulla.     

Influence of long-term hypoxia on tyrosine hydroxylase in the rat carotid body and adrenal gland.

Tyrosine hydroxylase (TH) protein was measured in the carotid body and adrenal gland of rats exposed to normobaric hypoxia (10% O2 in nitrogen) for 3, 7, 14 or 22 days. After 22 days of hypoxia, a gradual increase in TH protein content was observed in the carotid body and in the adrenal gland, reaching 168% and 148% of the normoxic controls, respectively. To determine if the increase in TH protein content in the carotid body could alter catecholamine biosynthesis, in vitro TH activity and catecholamine turnover were measured in rats submitted to hypoxia for 14 days. TH activity was increased by 11.2-fold, while the turnover of dopamine and norepinephrine was increased by 14.8- and 5.4-fold, respectively. The data show that long-term hypoxia exerts a stimulatory influence on TH protein in the carotid body and adrenal gland in addition to an elevation in dopamine and norepinephrine biosynthesis in the carotid body.     

Sensitive non-radioisotopic in situ hybridization histochemistry: demonstration of tyrosine hydroxylase gene expression in rat brain and adrenal

An alkaline phosphatase-labelled anti-sense oligonucleotide probe specific for tyrosine hydroxylase (TOH) mRNA has been used for visualisation of TOH mRNA in the rat brain and adrenal gland. Both ribonuclease pre-treatment and the use of excess non-labelled probe abolished the specific hybridization signal. Furthermore the TOH mRNA-positive signal was only found in cells known from earlier studies to react with anti-TOH antibodies. To determine if the alkaline phosphatase-labelled probe could be used in a semiquantitative manner for measurement of the density of TOH mRNA signal, we used reserpine pre-treatment which induces TOH mRNA expression. The results revealed a significant increase in TOH mRNA signal in locus coeruleus and substantia nigra neurons, and in adrenal medulla chromaffin cells. The increased signal in these areas agreed with the increase in TOH mRNA signal previously observed by Northern analysis and suggests that this type of alkaline phosphatase-labelled probe allows sensitive detection of changes in TOH gene expression.     

Effect of chronic cold exposure on tyrosine hydroxylase mRNA in rat adrenal gland

The effect of chronic stress on the levels of tyrosine hydroxylase (TH) RNA in rat adrenal gland was investigated by RNA-DNA hybridization using a cloned TH cDNA probe. Results of dot-blot hybridization experiments and northern analysis demonstrate that exposure of animals to cold for 1 week results in a 4-5-fold increase in the relative abundance of TH mRNA. This increase in TH mRNA level may underlie the increase in adrenal TH activity that is known to occur when rats are exposed to such cold stress.     

Nicotinic stimulation of catecholamine synthesis and tyrosine hydroxylase phosphorylation in cervine adrenal medullary chromaffin cells

The synthesis and secretion of catecholamines by the adrenal medulla is of major importance in the stress response. Tyrosine hydroxylase, the rate-limiting enzyme for catecholamine biosynthesis, has been extensively studied in adrenal medullary chromaffin cells from a number of species. Cervine chromaffin cells are of interest because the deer is known to be a relatively stress-prone reactive species. We report the first characterisation of tyrosine hydroxylase regulation in cervine chromaffin cells. Nicotinic receptor activation resulted in a time- and concentration-dependent increase in catecholamine synthesis, which was significantly reduced by the extracellular signal-regulated kinase (ERK)1/2 signalling pathway inhibitor PD98059 and the calcium/calmodulin protein kinase II inhibitor KN-93, but not by H89 or bisindolylmaleimide I, inhibitors of protein kinase A and C, respectively. Nicotinic stimulation also increased the phosphorylation of ERK1/2 and tyrosine hydroxylase. This latter response occurred on serine residues 19, 31 and 40 of the enzyme. The nicotinic-induced phosphorylation of ERK1/2 and serine 31 of tyrosine hydroxylase was suppressed by PD98059 but not bisindolylmaleimide I. These data indicate that nicotinic stimulation of tyrosine hydroxylase involves the phosphorylation of serine 31 via an ERK1/2-dependent, protein kinase C-independent pathway. Protein kinase C activation by phorbol 12-myristate 13-acetate also caused an ERK1/2-dependent increase in the serine 31 phosphorylation of tyrosine hydroxylase but, in contrast to the nicotinic response, was not accompanied by an increase in enzyme activity. Thus, ERK1/2-mediated serine 31 phosphorylation of tyrosine hydroxylase appears necessary but not sufficient for nicotinic activation of catecholamine synthesis in cervine chromaffin cells. These data present potentially important similarities and differences between the regulation of catecholamine synthesis in cervine and the more widely studied bovine adrenal medulla.     

