Role of opiates in striatal D-1 dopamine receptor supersensitivity induced by chronic L-dopa treatment

Repeated administration of L-dihydroxyphenylalanine (L-dopa) to rats lesioned with monolateral intranigral injections of 6-hydroxydopamine counteracted the increased density of striatal [3H]spiroperidol binding sites induced by the lesion. On the contrary, the treatment with L-DOPA further enhanced the hypersensitivity of adenylate cyclase to dopamine stimulation that follows striatal denervation. In addition, the apomorphine-induced rotations were strongly potentiated. The latter effect was antagonized by morphine given acutely shortly before the dopamine agonist. On the other hand, the efficacy of [D-Ala2]-methionine enkephalinamide to inhibit striatal adenylate cyclase was decreased in 6-hydroxydopamine-lesioned rats chronically treated with L-dopa. Moreover, in these animals, when naltrexone was given chronically together with L-dopa, the supersensitivity of the enzyme to dopamine stimulation did not develop. Finally, in 6-hydroxydopamine-lesioned rats, chronic morphine, similarly to L-dopa, further enhanced the responses of adenylate cyclase to dopamine stimulation. These data suggest that prolonged indirect activation of striatal opiate receptors and their consequent desensitization could be among the causes of the hyperactivity of D-1 dopamine receptors that follows chronic L-dopa treatment.     

Repeated amphetamine pretreatment alters the responsiveness of striatal dopamine-stimulated adenylate cyclase to amphetamine-induced desensitization

The repeated daily administration of moderate doses of amphetamine results in an augmentation of the behavioral response to subsequent amphetamine challenge. One feature of the augmentation is a shift in the type of perseverative behaviors to those generally associated with higher acute doses of the drug. Consistent with these observations, rats pretreated with six daily injections of amphetamine (3 mg/kg) exhibited primarily oral stereotypies to a challenge dose of 2.5 mg/kg of amphetamine, whereas control animals exhibited focused sniffing and repetitive head movements. Previously we found that the acute administration of amphetamine or methylphenidate only at doses which induce oral stereotypies promotes a rapid desensitization of striatal dopamine-stimulated adenylate cyclase. We therefore examined the effects of repeated amphetamine pretreatment on this index of D1 dopamine receptors. The administration of 2.5 mg/kg of amphetamine produced a 2-fold shift to the right in the concentration-response curve for dopamine-stimulated adenylate cyclase in animals pretreated with amphetamine, but not in saline pretreated controls. No effect of the chronic amphetamine pretreatment on dopamine stimulated cyclase in the absence of amphetamine challenge was observed. The binding of [3H]cis-flupenthixol to striatal D1 dopamine receptors was not affected by acute or chronic amphetamine. These results suggest a relationship between stimulant-induced desensitization of striatal D1 dopamine receptors and the induction of oral stereotypies.     

Effects of ethanol administration and withdrawal on neurotransmitter receptor systems in C57 mice

C57BL/6 mice were treated with 7% (v/v) ethanol in a Bio-Serve liquid diet for 7 days. Some animals were then allowed to withdraw from ethanol for a period of 24 hr. The severity of the ethanol withdrawal was assessed by monitoring behavioral changes and by quantitating the decrease in body temperature that occurred during the first 16 hr of withdrawal. Animals withdrawn from ethanol for 24 hr showed a decreased hypothermic response to apomorphine suggesting that changes in dopaminergic systems had occurred. This possibility was further examined in homogenates of striatum by measuring dopamine-stimulated adenylate cyclase activity and the binding of [3H]spiroperidol. However, there were no changes observed in either basal- or dopamine-stimulated adenylate cyclase activity or in the density or affinity or receptors for [3H]spiroperidol. The affinity of apomorphine for the dopamine receptor was also unchanged. In other experiments, alpha and beta adrenergic receptor-mediated increases in cyclic AMP accumulation were assessed in slices of cerebral cortex. There was no change in cyclic AMP accumulation due to either alpha or beta adrenergic receptors. There was, however, a significant decrease in the density of beta adrenergic receptors in both the ethanol-treated mice and in the withdrawn mice. This decrease was restricted to the beta-2 receptor subtype with no change being observed in the density of beta-1 adrenergic receptors. Ethanol administration was also associated with a significant increase in the density of muscarinic cholinergic receptors in the hippocampus and cerebral cortex. The effect was not observed in animals allowed to withdraw for 24 hr.     

