Hypothalamic-pituitary-adrenal axis targets for the treatment of epilepsy.

Stress is a common seizure trigger in persons with epilepsy. The body's physiological response to stress is mediated by the hypothalamic-pituitary-adrenal (HPA) axis and involves a hormonal cascade that includes corticotropin releasing hormone (CRH), adrenocorticotropin releasing hormone (ACTH) and the release of cortisol (in humans and primates) or corticosterone (in rodents). The prolonged exposure to stress hormones may not only exacerbate pre-existing medical conditions including epilepsy, but may also increase the predisposition to psychiatric comorbidities. Hyperactivity of the HPA axis negatively impacts the structure and function of the temporal lobe of the brain, a region that is heavily involved in epilepsy and mood disorders like anxiety and depression. Seizures themselves damage temporal lobe structures, further disinhibiting the HPA axis, setting off a vicious cycle of neuronal damage and increasing susceptibility for subsequent seizures and psychiatric comorbidity. Treatments targeting the HPA axis may be beneficial both for epilepsy and for associated stress-related comorbidities such as anxiety or depression. This paper will highlight the evidence demonstrating dysfunction in the HPA axis associated with epilepsy which may contribute to the comorbidity of psychiatric disorders and epilepsy, and propose treatment strategies that may dually improve seizure control as well as alleviate stress related psychiatric comorbidities.

Immunosuppression with mycophenolate mofetil attenuates the development of hypertension and albuminuria in deoxycorticosterone acetate-salt hypertensive rats.

1. The interplay between the immune and renin-angiotensin systems is emerging as a crucial factor in the development and progression of hypertension. The aim of the present study was to determine the involvement of immune cells in the hypertension and renal injury produced by a non-angiotensin II-dependent form of hypertension, namely deoxycorticosterone acetate (DOCA)-salt-induced hypertension, in rats. 2. Male Sprague-Dawley rats underwent uninephrectomy and received either a sustained-release pellet of DOCA s.c. and 0.9% NaCl (saline) to drink for 21 days or a placebo pellet and water to drink for 21 days. Additional groups of DOCA-salt- and placebo-treated rats were treated concurrently with the immune suppressant mycophenolate mofetil (MMF; 30 mg/kg per day). Rats were placed in metabolic cages for 24 h urine collection prior to and at weekly intervals during the 21 day experimental period. 3. Mycophenolate mofetil significantly attenuated the development of hypertension in DOCA-salt rats compared with untreated DOCA-salt hypertensive rats (mean arterial pressure by telemetry on Day 18,146 ± 7 vs 180 ± 3 mmHg, respectively; P < 0.001), as well as proteinuria (87 ± 27 vs 305 ± 63 mg/day, respectively, on Day 21) and albuminuria (51 ± 15 vs 247 ± 73 mg/day, respectively, on Day 21). Creatinine clearance was better preserved in MMF-treated DOCA-salt rats compared with untreated DOCA-salt rats (0.74 ± 0.07 vs 0.49 ± 0.09 mL/min, respectively; P < 0.05), but was still significantly reduced compared with that in the placebo group (1.15 ± 0.12 mL/min; P < 0.05). Finally, MMF treatment significantly attenuated the DOCA-salt-induced rise in renal cortical T-lymphocyte and macrophage infiltration (P < 0.05). 4. These data indicate that immune cells play a deleterious role in both the hypertension and renal injury associated with DOCA-salt hypertension.

Endothelin-1 contributes to the sexual differences in renal damage in DOCA-salt rats.

We investigated whether gender differences in renal damage in DOCA-salt hypertension are associated with effects of ovarian hormones and/or endothelin-1 (ET-1). Renal injuries and renal pre-pro-ET-1 mRNA expression were enhanced in male and female ovariectomized (OVX) DOCA rats versus female DOCA rats. Treatment with estrogen plus progesterone or progesterone, but not estrogen alone, attenuated renal damage and pre-pro-ET-1 mRNA expression in OVX DOCA rats. The ETA antagonist BMS182874 greatly ameliorated renal damage in male and OVX DOCA rats. In conclusion, the ovarian hormones have a protective role on the renal structural alterations in female DOCA rats by modulating effects of ET-1, via ETA receptors.

The acute anticonvulsant effects of deoxycorticosterone in developing rats: role of metabolites and mineralocorticoid-receptor responses.

