P2X7 receptor activation in rat brain cultured astrocytes increases the biosynthetic release of cysteinyl leukotrienes.

Astrocytes have been recognized as important elements in controlling inflammatory as well as immune processes in the central nervous system (CNS). Recently, glial cells have been shown to produce cysteinyl leukotrienes (CysLTs) which are known lipid mediators of inflammation and whose extracellular concentrations rise under different pathological conditions in the brain. In the same conditions also extracellular concentrations of ATP dramatically increase reaching levels able to activate P2X7 ionotropic receptors for which an emerging role in neuroinflammation and neurodegeneration has been claimed. RTPCR analysis showed that primary cultures of rat brain astrocytes express P2X7 receptors. Application of the selective P2X7 agonist benzoyl benzoly ATP (BzATP) markedly increased [Ca2+]i which was mediated by a calcium influx from the extracellular milieu. The P2X7 antagonist, oATP, suppressed the BzATP-induced calcium increase. Consistent with the evidence that increased calcium levels activate the leukotriene biosynthetic pathway, challenge of astrocytes with either the calcium ionophore A23187 or BzATP significantly increased CysLT production and the cell pre-treatment with EGTA abolished these effects. Again the P2X7 antagonist prevented the BzATP-mediated CysLT efflux, whereas the astrocyte pretreatment with MK-571, a CysLT1 receptor antagonist, was ineffective. The astrocyte pre-treatment with a cocktail of inhibitors of ATP binding cassette (ABC) proteins reduced the BzATP-mediated CysLT production confirming that ABC transporters are involved in the release of CysLTs. The astrocyte P2X7- evoked rise of CysLT efflux was abolished in the presence of MK-886, an inhibitor of 5-lipoxygenase activating protein (FLAP) whose expression, along with that of 5-lipoxygenase (5-LO) was reported by Northern Blot analysis. The stimulation of P2X7 induced an up-regulation of FLAPmRNA that was reduced by the antagonist oATP. These data suggest that in rat brain cultured astrocytes P2X7ATP receptors may participate in the control of CysLT release thus further supporting a role for extracellular ATP as an integral component of the inflammatory brain response.

AIT-082 and methylprednisolone singly, but not in combination, enhance functional and histological improvement after acute spinal cord injury in rats.

Extracellular non-adenine based purines are neuroprotective. Preliminary studies indicate that administration of the synthetic purine 4-[[3-(1,6 dihydro-6-oxo-9-purine-9-yl)-1-oxypropyl] amino] benzoic acid (AIT-082, leteprinim potassium) to rats immediately after acute spinal cord injury (SCI), improves functional outcome. The effects of potential new agents are often compared to methylprednisolone (MPSS). We evaluated the effects of AIT-082 and MPSS, separately and in combination, on the functional and morphological outcome of acute SCI in adult rats. After standardized T11-12 spinal cord compression rats were given intraperitoneally one of the following: vehicle (saline); MPSS (30 mg/kg or 60 mg/kg body weight, first dose 15 min after crush); AIT-082 (60 mg/kg body weight daily, first dose 15 min after crush); or AIT-082 plus MPSS. After 1, 3, or 21 days, the rats were perfused for histological analysis. AIT-082 administrations significantly reduced locomotor impairment from 121 days post-operatively. At 1 and 3 days post injury, AIT-082-treatment reduced tissue swelling, tissue loss and astrogliosis at the injured cords but did not alter the extent of hemorrhage and the number of macrophages and/or microglia. MPSS reduced hemorrhage and the number of macrophages and/or microglia, but did not alter astrogliosis. At 21 days, either AIT-082 or MPSS administration improved function and morphology similarly (less tissue loss and astrogliosis). In contrast, administration of AIT-082 and MPSS together abolished the beneficial effects observed when either drug was given individually. These results suggest that MPSS and AIT-082 may exert their beneficial effects through different and potentially antagonistic pathways.

The role of 3'-untranslated region (3'-UTR) mediated mRNA stability in cardiovascular pathophysiology.