Modulation of tyrosine hydroxylase gene expression in rat brain and adrenals by exposure to cold

The long-term changes in tyrosine hydroxylase (TH) activity induced by chronic exposure to cold in brain noradrenergic neurons of the locus coeruleus (LC) were analyzed and compared to those measured in a peripheral tissue such as adrenals. This analysis was made possible at the level of one single tissue corresponding to one animal by the use of sensitive methods that allow assay of TH activity, protein, and mRNA levels in parallel from the same homogenate. The three parameters were measured in brain structures and adrenals of rats maintained at 4 degrees C during 4 days and were compared to those of control animals kept at normal housing temperature (22 degrees C). LC of rats exposed to cold contained 200% more TH mRNA than controls. The amount of TH protein in this area rose to as much as 164% that of controls. Similarly, the activity of the enzyme increased to 140% of the normal value. Thus, these observations show that 1) the increase in TH mRNA was much higher than the increase in protein levels, and that 2) the newly synthesized molecules have about the same activity as that present under normal conditions. In contrast to the LC, no variation of these parameters was observed in the substantia nigra. In the adrenals, the variations in the different parameters were qualitatively similar to that observed in the LC, although they were quantitatively higher: TH mRNA, TH protein, and TH activity levels were respectively 330%, 182%, and 167% that of control adrenals. Altogether, these results demonstrate that exposure to cold induces an alteration in TH synthesis in brain noradrenergic neurons as well as in adrenals.     

Metyrapone-induced glucocorticoid depletion modulates tyrosine hydroxylase and phenylethanolamine N-methyltransferase gene expression in the rat adrenal gland by a noncholinergic transsynaptic activation

The hypothalamic corticotropin-releasing hormone system and the sympathetic nervous system are anatomically and functionally interconnected and hormones of the hypothalamic-pituitary-adrenocortical axis contribute to the regulation of catecholaminergic systems. To investigate the role of glucocorticoids on activity of the adrenal gland, we analysed plasma and adrenal catecholamines, tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) mRNA expression in rats injected with metyrapone or dexamethasone. Metyrapone-treated rats had significantly lower epinephrine and higher norepinephrine production than control rats. Metyrapone increased TH protein synthesis and TH mRNA expression whereas its administration did not affect PNMT mRNA expression. Dexamethasone restored plasma and adrenal epinephrine concentrations and increased PNMT mRNA levels, which is consistent with an absolute requirement of glucocorticoids for PNMT expression. Adrenal denervation completely abolished the metyrapone-induced TH mRNA expression. Blockage of cholinergic neurotransmission by nicotinic or muscarinic receptor antagonists did not prevent the metyrapone-induced rise in TH mRNA. Finally, pituitary adenylate cyclase activating polypeptide (PACAP) adrenal content was not affected by metyrapone. These results provide evidence that metyrapone-induced corticosterone depletion elicits transsynaptic TH activation, implying noncholinergic neurotransmission. This may involve neuropeptides other than PACAP.     

Effects of desipramine treatment on tyrosine hydroxylase gene expression in cultured neuroblastoma cells and rat brain tissue

Activity and expression of tyrosine hydroxylase (TH), the rate-limiting enzyme for catecholamine synthesis, are modified in response to antidepressant-treatment. We examined effects of the selective norepinephrine-transporter (NET) inhibitor antidepressant desipramine (DMI) on expression of TH in human neuroblastoma cells (SK-N-BE[2]M17) and in rat brain regions. TH mRNA levels were determined by Northern blot in vitro, and by in situ hybridization ex vivo; TH protein levels were measured by western blot. Brief exposure of neuroblastoma cells to 0 vs. 5, 50, or 500 nM of DMI had little effect on TH mRNA levels, but exposure to 50 and 500 nM DMI for 14 days increased the mRNA by up to 72%, with a continuous rise from 3 to 14 days of exposure to 500 nM DMI. In contrast, 500 nM DMI led to an initial slight increase, followed by a continuous and sustained decrease in TH protein level by up to 53%, from day 3 to day 14. Daily treatment of rats with DMI (10 mg/kg, i.p.) for 3 or 14 days significantly increased postmortem cerebral TH mRNA in the locus coeruleus (LC) area by 47-68%. Again, TH protein concentrations in LC decreased at 3 and 14 days, by 25-40%, with transient significant reduction in amygdala tissue after 3 days of treatment that were not sustained. These findings indicate that DMI exerts complex, typically opposite and perhaps compensatory, gradually evolving effects on the expression of TH protein (decreases) and its message (increases), possibly in response to increased synaptic availability of NE.     