PURPOSE: The mechanisms that mediate the acute anticonvulsant effects of deoxycorticosterone (DOC) were investigated in young rats. METHODS: Fifteen-day-old rats were pretreated with a variety of compounds, including (a) agonists of the receptors that bind DOC (mineralocorticoid receptors); (b) the DOC 5alpha- and 5alpha-3alpha-reduced metabolites, plus agonists that bind the receptors of the 5alpha-reduced metabolite of DOC (progesterone receptors); and (c) DOC itself in the presence and absence of metabolism and receptor blockers. Fifteen minutes later, pentylenetetrazol (PTZ) was administered, and maximal pentylenetetrazol (MMT) seizure responses were scored. RESULTS: Agonists of mineralocorticoid receptors increased the latency to forelimb flexion in PTZ seizures and sometimes suppressed the seizures completely. At low, nonconvulsant doses, spironolactone (a mineralocorticoid-receptor antagonist) blocked the anticonvulsant effects of a nonsedating, but not a sedating, dose of DOC. These data suggest the possible direct involvement of mineralocorticoid receptors in the anticonvulsant effects of DOC. At low, nonconvulsant doses, finasteride (which blocks the metabolism of DOC) partially blocked the protective effects of DOC, suggesting the contribution of metabolites to the anticonvulsant actions of DOC. Dihydrodeoxycorticosterone (DHDOC)-the first metabolite of DOC, an agonist at progesterone receptors, and an allosteric modulator of the gamma-aminobutyric acid (GABA)(A) receptor-and tetrahydrodeoxycorticosterone, a secondary metabolite of DOC and an allosteric modulator of the GABA(A) receptor, both blocked MMT seizures. CONCLUSIONS: These findings suggest that both DOC and its metabolites may contribute to the anticonvulsant effects seen in young rats, perhaps acting via interactions with several different receptors.

The effect of Sutherlandia frutescens on steroidogenesis: confirming indigenous wisdom.

Sutherlandia frutescens (Cancer bush), a Southern African indigenous plant, is traditionally used to treat stress related maladies linked to the endocrine system. Extracts of the shrub were used to investigate the claimed stress-relieving properties of the shrub. Dysregulation of the stress response is associated with elevated glucocorticoid levels. A model of chronic intermittent immobilization stress was investigated in 40 adult male Wistar rats to determine the effect of Sutherlandia. Immobilization stress resulted in increased corticosterone levels in the control group while rats receiving Sutherlandia extract showed significantly decreased corticosterone levels (P < 0.005). Since the biosynthesis of glucocorticoids in the adrenals is catalyzed by the cytochrome P450-dependent enzymes, the influence of Sutherlandia extracts on adrenal steroidogenesis was determined in ovine adrenocortical microsomes and mitochondria, using spectral binding and enzyme conversion assays. Water extracts showed inhibition of substrate binding to cytochrome P450 21-hydroxylase (CYP21) by 38% and cytochrome P450 11beta-hydroxylase (CYP11B1) by 60%. The conversion of progesterone and pregnenolone was inhibited by 34% and 30%, respectively. Subsequent extractions with chloroform and methanol showed inhibition of substrate binding and conversion with hydrophobic compounds exhibiting a greater inhibitory effect on deoxycorticosterone binding to CYP11B1 (30%) and on progesterone binding to CYP21 (50%). The inhibition of binding of pregnenolone to CYP17 by the chloroform extract was 62%, with negligible inhibition by the methanol extract. The chloroform extract showed a greater inhibitory effect than the methanol extract on progesterone and pregnenolone metabolism (20%-50%).

A rapid method for obtaining finasteride, a 5alpha-reductase inhibitor, from commercial tablets.

To study the effects of allopregnanolone (AP) depletion on stress-induced dopamine changes in cortical dopamine, the 5alpha-reductase inhibitor finasteride on a gram-scale is required. Two procedures for the extraction of finasteride from tablets are outlined (method A and B). In method A, a suspension of powdered tablets was preliminary extracted with chloroform and the extracts dried and evaporated. The resulting residue was then purified on column chromatography. Method B involves a direct chromatographic separation of the powdered tablets. In terms of isolated yields, the second procedure works well, is cheaper, and less time-consuming. The efficiency of the method was tested by measuring progesterone, AP and THDOC content in plasma and cerebral cortex of rats. The protocol enables the prompt availability of sufficient amount of finasteride in experimental grade, useful in examining the role of endogenous cerebrocortical AP in brain homeostasis.