Age dependence of adenylate cyclase in guinea pig ureter homogenate

Adenylate cyclase activity was measured in ureteral homogenates from guinea pigs of five age groups, ranging from neonates to older adults (retired breeders). Isoproterenol stimulation of adenylate cyclase activity is lost completely in ureters from 90 day and older animals. Forskolin activation also declines rapidly with increasing age; there is a significant (39%) decrease in activation at 30 days when compared to 10-day values with a further decline at 90 days. At 90 days, NaF, GTP and 5'-guanylimido-diphosphate stimulation of adenylate cyclase decreases by only 1, 9 and 23%, respectively, from the 10-day values. Prostaglandin E1 stimulation over 5'-guanylimido-diphosphate control does not decrease with age. 5'-Guanylimido-diphosphate in the presence of ethanol, activates adenylate cyclase to a greater extent in the older animals. These data may suggest that the decreased ability to activate adenylate cyclase with age may be due to a loss of the efficiency of coupling in the more rigid aged membranes. Other explanations include loss of catalytic function with age. The loss of adenylate cyclase activity in aging correlates well with our previously reported study showing that isoproterenol causes a greater decrease in contractility of ureters from younger than from older guinea pigs.     

Desensitization of adenosine and dopamine receptors in rat brain after treatment with adenosine analogs

Maximally tolerated doses of N6-[(R)-1-methyl-2-phenylethyl] adenosine (0.50 nmol/hr/2 wk), 5'-N-ethylcarboxamide adenosine (NECA, 0.04 nmol/hr/2 wk) or deoxycoformycin (5 nmol/hr/1 wk) were administered i.c.v. to rats using mini-osmotic pumps. Adenosine receptor function was subsequently assayed using both ligand binding and adenylate cyclase assays. Binding to A1 receptors was quantitated using [3H]N6-[(R)-1-methyl-2-phenylethyl]adenosine, a selective agonist ligand at A1 receptors. Differences in the binding of this ligand and that of [3H]NECA, which binds to A1 and A2 receptors with similar affinities, were used to quantitate A2 receptors. None of the treatments affected A1 receptor function as assessed by both ligand binding and adenylate cyclase assays. A2 receptor binding and A2 receptor-mediated stimulation of adenylate cyclase were blunted in striatal membranes from NECA- and deoxycoformycin-treated rats but unaffected in striatal membranes from N6-[(R]-1-methyl-2-phenylethyl]adenosine-treated rats. All three pretreatments attenuated D1 dopamine receptor-mediated stimulation of adenylate cyclase in striatal membranes. These results suggest that 1) the A2 adenosine receptor system is susceptible to desensitization and 2) different mechanisms are involved in the NECA- and deoxycoformycin-induced desensitization of A2 adenosine receptor and D1 dopamine receptor systems. It is suggested that the D1 dopamine receptor desensitization is, in fact, due to the tonic stimulation of adenosine A1 receptors.     

Altered behavioral response to a D2 agonist, LY141865, in spontaneously hypertensive rats exhibiting biochemical and endocrine responses similar to those in normotensive rats

