Sexually dimorphic development and binding characteristics of NMDA receptors in the brain of the platyfish.

This study investigated age- and gender-specific variations in properties of the glutamate N-methyl-d-aspartate receptor (NMDAR) in a freshwater teleost, the platyfish (Xiphophorus maculatus). Prior localization of the immunoreactive (ir)-R1 subunit of the NMDAR protein (R1) in cells of the nucleus olfactoretinalis (NOR), a primary gonadotropin-releasing hormone (GnRH)-containing brain nucleus in the platyfish, suggests that NMDAR, as in mammals, is involved in modulation of the platyfish brain-pituitary-gonad (BPG) axis. The current study shows that the number of cells in the NOR displaying ir-R1 is significantly increased in pubescent and mature female platyfish when compared to immature and senescent animals. In males, there is no significant change in ir-R1 expression in the NOR at any time in their lifespan. The affinity of the noncompetitive antagonist ((3)H)MK-801 for the NMDAR is significantly increased in pubescent females while maximum binding of ((3)H)MK-801 to the receptor reaches a significant maximum in mature females. In males, both MK-801 affinity and maximum binding remain unchanged throughout development. This is the first report of gender differences in the association of NMDA receptors with neuroendocrine brain areas during development. It is also the first report to suggest NMDA receptor involvement in the development of the BPG axis in a nonmammalian vertebrate.

Antipsychotic drug effects on glutamatergic activity.

Previous work from this laboratory indicated that some antipsychotic drugs possess unique action at N-methyl-D-aspartate (NMDA) receptors. A functional neurochemical assay showed that, at concentrations similar to those found in the cerebrospinal fluid (CSF) of schizophrenics, antipsychotic drugs augment NMDA activity while, at higher concentrations, NMDA activity is suppressed. Using similar analysis, the present paper reports that this pattern of response is also shown by the antipsychotic drugs thioridazine and chlorpromazine. In contrast, promazine, which is structurally similar to chlorpromazine but lacking both D2-effects and antipsychotic potency, had no influence on NMDA receptors. In addition, sulpiride and metoclopramide, drugs with high affinity for D2-dopamine receptors but with weak or no antipsychotic efficacy, also lack effects at the NMDA receptor. Thus, the drugs with clinical efficacy that were tested in the present and previous studies all share unique influence on NMDA receptors. Further work with other antipsychotic agents will be necessary to determine if influence on NMDA receptors contributes to antipsychotic effectiveness.

Glutamate agonist activity: implications for antipsychotic drug action and schizophrenia.

Antagonist action at dopamine D2 receptors appears to explain many, but not all of the effects of antipsychotic drugs. Because of the interactions of dopamine with glutamate, and the implication of the latter in the etiology of schizophrenia, possible effects of antipsychotic drugs on glutamate receptors were assessed in the present experiments. These studies showed that, at clinically relevant concentrations, the conventional neuroleptic haloperidol and the atypical antipsychotic clozapine had potent augmenting influences on the NMDA receptor. These data suggest that unique action at glutamate receptors may contribute to antipsychotic efficacy and emphasize the potential importance of glutamatergic dysfunction in the etiology of schizophrenia.

Taurine prevents haloperidol-induced changes in striatal neurochemistry and behavior.

Repeated daily administration of haloperidol produces changes in striatal neurochemistry (decreased dopamine synthesis, upregulation of D2 receptors) and behavior (increasing catalepsy). Coadministration of taurine greatly attenuated these neuroleptic-induced changes. Possible mechanisms of taurine's mitigating effects are its attenuating influences on glutamatergic transmission and its actions as a GABAA agonist. The possibility was discussed of adding taurine to chronic antipsychotic regimens to block the side-effects typically accompanying such therapy.

Anti-glutamatergic effects of clozapine.

Clozapine (Cz) is unique in its efficacy with treatment refractory patients and its freedom from motor side effects. The present work shows that Cz, even after dopamine depletion, suppresses responses evoked via the monosynaptic glutamatergic corticostriatal pathway. In addition, Cz is effective in displacing [3H]MK-801 from striatal homogenates. These data indicate that Cz is a glutamate antagonist. It is unclear, however, if this pharmacological action could explain Cz's lack of motor effects and it's antipsychotic potency.