Knowledge of transcription and translation has advanced our understanding of cardiac diseases. Here, we present the hypothesis that the stability of mRNA mediated by the 3'-untranslated region (3'-UTR) plays a role in changing gene expression in cardiovascular pathophysiology. Several proteins that bind to sequences in the 3'-UTR of mRNA of cardiovascular targets have been identified. The affected mRNAs include those encoding beta-adrenergic receptors, angiotensin II receptors, endothelial and inducible nitric oxide synthases, cyclooxygenase, endothelial growth factor, tissue necrosis factor (TNF-alpha), globin, elastin, proteins involved in cell cycle regulation, oncogenes, cytokines and lymphokines. We discuss: (a) the types of 3'-UTR sequences involved in mRNA stability, (b) AUF1, HuR and other proteins that bind to these sequences to either stabilize or destabilize the target mRNAs, and (c) the potential role of the 3'-UTR mediated mRNA stability in heart failure, myocardial infarction and hypertension. We hope that these concepts will aid in better understanding cardiovascular diseases and in developing new therapies.

Nifedipine induces apoptosis in cultured vascular smooth muscle cells from spontaneously hypertensive rats.

Apoptosis (programmed cell death) of smooth muscle cells (SMC) in blood vessels is an essential process involved in the control of vessel wall structure. Several antihypertensive drugs currently used in therapy may exert their pharmacological effects by promoting SMC apoptosis. The biochemical events which regulate SMC apoptosis in the vessel wall are complex, and not well understood. We therefore investigated whether treatment of cultured SMC from normotensive Wistar-Kyoto rats (WKY) and from spontaneously hypertensive rats (SHR) with selected antihypertensive drugs would induce SMC apoptosis. We treated aortic SMC from WKY and SHR in vitro with the L-type Ca2+ channel antagonist, nifedipine; with the nitric oxide donor, sodium nitroprusside (SNAP); with forskolin (an activator of adenylyl cyclase); or with thapsigargin (a selective inhibitor of the sarcoplasmic reticulum (SR), Ca2+-ATPase); and compared their apoptosis-promoting effects in SMC derived from the two strains of rats. SMC were derived from the thoracic aorta of 3-4-week-old WKY and SHR, and were used in passages 7-10. Apoptotic cells were detected by in-situ end labeling using the terminal deoxynucleotide transferase-mediated dUTP-nick end-labeling (TUNEL) method, and by morphological examination. We found that: 1) Treatment of cultured aortic SMC with the L-type Ca2+ channel antagonist, nifedipine (5 X 10(-5) M) for 24 hours induced a significantly higher level of apoptosis in SHR cells than in SMC from WKY. Cells from WKY, following exposure to nifedipine for 72 hours, exhibited a similar response to the cells from SHR treated for 24 hours. This was detectable by both morphological criteria as well as DNA labeling by the TUNEL technique. 2) Similar treatment of these cells with thapsigargin (1 x 10(-7) M) led to morphological alterations characteristic of apoptotic cells in SMC from both WKY and SHR, and cells from SHR but not WKY were labeled by the TUNEL technique at 24 hours. The TUNEL method did however identify cells from both WKY and SHR as apoptotic after 48 and 72 hours of treatment. 3) The addition of SNAP, or forskolin to the cultured SMC induced significant, but low levels of apoptosis in WKY SMC only. This selective apoptosis-promoting effect of nifedipine in SHR SMC may result from differences in the control of intracellular Ca2+ between the two strains of cells, or it may indicate that the signaling pathways which regulate apoptosis are different in SMC from the normotensive and the hypertensive rats. Our findings imply that SMC apoptosis may be a selective target for pharmacological intervention in hypertension.

Vascular beta-adrenoceptor function in hypertension and in ageing.