LY141865, a dopamine agonist selective for D2 dopamine receptors, caused hypomotility at low doses (0.01 and 0.1 mg/kg) and hypermotility at high doses (1 and 10 mg/kg) after i.p. injection into normotensive rats (WKY). In age-matched hypertensive rats (SHR), LY141865 caused hypomotility (not hypermotility) at all of these doses. The basal locomotor activity was higher in SHR than in WKY, but striatal concentrations of dopamine and its metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, and of serotonin and its metabolite, 5-hydroxyindoleacetic acid, were not different between the two groups of rats. The specific binding of a dopamine receptor radioligand, tritiated pergolide, in striatum and mesolimbic regions, did not differ in SHR compared with WKY. In contrast to the lack of locomotor stimulation in SHR, other dopaminergic responses to LY141865 occurred in SHR as well as WKY. For instance, LY141865 decreased striatal and mesolimbic concentrations of 3,4-dihydroxyphenylacetic acid and homovanillic acid, increased striatal and mesolimbic concentrations of acetylcholine, decreased hypothalamic concentrations of epinephrine, increased serum corticosterone concentration and decreased serum prolactin concentration in SHR as in WKY. Because radioligand-labeled dopamine receptors and several LY141865 responses mediated by dopamine receptors did not differ appreciably in SHR compared with WKY, the lack of behavioral hypermotility in response to LY141865 in SHR may be due to abnormalities in some synaptic mechanisms beyond dopamine receptor activation that are involved in the expression of increased locomotion in response to the dopamine agonist.     

Effect of chronic haloperidol treatment on stimulated synaptic overflow of dopamine in the rat striatum.

In vivo voltammetry was used to assess the change in stimulated striatal dopamine overflow in response to various treatments with the dopamine receptor antagonist haloperidol. Dopamine overflow was induced with stimulating electrodes implanted in the medial forebrain bundle of anesthetized rats while dopamine concentrations were monitored with Nafion-coated, carbon-fiber microelectrodes implanted in the striatum. An acute challenge of haloperidol (0.5 mg kg-1, i.p.) given to naive animals caused stimulated overflow to increase at all stimulation frequencies (10-60 Hz), with the greatest change, 5-fold, occurring at 30 Hz. These results have been compared to those obtained in a different group of rats given daily injections of haloperidol (0.5 mg kg-1, s.c.) for 30 consecutive days. On the 30th day, dopamine striatal tissue levels and uptake kinetics were not altered by this treatment, but 3,4-dihydroxyphenylacetic acid tissue levels were elevated almost 2-fold. A challenge dose of haloperidol (0.5 mg kg-1, i.p.) administered to the animals treated with chronic haloperidol did not elicit a change in stimulated dopamine overflow. In two other groups, rats were withdrawn from 30-day haloperidol treatment for 3 days or 14 days before experimentation. Stimulated dopamine overflow concentrations in both groups were not significantly different from naive animals. When the withdrawn animals were given a haloperidol challenge (0.5 mg kg-1, i.p.), 15- and 12-fold increases in overflow for 3-day and 14-day withdrawal groups, respectively, were observed at a stimulation frequency of 30 Hz. Thus, chronic treatment with haloperidol induces long-lasting effects on the capacity of dopamine receptors to modulate dopamine release.     

Chronic ethanol exposure of PC 12 cells alters adenylate cyclase activity and intracellular cyclic AMP content

Effects of chronic ethanol exposure on adenylate cyclase activity and intracellular cyclic AMP content were investigated in PC 12 cells. Stimulation of cyclic AMP content by 2-chloroadenosine (2-CADO) and forskolin were reduced after a 4-day exposure of the cells to 150 mM ethanol. Although chronic ethanol treatment did not alter 2-CADO- and NaF-stimulated adenylate cyclase activities in a cell-free preparation, 2-CADO-, forskolin, and NaF-stimulated adenylate cyclase activities were reduced when assays were carried out in saponin-treated cells. Chronic ethanol exposure also increased the ability of in vitro ethanol to stimulate adenylate cyclase activity and cyclic AMP content. The in vitro addition of tert-butanol caused a dose-dependent increase in adenylate cyclase activity, and a 4-day treatment of PC 12 cells with 50 mM tert-butanol reduced 2-CADO- and forskolin-stimulated cyclic AMP contents. Chronic treatment with tert-butanol, however, did not alter the stimulatory effects of in vitro ethanol. Similarly, a 4-day exposure to 125 mM ethanol using serum-free defined media decreased 2-CADO- and forskolin-stimulated cyclic AMP content without changing the stimulatory effects of in vitro ethanol. The increased sensitivity of adenylate cyclase to in vitro ethanol also was not observed when chronic ethanol treatment was carried out with media containing delipidated serum. The increased sensitivity to ethanol, however, was restored when the chronic ethanol treatment was carried out with serum-free defined media supplemented with serum lipoproteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alterations in opiate receptor function after chronic ethanol exposure