Acute administration of haloperidol enhances dopaminergic transmission.

It has been demonstrated that haloperidol, by blocking presynaptic D2 receptors, enhances dopamine release. However, it is generally assumed that augmentation of dopaminergic transmission is precluded by haloperidol's postsynaptic actions. This assumption was assessed in the present experiment by testing the effects of acute haloperidol administration on the activity of the striatum. Somatosensory stimulation was used to evoke striatal field potentials in rats anesthetized with either urethane or sodium pentobarbital. Haloperidol suppressed striatal responses in a dose-dependent fashion. Enhanced dopaminergic activity, caused by i.v. cocaine or substantia nigra stimulation, similarly suppressed striatal responses. Rather than blocking the effects of cocaine in the striatum, coadministration of haloperidol was additive, causing increased suppression. Substantia nigra lesions that depleted striatal dopamine blocked the suppressive effects of haloperidol. Administration of R-(+)-8-chloro-2,3,4,5-tetrahydro-3- methyl-5-phenyl-1H-3-benzazepine-7-ol to prevent postsynaptic actions of dopamine mediated by D1 receptors did not attenuate haloperidol-induced suppression. The present data suggest that acute administration of haloperidol actually augments dopaminergic activity due to relatively strong presynaptic actions and relatively weak postsynaptic blocking effects. It is plausible that this initial increase in dopaminergic transmission may participate in producing the neural changes evoked by chronic haloperidol administration that allow this drug to have antipsychotic potency.

Clozapine's mechanisms of action: non-dopaminergic activity rather than anatomical selectivity.

The mechanisms of clozapine's unusual actions were investigated in anesthetized rats. Sensory field potentials were recorded in the nucleus accumbens, striatum and somatosensory cortex. Both haloperidol and clozapine suppressed responses in the striatum and accumbens. Clozapine's but not haloperidol's subcortical actions were unaffected by dopamine depletion. In addition, the cortical effects of the two drugs differed. These data indicate that clozapine has non-dopaminergic activity but no particular specificity for limbic basal ganglia structures. While it is known that clozapine acts as a serotonergic and cholinergic antagonist, several considerations suggest that action on still another transmitter system, possibly glutamate, needs to be investigated.

Alterations in cardiac beta-adrenoceptor responsiveness and adenylate cyclase system by congestive heart failure in dogs.

The effects of congestive heart failure on the physiological and biochemical functions of the cardiac beta-adrenoceptor-coupled adenylate cyclase system were studied in dogs with right heart failure produced by progressive pulmonary artery constriction and tricuspid avulsion. The cardiac inotropic response to dobutamine was attenuated in congestive heart failure, as determined by the right and left ventricular dP/dt responses. Adrenergic beta-receptor density, measured by [3H]dihydroalprenolol binding, was reduced in membrane fractions of the failing right ventricle, but not in the left ventricle. The functional activity of the adenylate cyclase system was studied in vitro by measuring the net cyclic AMP production following additions of isoproterenol, 5'-guanylylimidodiphosphate (Gpp(NH)p), forskolin, or manganese chloride, which act either directly on the beta-adrenergic receptors or on one of the post-receptor components of the adenylate cyclase system. Congestive heart failure reduced the net production of cyclic AMP by isoproterenol, Gpp(NH)p, and forskolin in both the right and left ventricles, but did not alter the effect of manganese chloride. Thus, beta-receptor down-regulation is chamber-specific, occurring only in the hemodynamically stressed right ventricle. In contrast, the post-receptor defect of the adenylate cyclase system occurred in both ventricles of the heart failure dogs. This decreased activation of adenylate cyclase by beta-agonists may be responsible, at least in part, for the diminished cardiac inotropic response to catecholamines in congestive heart failure.

Preserved cardiac beta-adrenergic sensitivity in early renovascular hypertension.