Functional beta-adrenoceptors (beta-AR) have been identified and characterized in blood vessels under in vivo conditions as well as in vascular smooth muscle cells (SMC) grown in culture. Agonist occupancy of beta-AR activates adenylyl cyclase (AC) via the stimulatory guanine nucleotide-binding protein (Gs) and leads to elevations in intracellular adenosine 3'5'-cyclic monophosphate levels (cAMP). Increased cAMP activates the cAMP-dependent protein kinase (PKA), with subsequent phosphorylation of various target proteins. This beta-AR pathway interacts with several other intracellular signalling pathways via cross-talk, so that activation by beta-AR agonists may also modulate other second messengers and protein kinases. SMC beta-AR play an important role in SMC function. In intact blood vessels they mediate SMC relaxation by various intracellular mechanisms, ultimately causing a decrease in intracellular Ca2+ levels. In cultured SMC, activation of the beta-AR pathway results in inhibition of cellular proliferation, the development of SMC polyploidy, and SMC apoptosis. Blood vessels from hypertensive animals are characterized by an increase in SMC cell mass, a greater incidence of SMC polyploidy in the aorta, and an impairment in the beta-agonist-mediated SMC relaxation. Some of these changes may result from an attenuation of beta-AR function due to agonist-induced receptor desensitization caused by the uncoupling of receptors from the Gs-AC system. The phosphorylated beta-AR may in turn trigger new signals and activate different intracellular pathways. However, the details of these mechanisms are still unresolved. Since functional beta-AR play such a prominent and multi-faceted role in SMC function, it is important to understand how these diverse physiological effects are mediated by this receptor system, and how they contribute to the development of hypertension. With ageing, a decrease in beta-AR-Gs-AC coupling is observed, and this is implicated in the reduced responsiveness of SMC. The similarities in SMC beta-AR functional changes in hypertension and in ageing suggest that the underlying mechanisms are also analogous.

Cross-over study comparing effects of treatment with an angiotensin converting enzyme inhibitor and an angiotensin II type 1 receptor antagonist on cardiovascular changes in hypertension.

OBJECTIVE: To compare the blood-pressure-lowering effects of an angiotensin converting enzyme inhibitor, perindopril, with those of an angiotensin II type 1 receptor antagonist, L-158,809, for adult spontaneously hypertensive rats. DESIGN: A cross-over design was used, to treat adult spontaneously hypertensive rats with one drug for 10 weeks, and then with the other for 5 weeks. METHODS: Adult, male spontaneously hypertensive rats (aged 15 weeks) were treated daily by gavage for 10 weeks with perindopril (P group) or L-158,809 (L group), then treatment was crossed over so that rats in the P group were treated with L-158,809 (P/L group) and rats in the L group were treated with perindopril (L/P group) for 5 weeks. Blood pressure was measured weekly. Plasma angiotensin converting enzyme activity, renal angiotensin receptor density, and arterial structure and functioning were measured after the single and crossover treatment periods. RESULTS: Treatment lowered the blood pressure from 206 +/- 2 mmHg in rats in the control group, to 126 +/- 2 in rats in the P group and 150 +/- 2 in rats in the L group. After the cross-over period, blood pressure decreased further from 150 +/- 2 to 129 +/- 3 mmHg in rats in the L/P group, whereas blood pressure of spontaneously hypertensive rats in the P/L group increased from 126 +/- 2 to 148 +/- 2 mmHg. Perindopril treatment almost abolished plasma angiotensin converting enzyme activity, whereas L-158,809 treatment had no effect. Renal angiotensin II receptor density was decreased versus baseline in rats in the P and L groups. The affinity of binding was decreased versus baseline in rats in the L group. A positive correlation to blood pressure was found for mesenteric artery wall thickness and wall: lumen ratio. Concentration for half-maximal effect for the response of mesenteric arteries from rats in the P group to norepinephrine was lower than that of the control group rats. Angiotensin II potentiated the norepinephrine-stimulated contraction of arteries from rats in the control and P groups, but not that of arteries from rats in the groups treated with L-158,809. CONCLUSION: Perindopril was more effective than was L-158,809 at lowering the blood pressure of adult spontaneously hypertensive rats, and at altering the structure and functioning of the arteries.

Effects of electroconvulsive therapy and desipramine on neuroendocrine responses to the clonidine challenge test.