The dose-response curve for morphine-induced stimulation of striatal dopamine metabolism was shifted to the right in mice which had been withdrawn for 24 hours after chronic consumption of an ethanol-containing liquid diet. The apparent ED50 for morphine was increased by 33% in ethanol-treated mice. Concomitant with the shift in the dose-response curve, the affinity for dihydromorphine of the high-affinity caudate morphine receptor was decreased in ethanol-treated mice. The change in receptor properties after ethanol treatment included a decreased sensitivity of the receptor to the effects of sodium ion on morphine binding. The results suggest: 1) that the effect of morphine on dopamine metabolism in the mouse striatum is, at least in part, mediated by receptors that exhibit a high affinity for dihydromorphine: and 2) that ethanol treatment and withdrawal may induce specific changes in these particular opiate receptors.     

K+ channel and adenylate cyclase involvement in regulation of Ca2+-evoked release of [3H]dopamine from synaptosomes

In superfused striatal synaptosomes, previously unexposed to Ca2+ during isolation and superfusion, 1.25 mM Ca2+ evokes the release of [3H]dopamine. This Ca2+-evoked release is produced without elevating K+ (4.5 mM) before or after Ca2+ exposure, can be blocked by the Na+ channel antagonist tetrodotoxin, and modulated by dopamine (D2) receptor agonists and antagonists. We now present evidence that functional K+ channels regulate Ca2+-evoked [3H]dopamine release and may be necessary for the dopamine (D2) modulation of this release. The K+ channel blocker tetraethyl ammonium (TEA) could partially prevent D2 agonist (LY-171555) inhibition of Ca2+-evoked release in both olfactory tubercle and striatal synaptosomes. Another K+ channel blocker, 4-aminopyridine, also partially blocked dopamine (D2) agonist inhibition of release. When both 5 mM tetraethyl ammonium and 0.1 mM 4-aminopyridine were employed, by, dopamine (D2) inhibition of Ca2+-evoked [3H]dopamine release was prevented. However, with both K+ channel blockers present, only the initial portion of the release could be blocked by tetrodotoxin. These results are consistent with what might be expected if K+ channels were linked to dopamine (D2) receptors. In additional experiments we found that stimulation of adenylate cyclase by 1 microM forskolin with 0.25 mM 3-isobutyl-1-methylxanthine present potentiated Ca2+-evoked [3H]dopamine release but that this combination did not affect dopamine (D2) inhibition of [3H]dopamine release. Furthermore, although the protein alkylator n-ethylmaleimide could block dopamine (D2) inhibition of release, pertussis toxin, a specific inactivator of the inhibitory protein regulating adenylate cyclase, had little effect on dopamine (D2) inhibition. Therefore, dopamine (D2) inhibition of dopamine release may not be coupled to adenylate cyclase activity.     

The effect of chronic ethanol consumption and withdrawal on mu-opioid and dopamine D(1) and D(2) receptor density in Fawn-Hooded rat brain