To determine the mechanism of blunted sympathetic reflex responses in early renovascular hypertension, we measured inotropic and chronotropic responses of the heart to beta-adrenergic stimulation in vivo and myocardial beta-adrenergic receptor number and adenylate cyclase activity in 10 dogs during an early stage of one-kidney renal hypertension. Mean aortic pressure was higher in the hypertensive dogs (152 +/- 4 mm Hg) than in eight sham-operated dogs (122 +/- 1 mm Hg; p less than 0.001), but heart rate, cardiac output, and left atrial pressure did not differ between the two groups. Blood pressure reduction with a direct-acting vasodilator, pinacidil, resulted in marked increases in heart rate (+97 +/- 12 beats/min) and rate of change of left ventricular pressure (dP/dt; +1447 +/- 367 mm Hg/sec) in normotensive dogs but only blunted heart rate (+54 +/- 12 beats/min) and minimal left ventricular dP/dt (+376 +/- 264 mm Hg/sec) responses in hypertensive dogs. In contrast, intravenously administered isoproterenol produced similar increases in heart rate and left ventricular dP/dt in the two groups. These two groups also did not differ in either left ventricular beta-adrenergic receptor number and affinity or basal, isoproterenol-stimulated, and fluoride-stimulated adenylate cyclase activity. Thus, despite blunted reflex responses to blood pressure reduction, hypertensive dogs showed neither reduction in chronotropic and inotropic responses to direct beta-adrenergic stimulation nor beta-adrenergic desensitization of the myocardium, as assessed by beta-adrenergic receptor number and adenylate cyclase activity. Blunted reflex responses in this model of early hypertension must be due to factors operating at some locus other than the beta-adrenergic receptor-adenylate cyclase complex.

Differential regulation of beta-adrenergic receptor-coupled adenylate cyclase by thyroid hormones in rat liver and heart: possible role of corticosteroids.

Thyroid hormone regulation of beta-adrenergic receptor-coupled adenylate cyclase activity was studied in rat liver and heart particulate fractions. Thyroidectomy (Tx) increased isoproterenol-stimulated cAMP accumulation in the liver and decreased it in the heart. Administration of L-thyroxine (L-T4) or L-3,3',5-triiodothyronine (L-T3) reversed these changes in both liver and heart. The changes observed in liver beta-receptor-coupled adenylate cyclase activity after Tx were similar to those reported after adrenalectomy (ADX). Thus the hypothesis was considered that these changes with altered thyroid status are produced indirectly through alteration in adrenal corticosteroids. Hydrocortisone in Tx rats decreased liver isoproterenol-stimulated adenylate cyclase activity but had no significant effect on the heart. Serum corticosterone levels were decreased significantly (by 34%) in Tx rats, as compared to euthyroid rats. Administration of L-T4 to Tx rats doubled the serum corticosterone levels. In Tx-ADX rats, L-T4 had no significant effect on liver beta-receptor-coupled adenylate cyclase. However, L-T4 significantly increased heart beta-receptor-coupled adenylate cyclase in these animals. Dexamethasone, but not deoxycorticosterone, decreased liver isoproterenol-stimulated cAMP accumulation in Tx animals to the same extent as was observed with L-T4 and hydrocortisone. Thus overall the results indicate that in the liver, as opposed to the heart, thyroid hormones regulate beta-adrenergic receptor-coupled adenylate cyclase indirectly through corticosteroids. Glucocorticoid rather than mineralocorticoid activity seems to be responsible for this regulation.

Dobutamine-induced cardiac adaptations: comparison with exercise-trained and sedentary rats.