Inhibition of cholinesterase elicits muscarinic receptor-mediated synaptic transmission in the rat adrenal medulla

To determine the role of acetylcholinesterase in cholinergic synaptic transmission in the adrenal medulla in vivo, we applied a dialysis technique to the adrenal medulla of anesthetized rats and examined the effect of acetylcholinesterase inhibitor on the contribution of nicotinic and muscarinic receptors to catecholamine release. Exogenous acetylcholine-induced epinephrine release was inhibited by atropine (a muscarinic receptor antagonist) as well as hexamethonium (a nicotinic receptor antagonist). Endogenous acetylcholine (nerve stimulation)-induced epinephrine release was inhibited by hexamethonium but not atropine. In the presence of neostigmine (an acetylcholinesterase inhibitor), both exogenous and endogenous acetylcholine-induced catecholamine release was enhanced. In either case, epinephrine release was inhibited by atropine as well as hexamethonium. In the presence of eserine (another acetylcholinesterase inhibitor), endogenous acetylcholine-induced epinephrine release was also inhibited by atropine. Exogenous or endogenous acetylcholine-induced norepinephrine release was primarily inhibited by hexamethonium regardless of whether neostigmine was absent or present. In the rat adrenal medulla, the inhibition of acetylcholinesterase not only enhanced cholinergic synaptic transmission but also elicited muscarinic receptor-mediated synaptic transmission for epinephrine release.     

Regional circadian variation of acetylcholine muscarinic receptors in the rat brain

The level of binding of a labeled acetylcholine muscarinic antagonist (quinuclidinyl benzilate) to different cerebral membranes has been measured. Of the regions examined, circadian rhythmicity of binding could only be detected significantly in the hippocampus and the hypothalamus and not in the cerebral cortex, striatum, or cerebellum.     

Adolescent nicotine treatment changes the response of acetylcholine systems to subsequent nicotine administration in adulthood

Nicotine alters the developmental trajectory of acetylcholine (ACh) systems in the immature brain, with vulnerability extending from fetal stages through adolescence. We administered nicotine to adolescent rats (postnatal days PN30–47) and then examined the subsequent response to nicotine given in adulthood (PN90–107), simulating plasma levels in smokers, and performing evaluations during nicotine treatment (PN105) and withdrawal (PN110, PN120 and PN130), as well as assessing persistent changes at 6 months of age (PN180). We measured nicotinic acetylcholine receptor (nAChR) binding, choline acetyltransferase (ChAT) activity, a marker for ACh terminals, and hemicholinium-3 (HC3) binding to the choline transporter, an index of ACh presynaptic activity. By itself, adolescent nicotine exposure evoked sex-selective deficits in cerebrocortical HC3 binding while elevating ChAT in young adulthood in striatum and midbrain. Nicotine given in adulthood produced profound nAChR upregulation lasting 2 weeks after discontinuing treatment, and decrements in cerebrocortical and striatal HC3 binding emerged during withdrawal, indicative of reduced ACh synaptic activity. For all three parameters, adolescent nicotine altered the responses to nicotine given in adulthood, producing both sensitization and desensitization that depended on sex and brain region, effects that parallel the disparate behavioral outcomes reported for these treatments. The interaction seen here for the impact of adolescent nicotine exposure on adult nicotine responses was substantially greater than that found previously for the effects of prenatal nicotine exposure on adult responses. Our findings thus reinforce the importance of adolescence as a critical period in which the future responsiveness to nicotine is programmed.     

Decreased expression and impaired function of muscarinic acetylcholine receptors in the rat hippocampus following transient forebrain ischemia

In this study, we investigated whether transient cerebral ischemia affects the function and molecular expression of specific muscarinic cholinergic receptors. Our results show that in contrast to the GABA-B and A1 adenosine receptor systems, the ability of muscarinic receptors to attenuate evoked excitatory responses at vulnerable CA1 synapses is significantly attenuated by 18 h following reperfusion. This attenuation in efficacy was restricted to the vulnerable CA1 subfield, as no significant change in muscarinic receptor-mediated attenuation of evoked responsiveness was observed within post-ischemic dentate granule cell synapses. Expression analysis revealed that the mRNA and immunoreactive protein levels for individual types of muscarinic receptors respond differently and uniquely to transient cerebral ischemia insult. Of particular interest is the m4 subtype of receptor, whose mRNA and protein expression levels were significantly diminished within the hippocampus by 12 and 24 h following reperfusion, respectively. As the m4 muscarinic receptor localizes to presynaptic terminals within the hippocampus, a decrease in its expression could account for the impaired functional responsiveness of the muscarinic receptor system following ischemic insult. Taken together, these results demonstrate that transient forebrain ischemia leads to dynamic alterations in the gene expression, protein prevalence, and functionality of muscarinic receptors in the post-ischemic hippocampus at times preceding the degeneration of the vulnerable neurons.