Previous studies have implicated the dopamine and opioid systems in the induction and maintenance of ethanol consumption. This study investigated, in alcohol-preferring Fawn-Hooded (FH) rats, whether chronic free-choice ethanol consumption and subsequent withdrawal cause alterations in central mu-opioid, dopamine D(1), and D(2) receptor density using autoradiography. FH rats were given a free choice between a 5% ethanol solution and tap water (n = 25) and displayed a mean ethanol consumption of 5.6 g/kg/day. A parallel group of FH rats (n = 5) only had access to tap water. Rats were then withdrawn from ethanol for 0, 1, 2, 5, or 10 days and killed by cervical dislocation and decapitation. Increases in mu-opioid receptor density were observed in the nucleus accumbens and ventral tegmental area upon withdrawal compared with the ethanol naive group. In the lateral amygdala, binding in all withdrawal groups was significantly different from the ethanol naive FH rats, and also from the chronic ethanol rats. An increase in dopamine D(1) receptor density was observed in the substantia nigra, pars reticulata in the 5- and 10-day withdrawal groups compared with ethanol naive. Accumbal dopamine D(2) receptor density (+25-30%) increased in the 10-day withdrawal group compared with both naive and chronic ethanol groups. These findings demonstrate that the opioid and dopamine systems are susceptible to modulation by chronic ethanol consumption and withdrawal in the FH rat. Furthermore, although acute ethanol withdrawal results in modulation of mu-opioid receptors, effects on dopamine receptors are delayed and only become evident 5 to 10 days after withdrawal.     

Repeated amphetamine administration alters the interaction between D1-stimulated adenylyl cyclase activity and calmodulin in rat striatum

The effect of repeated intermittent treatment with amphetamine on the responses of rat striatal adenylyl cyclase to the D1 dopamine receptor agonist, SKF38393 [(+-)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride], and potentiative interactions with the endogenous Ca(++)-binding protein, calmodulin, were investigated. Female Sprague-Dawley rats were treated with saline or an escalating dose regimen of amphetamine for 4 weeks and withdrawn from treatment for either 2 or 4 weeks. Thirty minutes before sacrifice, rats in both groups were given a challenge dose of either saline or 1.0 mg/kg of amphetamine. In striatal membranes from rats chronically treated with saline and withdrawn 4 weeks, calmodulin increased SKF38393-stimulated adenylyl cyclase activity by 195% over that seen in the presence of GTP alone. In contrast, this pronounced potentiative interaction was absent in rats chronically treated with amphetamine. The lack of potentiation by calmodulin was independent of length of withdrawal from the drug or amphetamine challenge. Although the repeated amphetamine treatment abolished the potentiative response to calmodulin, this treatment significantly increased the calmodulin content in the striatal cytosol by 40%. Heightened responsiveness of several dopamine-related neurochemical activities was evident after an amphetamine challenge to rats that had been treated repeatedly with amphetamine. A challenge dose of 1 mg/kg of amphetamine decreased SKF38393-stimulated adenylyl cyclase activity in amphetamine-treated but not saline-treated rats. The degree of desensitization induced by amphetamine challenge was heightened with increased length of withdrawal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological differences between the D-2 autoreceptor and the D-1 dopamine receptor in rabbit retina

The effect of dopamine receptor agonists and antagonists was studied on the calcium-dependent release of [3H]dopamine elicited by field stimulation at 3 Hz for a duration of 1 min (20 mA, 2 msec) from the rabbit retina in vitro and on adenylate cyclase activity in homogenates of rabbit retina. The relative order of potency of dopamine receptor agonists to inhibit the stimulation-evoked [3H]dopamine release was pergolide greater than bromocriptine greater than apomorphine greater than LY 141865 greater than N,N-di-n-propyldopamine greater than or equal to dopamine. The relative order of potencies of dopamine receptor antagonists to increase [3H]dopamine release was: S-sulpiride greater than or equal to domperidone greater than or equal to spiroperidol greater than metoclopramide greater than fluphenazine greater than or equal to R-sulpiride. alpha-Flupenthixol (0.01-1 microM) and (+)-butaclamol (0.01-1 microM) did not increase [3H]dopamine overflow when added alone, but they antagonized the concentration-dependent inhibitory effect of apomorphine (0.1-10 microM). These results suggest that the dopamine inhibitory autoreceptor involved in the modulation of dopamine release from the rabbit retina possesses the pharmacological characteristics of a D-2 dopamine receptor. Maximal stimulation by 30 microM dopamine resulted in a 3-fold increase in adenylate cyclase activity with half-maximal stimulation occurring at a concentration of 2.46 microM. Apomorphine and pergolide elicited a partial stimulation of adenylate cyclase activity. However, at low concentrations both compounds were more potent than dopamine. N,N-di-n-Propyl-dopamine was 30 times less potent than dopamine, and bromocriptine was unable to stimulate adenylate cyclase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential regulation of right and left ventricular beta-adrenergic receptors in newborn lambs with experimental cyanotic heart disease.