To investigate dobutamine-induced cardiac adaptations, we compared dobutamine-treated with swim-trained and sedentary control rats. After 14 wk of treatment, heart rate was lower in the dobutamine-treated (279 +/- 6) and exercise-trained (287 +/- 4 beats/min) groups than in the control animals (305 +/- 3 beats/min; P less than 0.05). The exercised rats gained less weight (164 +/- 11 g; P less than 0.05) than the dobutamine-treated (238 +/- 16 g) and the control animals (231 +/- 12 g). Also, compared with the two other groups, the exercise group had higher relative heart weights (3.47 +/- 0.08 vs. 2.82 +/- 0.06 and 2.90 +/- 0.05 g/kg in the dobutamine and control groups, respectively; P less than 0.05) and lower epididymal fat pad weights (6.6 +/- 0.4 vs. 13.3 +/- 1.0 and 11.4 +/- 0.5 g/kg in the dobutamine and control groups, respectively; P less than 0.05). However, both the maximum heart rate produced by isoproterenol and the isoproterenol dose producing 50% of the peak heart rate response were similar among the three groups. Myocardial norepinephrine content, beta-adrenergic receptor number, and adenylate cyclase activation by isoproterenol, NaF, 5'-guanylyl imidodiphosphate, and forskolin also did not differ. Thus, although there were differences between the dobutamine-treated and the exercised rats, the two groups were similar in that they developed bradycardia that was not due to cardiac adrenergic desensitization.

Dopamine-stimulated adenylate cyclase and tyrosine hydroxylase in diabetic rat retina.

The effects of streptozotocin-induced diabetes on the retinal dopaminergic system have been examined in Long-Evans (pigmented) rats. Tyrosine hydroxylase activity was significantly decreased while dopamine-stimulated adenylate cyclase was increased in 2-month-diabetic rats. The observed increase in dopamine-stimulated adenylate cyclase activity in diabetic retinae may be related to neurotransmitter receptor changes because postreceptor activation of adenylate cyclase by guanylyl imidodiphosphate was not altered.

Age-related reduction of beta-adrenoceptor sensitivity in rat heart occurs by multiple mechanisms.

Myocardial responsiveness to catecholamines was evaluated by measuring isoproterenol-stimulated adenylate cyclase activities in myocardial particulate fractions from Fischer 344 rats of 3, 12, and 24 months of age. Dose-response curves of isoproterenol revealed a progressive increase in the activation constant (Kact) with advancing age. In addition, the maximal velocity (Vmax) for 12- and 24-month-old groups was about 20-25% lower than for the 3-month-old group. Analysis of receptor-binding data and nonreceptor-mediated enzyme activities suggests that the age-related decrease in Vmax for isoproterenol may result from a loss of myocardial beta-receptors, whereas the increase in Kact is probably due to a deficit in the postreceptor components of the receptor-cyclase complex.

Measurement of cytosolic free calcium concentration in isolated rat ventricular myocytes with quin 2.

Cytosolic free calcium concentration was determined in isolated ventricular myocytes from adult rats with the calcium-sensitive indicator, quin 2. The fluorescence signal from resting cells indicated that cytosolic free calcium concentration was 181 +/- 18 nM (mean +/- SEM, n = 18). Inhibition of the sodium-potassium pump with strophanthidin (0.1 mM) resulted in an increase of cytosolic free calcium concentration from 186 +/- 17 to 736 +/- 129 nM (n = 6). The results indicate that it is possible to measure cytosolic free calcium concentration in cardiac muscle cells that have been isolated enzymatically. Moreover, they confirm the observation that inhibition of the sodium-potassium pump increases cytosolic free calcium concentration, presumably via the sodium-calcium exchange mechanism.

Decreased beta-adrenergic receptors in rat heart in streptozotocin-induced diabetes: role of thyroid hormones.

The effect of streptozotocin-induced diabetes on the beta-adrenergic receptor-coupled adenylate cyclase was studied in rat heart particulate fractions. Streptozotocin treatment decreased the number of myocardial beta-adrenergic receptors by 34% with no change in the apparent affinity of these receptors for [3H]dihydroalprenolol. The maximal isoproterenol-activated accumulation of cAMP in streptozotocin-treated rat hearts was decreased by only 10%. Insulin administration to streptozotocin-treated rats increased the number of myocardial beta-adrenergic receptors to near or above control levels. Administration of L-T4 to streptozotocin-treated rats had the same effect. Total T4, free T4, and total T3 levels were all significantly decreased in the diabetic animals. Administration of insulin to streptozotocin-treated rats increased the serum thyroid hormone levels toward or above the levels found in control animals. Streptozotocin-induced diabetes had no significant effect on cardiac beta-adrenergic receptor number in thyroidectomized rats. Insulin did not elevate cardiac beta-adrenergic receptor number in thyroidectomized diabetic rats. The decrease in the number of myocardial beta-adrenergic receptors occurring in diabetes mellitus is probably mediated through thyroid hormones.