OBJECTIVE: To investigate responses to the clonidine challenge test in depression, and after electroconvulsive therapy (ECT) or desipramine treatment for depression, in order to determine the usefulness of noradrenergic responses to clonidine as a state or trait marker in depression. PATIENTS: Twenty-six patients with depression and 15 control subjects. SETTING: The psychiatric ward of St. Joseph's Hospital in Hamilton. INTERVENTIONS: In the patients with depression: clonidine challenge pre- and post-treatment with ECT or desipramine. In the controls: 2 clonidine challenge tests 4 to 8 weeks apart. OUTCOME MEASURES: The primary measure was the growth hormone response to the clonidine challenge. Plasma norepinephrine, 3-methoxy-4-hydroxyphenylglycol (MHPG), cortisol, blood pressure, pulse and sedation levels were examined in subgroups of participants as secondary measures. RESULTS: The pre-treatment growth hormone response to clonidine was significantly more blunted in patients than in controls (p = 0.02). This response improved in both treatment groups after therapy and, although it remained decreased, there was no longer a significant difference in response between the patients and the controls. In the patients, a decreased growth hormone response to clonidine at baseline was correlated with response to treatment. Of the secondary measures, patient baseline norepinephrine levels were significantly elevated pre- and post-treatment, although there were no significant group-by-time challenge effects. MHPG levels were not significantly different pre- and post-treatment between patients and controls. Baseline blood pressure and pulse were elevated in the patients pre- and post-treatment. These differences were not statistically significant and did not change after treatment. Sedation levels were not significantly different among the groups at baseline. Clonidine-induced sedation occurred significantly earlier in the patients pretreatment and improved to the range of the controls after treatment. Pretreatment cortisol response was significantly more blunted in the patients who received ECT than in the controls; however, the group-by-time effect post-treatment was no longer significant. DISCUSSION: Treatment with either desipramine or ECT modified noradrenergic functioning in patients with depression, as assessed by growth hormone response to the clonidine challenge. In the patients, a decreased growth hormone response at baseline was correlated with clinical response. Changes between pre- and post-treatment measures suggest that this challenge test may not be sensitive enough to serve as a trait marker but may correlate with the state of depression in a subpopulation of these patients.

Antihypertensive effects of perindopril treatment in adult spontaneously hypertensive rats.

OBJECTIVE: To determine the effect of perindopril treatment and treatment withdrawal in the prevention of hypertension in adult male spontaneously hypertensive rats (SHR). ANIMALS AND METHODS: Beginning at 15 weeks of age, male SHR were treated with either distilled water (control) or different daily dosages of perindopril (1, 2 or 4 mg/kg) by gavage for 10 weeks, followed by 10 weeks of treatment withdrawal. Systolic blood pressure, heart rate and body weight of adult SHR were determined at regular intervals before, during and after the treatment withdrawal periods. At the end of the treatment withdrawal period, plasma and tissue samples were taken for measurement of noradrenaline levels. Angiotensin-converting enzyme (ACE) activity in the plasma from adult SHR and Wistar-kyoto (WKY) rats treated with perindopril 4 mg/kg for two weeks was measured by a radioassay method 6 and 24 h after treatment. RESULTS: Treatment with perindopril caused a dose-dependent lowering of blood pressure in SHR during the 10-week treatment. After withdrawal of the treatment, persistent lowering of blood pressure was found in SHR treated with higher dosages (2 or 4 mg/kg), but not in the 1 mg/kg group. There was no difference in the tissue level of noradrenaline among the control group and SHR previously treated with perindopril. In SHR and WKY treated with perindopril for two weeks, plasma level of ACE activity was reduced longer than 24 h compared with their respective controls. CONCLUSIONS: Chronic treatment of adult SHR with perindopril has a dose-dependent effect on the blood pressure of these animals both during and after withdrawal of treatment, but such a treatment had no long term effects on the noradrenaline levels in various tissues.

Expression of functional beta-adrenoceptors and polyploidy development in cultured vascular smooth muscle cells from spontaneously hypertensive rats.