Stress-induced deoxycorticosterone-derived neurosteroids modulate GABA(A) receptor function and seizure susceptibility.

Stress affects seizure susceptibility in animals and humans, but the underlying mechanisms are obscure. Here, we provide evidence that GABA(A) receptor-modulating neurosteroids derived from deoxycorticosterone (DOC) play a role in stress-related changes in seizure control. DOC, an adrenal steroid whose synthesis is enhanced during stress, undergoes sequential metabolic reduction by 5alpha-reductase and 3alpha-hydroxysteroid oxidoreductase to form 5alpha-dihydrodeoxycorticosterone (DHDOC) and allotetrahydrodeoxycorticosterone (THDOC), a GABA(A) receptor-modulating neurosteroid with anticonvulsant properties. Acute swim stress in rats significantly elevated plasma THDOC concentrations and raised the pentylenetetrazol (PTZ) seizure threshold. Small systemic doses of DOC produced comparable increases in THDOC and PTZ seizure threshold. Pretreatment with finasteride, a 5alpha-reductase inhibitor that blocks the conversion of DOC to DHDOC, reversed the antiseizure effects of stress. DOC also elevated plasma THDOC levels and protected mice against PTZ, methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate, picrotoxin, and amygdala-kindled seizures in mice (ED50 values, 84-97 mg/kg). Finasteride reversed the antiseizure activity of DOC (ED50, 7.2 mg/kg); partial antagonism was also obtained with indomethacin (100 mg/kg), an inhibitor of 3alpha-hydroxysteroid oxidoreductase. Finasteride had no effect on seizure protection by DHDOC and THDOC, whereas indomethacin partially reversed DHDOC but not THDOC. DHDOC, like THDOC, potentiated GABA-activated Cl- currents in cultured hippocampal neurons (< or =1 microm) and directly activated GABA(A) receptor currents (> or =1 microm), compatible with a role for DHDOC in the antiseizure activity of DOC. DOC is a mediator of the physiological effects of acute stress that could contribute to stress-induced changes in seizure susceptibility through its conversion to neurosteroids with modulatory actions on GABA(A) receptors including THDOC and possibly also DHDOC.

Effects of central alpha-adrenergic agonists on hormone-induced 3% NaCl and water intake.

Male rats received intracerebroventricular (ICV) renin (600 ng) or daily subcutaneous injections of deoxycorticosterone (5 mg) to induce 3% NaCl and water intake. Noradrenaline (NOR; 40-160 nmol) and clonidine (CLO; 5-20 nmol) injected ICV induced 70 to 100% inhibition of the intakes. Phenylephrine (PHE; 40-160 nmol) injected ICV induced 60 to 95% inhibition of the intakes. NOR and PHE induced a stronger inhibition on the 3% NaCl intake induced by renin than on the intake induced by deoxycorticosterone (DOC), and CLO did the opposite. CLO was always more effective than PHE to induce inhibition of the intakes. The results suggest that NOR inhibits hormone (angiotensin II, aldosterone)-induced NaCl intake by acting mainly on alpha 2-adrenergic receptors.

Increased responsiveness and decreased expression of G proteins in deoxycorticosterone hypertension.