Platelet alpha-adrenergic receptors in obesity: alteration with weight loss.

We studied platelet alpha-adrenergic receptor concentration and function in 19 subjects with simple obesity participating in a double-blind, controlled clinical trial of diet and anorexiants (phentermine, fenfluramine, or a combination of the two) or placebo. From wk 1 to wk 8, weight loss for the group as a whole was 4.9 +/- 0.7 kg (mean +/- SE). Concomitant with this weight loss, the platelet alpha-adrenergic receptor concentration rose from 85.7 +/- 5.8 to 113 +/- 5.8 fmol/mg protein. This increase moved the values for the obese subjects toward or beyond values in lean controls (100 +/- 10.5 fmol/mg protein). The response in the different treatment groups was similar. The receptor concentration increase was accompanied by a corresponding increase in alpha-adrenergic receptor-mediated platelet aggregation. For individual subjects the extent of weight loss over time generally correlated with percent receptor change. Altered adrenergic sensitivity occurring in obese subjects who are losing weight may have important implications in relation to external (therapeutic or inadvertent) administration of catecholamines.

Cerebral compensation for chronic noradrenergic denervation induced by locus ceruleus lesion: recovery of receptor binding, isoproterenol-induced adenylate cyclase activity, and oxidative metabolism.

The long term effects of specific noradrenergic denervation of rat cerebral cortex were considered in parallel studies of in vitro noradrenergic receptor binding and isoproterenol-induced adenosine 3':5'-monophosphate (cycle AMP) generation and of in vivo oxidative metabolism. Noradrenergic denervation was achieved by the local, unilateral injection of 6-hydroxydopamine into the locus ceruleus. Cerebral noradrenaline remained depleted throughout the 8-week duration of the study. Ligand-binding assays showed increased beta-adrenergic receptors 2 weeks after locus ceruleus lesion with recovery occurring by 4 weeks. There were no changes in alpha 1-adrenergic receptors. Isoproterenol-induced cyclic AMP generation increased at 2 weeks after lesion but recovered at 8 weeks. Dual wavelength reflection spectrophotometric measurements of cytochrome oxidase reduction/oxidation and local blood volume shifts, provoked in situ by direct cortical stimulation, also demonstrated abnormalities at 2 weeks with recovery by 4 weeks after lesion. Thus, in vivo and in vitro changes after locus ceruleus lesion are reversible and the time course of these changes is related temporally. These data suggest that cerebral cortex has adaptive capabilities which are activated to compensate for prolonged noradrenaline depletion. We also speculate that there may be a causal relationship between the in vitro and in vivo parameters studied.

Adrenergic and cholinergic receptors of cerebral microvessels.

The presence of alpha- and beta-adrenergic and muscarinic cholinergic receptors in cerebral microvessels of the rat and pig was assessed by ligand binding techniques. The results demonstrate the presence of specific binding to alpha 2- and beta-adrenergic receptors but no appreciable specific binding to alpha 1-adrenergic or muscarinic cholinergic receptors. beta-Adrenergic receptors of pig cerebral microvessels are similar to those of the brain and other organs in their binding characteristics to the tritiated ligand and in their stereospecificity of binding to the biologically active isomers of beta-adrenergic agonists. Further evidence derived from the differential potency of binding displacement by the various beta-adrenergic agonists and selective beta 1- and beta 2-adrenergic antagonists indicates that beta-adrenergic receptors of pig cerebral microvessels are mostly of the beta 2-subtype.