The main objective of this study was to investigate the role of beta-adrenoceptor activation in the development of polyploidy in these cells. Smooth muscle cells (SMCs) were cultured from 3- to 4-, 10- to 12-, and 28- to 30-week-old spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). Functional expression of beta-adrenoceptors was examined by treatment of cultured SMCs with a nonselective beta-adrenoceptor agonist, isoproterenol, or an adenylate cyclase activator, forskolin, or a membrane-permeable analog of adenosine 3',5'-cyclic monophosphate (cAMP), 8-bromo-cAMP, and the measurement of intracellular cAMP levels, using a radioimmunoassay. The effects of these treatments on polyploidy development were also studied by measuring the DNA content of SMCs, using scanning microdensitometry. Treatments with isoproterenol or forskolin increased intracellular cAMP levels in both strains of rats in all three age groups. Addition of the beta-adrenoceptor antagonist DL-propranolol inhibited the isoproterenol-stimulated response in SMCs from both SHR and WKY in all three age groups. The number of polyploid SMCs was significantly increased by treatments with isoproterenol, forskolin, or 8-bromo-cAMP in SMCs from 3- to 4-week-old WKY and SHR, but in the 10- to 12-week age group, an increase was found only in SMCs from WKY. Such treatments had no effect on the incidence of polyploid SMCs in the 28- to 30-week groups. We conclude that (i) beta-adrenoceptors expressed by the SMCs from WKY and SHR at all three ages are functional and are coupled via Gs proteins to the stimulation of adenylate cyclase, (ii) treatments that elevate intracellular cAMP levels (by activation of beta-adrenoceptors or of adenylate cyclase) lead to increased polyploid SMCs from WKY and SHR in the younger age group, confirming a role for the beta-adrenoceptor-adenylate cyclase pathway in the development of polyploidy in cultured SMCs from both of these strains of rats, and (iii) the absence of these treatment effects in the induction of polyploid SMCs in older age groups suggests that in these cells, other factors or processes may be involved in regulating the development of polyploid SMCs.

Effects of electroconvulsive therapy on peripheral adrenoceptors, plasma, noradrenaline, MHPG and cortisol in depressed patients.

BACKGROUND: The mechanism of the antidepressant action of electroconvulsive therapy (ECT) remains unknown. Based on previous work with antidepressant drugs and their effects on the noradrenergic system, we undertook this study to further determine the effects of ECT on selected indices of peripheral adrenoceptor function in depressed patients. METHODS: Binding parameters (Bmax and Kd) of platelet alpha 2- and leukocyte beta 2-adrenoceptors, plasma noradrenaline (NA), 3-methoxy-4-hydroxy-phenylglycol (MHPG) and cortisol levels were determined in 18 patients, prior to treatment and 14 days after the last of a series of ECTs, and compared with samples obtained from 18 matched control subjects. RESULTS: Platelet alpha 2-adrenoceptor sites were significantly elevated in untreated patients compared with controls (P < 0.03), but leukocyte beta 2-adrenoceptor numbers did not differ. Treatment with ECT led to a significant reduction in platelet alpha 2-adrenoceptor numbers, whereas leukocyte beta 2-adrenoceptor densities increased. Pre-ECT plasma NA, MHPG, and cortisol levels were elevated in patients, compared with controls, and decreased following ECT, but these differences were not statistically significant. Post-ECT plasma NA and beta 2-adrenoceptor numbers were significantly, negatively correlated (P < 0.05). CONCLUSIONS: These results suggest that platelet alpha 2-adrenoceptors are supersensitive in depressed patients and treatment with ECT results in down-regulation of these receptors, which may be interpreted as a primary therapeutic, "normalising' effect. The post-ECT changes in leukocyte beta 2-adrenoceptors are probably only secondary to the lower circulating plasma NA levels.

Effects of peroxide and superoxide on coronary artery: ANG II response and sarcoplasmic reticulum Ca2+ pump.