To determine whether chronic hypoxemia secondary to an intracardiac right-to-left shunt alters regulation of the myocardial beta-adrenergic receptor/adenylate cyclase system, we produced chronic hypoxemia in nine newborn lambs by creating right ventricular outflow obstruction and an atrial septal defect. Oxygen saturation was reduced to 65-74% for 2 wk. Eight lambs served as normoxemic controls. beta-receptor density (Bmax) and ligand affinity (KD) were determined with the radio-ligand [125I]iodocyanopindolol and adenylate cyclase activity determined during stimulation with isoproterenol, sodium fluoride (NaF), and forskolin. During chronic hypoxemia, Bmax decreased 45% (hypoxemic, 180.6 +/- 31.5 vs. control, 330.5 +/- 60.1 fmol/mg) in the left ventricle (exposed to hypoxemia alone) but was unchanged in the right ventricle (exposed to hypoxemia and pressure overload). KD was not different from control in either ventricle. Left ventricular isoproterenol-stimulated adenylate cyclase activity was decreased by 39% (30.0 +/- 4.3% increase vs. 44.1 +/- 9.5% increase) whereas right ventricular adenylate cyclase activity was unchanged. Stimulation of adenylate cyclase with NaF or forskolin was not different from control in either ventricle. Circulating epinephrine was increased fourfold whereas circulating and myocardial norepinephrine were unchanged. These data demonstrate a down-regulation of the left ventricular beta-adrenergic receptor/adenylate cyclase system during chronic hypoxemia secondary to an intracardiac right-to-left shunt.     

Cellular Action of Antidiuretic Hormone in Mice with Inherited Vasopressin-Resistant Urinary Concentrating Defects

Previous work has suggested that resistance to vasopressin in two strains of mice with nephrogenic deficiency of urinary concentration may entail a defect in the action of vasopressin at the cellular level. Several components involved in this action were therefore examined in vitro in renal medullary tissues from control mice (genotype VII +/+) and two genotypes with mild diabetes insipidus (DI +/+ nonsevere) and marked (DI +/+ severe) vasopressin-resistant concentrating defects. No significant differences were found in the affinity of adenylate cyclase for [8-arginine]-vasopressin (AVP), tested over a range of hormone concentration from 10(-10) to 10(-5) M. However, maximal stimulation of adenylate cyclase by saturating concentrations of AVP (intrinsic activity) was markedly decreased from control values in DI +/+ severe mice, and decreased to a lesser extent in DI +/+ nonsevere animals. A significant correlation was found between the activity of adenylate cyclase maximally stimulated by AVP in a given genotype, and the urine osmolality in the same animals. There were no significant differences in maximal stimulation of renal medullary adenylate cyclase in control experiments: not when stimulated nonspecifically by sodium fluoride, nor when stimulated by AVP in tissues from rats with induced water diuresis as compared to antidiuretic rats. Nor were there significant differences between VII +/+ and DI +/+ severe mice in the activity of renal cortical adenylate cyclase, either basal or when stimulated by parathyroid hormone. Furthermore, the abnormal genotypes did not differ significantly from control mice in the renal medullary activities of cyclic AMP phosphodiesterase or cyclic AMP-dependent protein kinase, nor in the content of microtubular subunits (assessed as colchicinebinding protein). The results are compatible with the view that impaired stimulation of renal medullary adenylate cyclase by vasopressin might be the sole or contributing cause of the vasopressin-resistant concentrating defect in the diseased mice; however, a causal relationship has not yet been proved.     