The sarcoplasmic reticulum (SR) Ca2+ pump in membranes isolated from arterial smooth muscle is damaged by reactive oxygen species (ROS). Because angiotensin II (ANG II) contracts arterial smooth muscle by mobilizing intracellular Ca2+ concentrations ([Ca2+])i, we determined the effects of ROS pretreatment on ANG II-induced contractions in coronary artery rings and [Ca2+]i transients in smooth muscle cells (SMC) cultured from them. This experimental design eliminates direct ROS interference in assay solutions, thus monitoring only the tissue damage. Pretreating the arteries with peroxide inhibited the ANG II contractions with the concentration for half-maximal activation (K0.5) = 74 +/- 5 microM. Peroxide (250 microM) inhibited the contractions to ANG II and cyclopiazonic acid (CPA, SR Ca(2+)-pump inhibitor) by 78.3 +/- 5.1 and 67.4 +/- 6.3%, respectively, but did not significantly affect the contractions by 60 mM KCl. Pretreating SMC with peroxide inhibited the ANG II-induced increase in [Ca2+]i with K0.5 = 24 +/- 3 microM for peroxide. Peroxide (100 microM) inhibited the increase in [Ca2+]i in response to ANG II and CPA by 78.9 +/- 5.1 and 38.3 +/- 4.9%, respectively. The SR Ca(2+)-pump activity was also measured as the Ca(2+)-dependent formation of 115-kDa acylphosphate. Pretreating SMC with 100 microM peroxide inhibited the acylphosphate levels by 36.3 +/- 3.2%. Peroxide (100 microM) pretreatment of SMC did not significantly affect their ANG II binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Platelet alpha 2-adrenergic receptors in depressed patients and healthy volunteers: the effects of desipramine.

Binding parameters (Bmax and Kd) of alpha 2-adrenergic receptors were studied in platelets from 14 depressed patients and 18 control subjects. Using 3H-clonidine (a partial alpha 2-adrenergic agonist) as the ligand and membranes, prepared from platelets isolated under physiological conditions, we found no significant differences in Bmax and Kd between medication free patients and control subjects. Platelet binding parameters in the depressed patients did not correlate with plasma levels of norepinephrine, epinephrine or MHPG. Age had a significant positive effect on platelet alpha 2-adrenergic receptor Bmax in both groups, and may have masked the patient-control differences. Treatment with desipramine for 28 days had no effect on the binding parameters in depressed patients when compared to pretreatment values. Adding desipramine to platelets of control subjects 'in vitro' did also not affect binding parameters. Our findings suggest that receptor binding studies with a partial alpha 2-adrenergic agonist in platelet membranes are not a useful model to test the hypothesis of a central supersensitive adrenergic system in depression.

Studies on leukocyte beta-adrenergic receptors in depression: a critical appraisal.

Alterations in beta-adrenergic receptors (BAR) of human mononuclear leukocytes (MNL) are considered to reflect changes in central noradrenergic function and have been studied in a number of diseases. This paper critically reviews the results of recent studies on MNL-BAR in depression, with particular emphasis on the biochemical and clinical methodologies used. Despite considerable differences in these methods, a number of laboratories report consistent decreases in MNL-BAR density and significantly reduced functional response in patients as compared to controls. These studies used MNL, isolated from patients who had a greater than 14 day drug washout, and BAR-densities were measured in membrane preparations, using full Scatchard analyses, and 125I-ICYP or 3H-DHA as the ligand. Functional response of MNL-BARs was assessed by the determination of isoproterenol-stimulated cyclic AMP accumulation. A comparison of methods used by these groups further indicates that additional biochemical parameters such as lymphocyte preparation and standardized experimental conditions for the binding assays are also important for obtaining consistent results. The clinical methods in rigorous study designs also include clearly stated inclusion/exclusion criteria for patients, and age-, and gender-matched patient-control populations. Whether the reduced MNL-BAR density and function is an inherited abnormality in depressed patients, or results from downregulation by elevated catecholamines is at present not known. Studies are needed to characterize further the changes in MNL-BARs in depression and to evaluate the effects of caetcholamines and hormones on this system. Based on critical assessment of the methods reviewed we propose specific biochemical and clinical guidelines, and recommend, that these be followed in future studies on MNL-BARs in this disease.

Lithium and rhythms of beta-adrenergic ([3H]CGP-12177) binding in intact rat retina, pineal gland, and hypothalamus.

A new assay technique for the determination of neurotransmitter binding in retinal fragments has been used to characterize and quantify beta-adrenergic receptors with the ligand [3H]CGP-12177. This assay allowed us to quantify beta-adrenergic receptors in the retina, pineal gland, and hypothalamus obtained from individual rats during a 10-hr period around the switch from light to dark under a 12-hr light/12-hr dark lighting cycle. A significant rhythm of beta-adrenergic binding was observed in the retina and pineal gland. These rhythms were abolished by chronic lithium treatment. In contrast to previous observations in whole brain preparations, lithium did not affect beta-adrenergic binding in brain tissue (hypothalamus) using this assay. Our data suggest that lithium may attenuate beta-adrenergic receptor down-regulation in pineal and retinal tissue. To the extent that this mechanism is important for the coding of information about light and dark in the environment, these observations might assist in our understanding of the clinical chronopharmacological properties reported for lithium.

Dysregulation of neuroendocrine crossroads: depression, circadian rhythms and the retina--a hypothesis.

The pathophysiology of depression and the mechanism of action of lithium and other antidepressant drugs involve alterations in circadian rhythms. These include changes in both the intrinsic rhythm of circadian oscillators and in the sensitivity of the retina to LIGHT. The retina in humans is the only photoreceptor for circadian entrainment. The retinal-hypothalamic-pineal axis is the essential pathway for neuronal entrainment of rhythms which use light as a phase cue. A common substance throughout this axis in many species is MELATONIN. Retinal melatonin has been implicated in regulation of the sensitivity of the retina to light. The hypothalamus, at THE NEUROENDOCRINE CROSSROADS, has a central role in the integration of neurotransmitters and hormones in circadian rhythms. DYSREGULATION of the hypothalamic-pituitary-adrenal, as well as -gonadal, axes has been documented in depressed patients. Abnormalities in circulating melatonin have also been found in patients with affective disorders. It is speculated that the availability of melatonin along the retinal-hypothalamic-pineal axis may have important implications in the genesis of affective disorders. More specifically--is there a latent biochemical defect which causes a phase shift and change in circadian rhythms of melatonin and/or other neurotransmitters in the retina which then alters the sensitivity of the retina to light (for the visible spectrum) which in turn desynchronizes all other biological rhythms thus disrupting mental well-being? We suggest that variations of retinal photosensitivity in humans can be measured by using a visual testing system, and that depressed patients might show changes in photosensitivity which could be corrected when treated with lithium and/or antidepressants. It is our working hypothesis that the primary defect in depression may be a change in retinal function, and that behavioural and neuroendocrine concomitants of this disorder are secondary events.

Lithium and circadian patterns of melatonin in the retina, hypothalamus, pineal and serum.

Lithium, a widely used substance for treatment of manic-depressive illness has been reported to alter the phase relationship of a variety of circadian rhythms which have been implicated in the aetiology of depression and manic-depressive disorder. Although its mechanism of action is not understood, the theraputic action of lithium has been related to its ability to alter circadian rhythms. Chronic lithium administration to rats resulted in lithium levels comparable to the human theraputic range. These lithium levels affected a broad range of biological variables by significantly modifying their circadian pattern of variation, notably during the dark period of an alternating 12h light/12h dark schedule. These included water intake, body weight, retina weight and pineal, serum, retina and hypothalamic melatonin measures. Retinal lithium levels were significantly higher than serum lithium levels and retinal melatonin levels were reduced by lithium. The data are interpreted as suggesting that lithium may exert its theraputic effects by influencing melatonin levels at several locations along the retinal-hypothalamic-pineal pathway, resulting in a modulation of the potential cue value of this physiological stimulus for synchronization of circadian rhythms. Such an effect of lithium could have important chronobiological implications for circadian rhythms which use light and dark as a phase cue.

3H-clonidine binding to membranes of platelets prepared under physiological conditions.