Ethanol enhancement of isoproterenol-stimulated melatonin and cyclic AMP release from cultured pineal glands

Norepinephrine stimulates the synthesis of melatonin in the pineal gland. The action of norepinephrine is believed to be mediated primarily by beta adrenergic receptors, and involves activation of adenylate cyclase. Ethanol, 25 to 50 mM, added to cultured pineal glands in vitro, enhanced isoproterenol-induced stimulation of cyclic AMP and melatonin production. The action of ethanol was observed only at doses of isoproterenol that produced a submaximal effect, and ethanol alone had no effect on cyclic AMP or melatonin release. Butanol, at a concentration of 2 mM, was as effective as 50 mM ethanol in increasing isoproterenol-stimulated cyclic AMP and melatonin release, indicating that the response to alcohols was not due simply to changes in osmolarity, and may reflect a hydrophobic interaction of the alcohols with the cell membrane. The effects of ethanol on pineal cyclic AMP and melatonin release were reversible after a 15-min preincubation, but not after a 2-hr preincubation, suggesting that, over a long incubation period, ethanol may sensitize the pineal beta adrenergic receptor-coupled adenylate cyclase system to isoproterenol. The findings in this study are consistent with earlier work showing that ethanol increases cerebral cortical beta adrenergic receptor-coupled adenylate cyclase activity, and demonstrate that the effect of ethanol on the receptor-effector system can result in an endocrinological response.     

Adaptive mechanisms of striatal D1 and D2 dopamine receptors in response to a prolonged reserpine treatment in mice

Mice receiving reserpine (1 mg/kg/day) during 5 days develop behavioral supersensitivity. To study the possible molecular correlates of these adaptive changes we compared binding parameters of D1 and D2 receptors and adenylate cyclase activity in striata from normal and reserpinized mice. Saturation curves using [3H]SCH 23390 showed no changes in maximum binding capacity (Bmax) or Kd of striatal D1 receptors taken from control or 5 days reserpine-treated mice. However, [3H]spiperone saturation curves showed a 31% increase in D2 receptors Bmax with no changes in Kd. Dopamine competition of [3H]SCH 23390 and [3H]spiperone binding in mouse striatum was also performed. Analysis of data by LIGAND showed that dopamine recognizes two subpopulations for D1 and for D2 receptors. The proportion of receptors in the high affinity state (D1high and D2high) were increased in reserpine-treated animals. The addition of 100 microM GTP produced a complete conversion of D1high and D2high receptors into their low-affinity states in striata from control and reserpinized mice. Five days of reserpine treatment increased basal adenylate cyclase activity of mouse striatum in the presence of Mn++ or Mg++ ions. Concentration curves with dopamine, NaF or forskolin revealed shifts to the left and higher maximum responses without changes in EC50 values in striata from reserpinized mice. Thus, a prolonged reserpine treatment produces marked changes in D1 and D2 receptors increasing the proportion of high affinity state subpopulations and the total Bmax of D2 receptors. Also, dopamine function may be enhanced through an increment of the catalytic component of striatal adenylate cyclase.     

Effect of Digoxin on Ventricular Remodeling and Responsiveness of beta-Adrenoceptors in Chronic Volume Overload