A reproducible binding assay has been established to measure alpha 2-adrenoreceptors on membranes of human platelets prepared under physiological conditions. Washed platelet suspensions were obtained from fresh blood by a modified method (Mustard et al., 1972) and membranes were prepared by mechanical homogenization. 3H-clonidine, a selective alpha 2-adrenoreceptor agonist was selected as the ligand in a concentration range of 2-64 nM. Specific binding of 3H-clonidine was defined as the difference between total bound radioactivity and that not displaced by excess cold clonidine (6.4 X 10(-5) M). Platelets from 14 healthy young male volunteers were analysed using this technique, and one high affinity binding site, with KD and Bmax values in the reported range was found (KD: 10.14 +/- 1.95 nM; Bmax: 428 +/- 53 fmol/mg protein (mean +/- S.E.M.)). This technique will be used in future studies that will examine alpha 2-adrenoreceptor changes in specific subgroups of depressed patients.

Lithium and twenty-four hour rhythms of serum corticosterone, prolactin and growth hormone in pigmented eye rats.

Lithium, a widely used substance for the treatment of manic-depressive illness, has been reported to alter the phase relationships of a variety of biological rhythms. We have previously found that lithium affects serum melatonin differently in albino compared to pigmented eye rats. The present study was undertaken to investigate the effect of lithium on pituitary rhythms in pigmented eye rats. Six point 24 hour maps were generated throughout a 12 hour light/12 hour dark lighting regime on separate groups of individually housed adult male Long Evans rats (with pigmented eyes), maintained for six weeks on ad lib water and either normal lab chow or lab chow supplemented with 50 mM/Kg of lithium chloride. Animals were sacrificed by rapid decapitation with care to ensure that blood samples were obtained from subjects in the resting, undisturbed state. Plasma lithium levels were 0.57 +/- 0.02 mEq/1. In comparison to normal controls, lithium treatment suppressed body weight by 19%, and increased water intake by 100%. Absolute corticosterone levels were not altered by lithium, but the 24-hour pattern was significantly changed. Growth hormone levels were significantly reduced by lithium treatment without alteration of the 24-hour pattern. Prolactin levels were significantly reduced by lithium and the normal 24-hour variation was attenuated. Comparison of these effects of lithium in pigmented eye rats with similar data from albino rats suggests that the effects on growth hormone, body weight and water intake were similar; however, the effects on prolactin and corticosterone differed.(ABSTRACT TRUNCATED AT 250 WORDS)

Erythrocyte lithium efflux in bipolar patients and control subjects: the question of reproducibility.

The reproducibility of in vitro erythrocyte lithium efflux and lithium efflux in the presence of selected membrane transport inhibitors (phloretin, ouabain, 4,4'-diisothiocyano-2,2'-disulphonic acid stilbene, and p-chloromercury-benzene sulphonate) was investigated in bipolar patients and age- and sex-matched control subjects. Efflux experiments were repeated three times in each patient-control pair within a period of 14 days. No differences were detected between patients and control subjects in any of the parameters measured. All components of lithium efflux showed wide day-to-day variation in the same subject in both patients and control subjects. Intersubject variability, however, was significantly greater than intrasubject variation. Since intraindividual variation of phloretin-inhibited lithium efflux was found to be considerable, and no real patient-control differences could be detected, the significance of this in vitro parameter in bipolar affective illness seems somewhat questionable and should be carefully reconsidered. The relevance of these findings to the putative cell membrane dysfunction in this disease is discussed.

Erythrocyte lithium transport in bipolar affective disorders. The effect of membrane transport inhibitors.

Erythrocyte lithium influx and efflux were investigated in vitro in patients with bipolar affective disorders and in age- and sex-matched healthy controls. To explore the components of lithium influx and efflux five selected inhibitors (ouabain, phloretin, p-chloromercury benzene sulfonate [PCMBS], 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene, and lanthanium chloride) were employed. The mean values of lithium influx were similar in both populations of erythrocytes. The addition of ouabain and phloretin reduced lithium influx, but this effect was comparable in both patients and controls. PCMBS had an accelerating effect, and this was more pronounced in patients. Total erythrocyte lithium efflux from lithium-containing erythrocytes was comparable in both patients and controls. The addition of phloretin reduced RBC lithium efflux, the magnitude of this parameter, however, was similar in patients and controls. Erythrocyte lithium efflux was accelerated in the presence of PCMBS, and this effect was greater in patients. The relevance of these findings to the postulated cell membrane defect in affective disorders is evaluated.