BACKGROUND: Digoxin improves baroreflex function and reduces neurohumoral activation in severe heart failure, but it is uncertain how digoxin affects ventricular remodeling and progression to left ventricular dysfunction. In addition, the effect of digoxin in in vitro beta-adrenoceptor density and function, and contractile reserve in vivo is not well understood. METHODS AND RESULTS: To study this, we compared digoxin with placebo treatment in rats with chronic volume overload induced by aortocaval fistula and in sham-operated control animals. Left ventricular end-diastolic cavity dimensions (LVDd) and wall thickness were measured weekly by in vivo transthoracic echocardiography, and left ventricular mass (LVM) and percent fractional shortening (%FS) were calculated. Six weeks after fistula creation, simultaneous echocardiographic and invasive hemodynamic evaluation at rest and in response to incremental dobutamine (1-10 μg/kg/min intravenously) were measured. Myocardial plasma membrane beta-adrenoceptor density and maximal adenylate cyclase responses (V(max)) to isoproterenol, 5'-guanylylimi dodiphosphate, and forskolin were measured in vitro. Volume overload induced progressive increases in LVDd and LVM over the 6-week study period. Percent fractional shortening at rest, and the change in %FS in response to dobutamine stress were dramatically reduced 6 weeks after fistula creation. Although 6-week fistula animals had unchanged beta-adrenoceptor density (B(max)) and binding affinity (K(d)) as compared with controls, maximal adenylate cyclase responses to stimulation in vitro (V(max)) were markedly reduced. Digoxin treatment prevented this loss of responsiveness of adenylate cyclase but did not affect beta-adrenoceptor density or affinity in vitro. Digoxin had no effect on LVDd, LVM, %FS, or the response to dobutamine infusion in vivo. CONCLUSIONS: Although digoxin prevented beta-adrenoceptor desensitization and improved in vitro myocardial adenylate cyclase response, the cardiac response to adrenergic stimulation in vivo was not significantly improved. These results suggest that the role of beta-adrenoceptor desensitization in the progression from volume overload hypertrophy to left ventricular dysfunction and heart failure may be less important than previously thought. Furthermore, although digoxin treatment did produce modest hemodynamic benefits, it did not prevent progressive remodeling in this model.     

Prostacyclin production and mediation of adenylate cyclase activity in the pulmonary artery. Alterations after prolonged hypoxia in the rat.

Prostacyclin is a critical mediator of structure and function in the pulmonary circulation, causing both the inhibition of vascular smooth muscle growth and vasodilation via the stimulation of adenylate cyclase. To examine the potential role of alterations in prostacyclin production or mechanism of action in chronic hypoxic pulmonary hypertension, we determined the effects of prolonged (7 d) in vivo hypoxia on in vitro prostacyclin synthesis and mediation of adenylate cyclase activity in rat main pulmonary arteries. In control arteries prostacyclin production exceeded that of prostaglandin (PG) E2 by 25-fold, with 42% originating from the endothelium. Studies utilizing indomethacin revealed that endogenous prostaglandins mediate at least 69% of basal adenylate cyclase activity. Prostacyclin-stimulated enzyme activity was enhanced by exogenous GTP, indicating that this is a receptor-mediated process involving G protein amplification. Comparable dose-related responses to prostacyclin and PGE2 suggest that these agents may activate a common receptor. After 7 d of in vivo hypoxia there was a 2.7-fold increase in in vitro prostacyclin production, with equivalent increases in synthesis in the endothelium and vascular smooth muscle. However, despite this increase there was no change in basal adenylate cyclase activity, and this was associated with attenuated sensitivity of the enzyme to prostacyclin stimulation. Concomitant diminution of the response to beta-adrenergic stimulation, with previously-demonstrated beta receptor downregulation and unaltered postreceptor-mediated activity, suggests that the blunted response to prostacyclin is due to receptor downregulation. Parallel studies of the thoracic aorta indicated that these changes are specific to the pulmonary artery. It is postulated that attenuation of the response of adenylate cyclase to prostacyclin may contribute to the structural changes and hypertension observed in the pulmonary vasculature of the rat with chronic hypoxia.     

Different effects of dietary chenodeoxycholic acid and ursodeoxycholic acid on colonic adenylate cyclase in the rat

The oral administration of dietary chenodeoxycholic acid (1%), but not of ursodeoxycholic acid (1%), to male Sprague Dawley rats results in a significant increase in the colonic adenylate cyclase activity without any influence on the colonic cyclic-AMP phosphodiesterase activity. No effect of chronic bile acid feeding on the response of colonic adenylate cyclase to prostaglandin E2 and vasoactive intestinal peptide is observed. These data emphasize a dependence of the cyclic-AMP adenylate cyclase activation on the chemical structure of the bile acid. This may be of pathophysiologic relevance with respect to the frequently observed diarrhea as a side effect of oral chenodeoxycholic, but not ursodeoxycholic acid therapy for cholesterol gallstone dissolution in man.