Deoxycorticosterone-salt (DOCA-salt) hypertension is characterized by elevated vasoconstriction to agonists that stimulate G protein-mediated activation of phospholipase C. However, the mechanisms responsible for the augmented responsiveness are unknown. This study tested the hypothesis that this augmented vascular responsiveness is due to elevated content of G(alpha)q, the G protein alpha-subunit that activates phospholipase C. Thoracic aortae from DOCA-salt hypertensive rats (systolic blood pressure 183 +/- 7 mm Hg) and normotensive controls (systolic blood pressure 115 +/- 2 mm Hg) were homogenized and G protein content determined. Western analysis revealed that G(alpha)i content was decreased in DOCA compared with control rats (1364 +/- 196 versus 2343 +/- 188 densitometry units, P < or = .05) with no differences observed for G(alpha)q or G(alpha)s. In addition, contractile responses in denuded femoral artery strips revealed a significant decrease in EC50 values in DOCA arteries to all of the agonists examined: aluminum fluoride (DOCA = 1.42, control = 2.34 mmol/L), mastoparan (DOCA = 0.51, control = 35 micromol/L), phenylephrine (DOCA = 0.08, control = 0.53 micromol/L), and serotonin (DOCA = 0.014, control = 0.04 micromol/L, EC20 values). Finally, arteries from DOCA rats contracted with aluminum fluoride had increased sensitivity to G protein antagonists but not to a phospholipase C inhibitor. The enhanced contractile responsiveness in the DOCA arteries may be mediated in part through decreased G(alpha)i levels. However, it is not caused by increased concentrations of G(alpha)q in the cell membrane or by increased phospholipase C sensitivity, and the increased constrictor response to G protein stimulators of phospholipase C appears to depend primarily on increased G protein sensitivity.

Chloride alters renal blood flow autoregulation in deoxycorticosterone-treated rats.

Renal blood flow autoregulation was studied in deoxycorticosterone acetate (DOCA)-treated rats. DOCA pellets (75 mg) were implanted in uninephrectomized rats, and the animals were then fed one of four diets: (1) normal sodium chloride level, (2) high in NaCl, (3) high in chloride, or (4) high in sodium. After 40 to 45 days of the DOCA-diet treatment, animals were subjected to renal blood flow autoregulation experiments; an inflatable aortic occluder was placed proximal to the renal artery, and renal blood flow (electromagnetic flow probe) was measured while renal perfusion pressure was reduced from normal (in that animal) to 20 mm Hg, in 10 mm Hg decrements to determine the lower threshold of autoregulation. Directly measured arterial blood pressure was higher in the DOCA-high NaCl group compared with the DOCA-normal NaCl group (127 +/- 3 mm Hg vs 103 +/- 4 mm Hg) during anesthesia. Significant elevation of lower autoregulatory thresholds were demonstrated in both the DOCA-high NaCl (98 +/- 7 mm Hg) and high chloride groups (94 +/- 3 mm Hg) compared with the DOCA-normal NaCl (77 +/- 4 mm Hg) and the DOCA-high sodium (76 +/- 5 mm Hg). Pressure-flow curves of the DOCA-high chloride groups were shifted significantly downward (reduced renal blood flow at all pressures) and rightward (elevated lower threshold) compared with the DOCA-normal NaCl and -high sodium groups. These data indicate that DOCA-treated rats consuming a diet high in chloride have altered renal blood flow autoregulatory mechanisms.

Enhanced induction of gastric carcinogenesis by N-methyl-N'-nitro-N-nitrosoguanidine in deoxycorticosterone acetate-NaCl hypertensive rats and its inhibition by potassium chloride.

The effect of s.c. administration of deoxycorticosterone acetate (DOCA) plus p.o. treatment with NaCl solution on gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine and the effect of p.o. potassium supplementation on the enhanced induction of gastric carcinogenesis in DOCA-NaCl rats were investigated in Wistar rats. After 25 weeks of p.o. treatment with the carcinogen, rats received s.c. injections of DOCA (50 mg/kg) twice a week and were given 1% NaCl solution with and without 1% KCl as drinking water. In Week 52, the blood pressure, the incidence of gastric cancer, and the number of cancers per rat were significantly greater in DOCA-NaCl rats than in the untreated group. Prolonged p.o. treatment of DOCA-NaCl hypertensive rats with potassium significantly reduced their blood pressure, the incidence of gastric cancers, and their number per rat. All gastric tumors were in the glandular portions of the stomach. The norepinephrine concentration in the gastric wall and the labeling indices of gastric mucosa were significantly greater in DOCA-NaCl hypertensive rats than in the untreated group, but p.o. potassium supplementation significantly reduced the norepinephrine concentration in the gastric wall and the labeling indices of the gastric mucosa in DOCA-NaCl rats. Thus, administration of DOCA and NaCl increased the norepinephrine concentration in the gastric wall and promoted gastric carcinogenesis, and p.o. potassium supplementation decreased the norepinephrine concentration in the gastric rats. Inasmuch as the norepinephrine concentration has been used as a marker of sympathetic nervous activity, these findings suggest that the sympathetic nervous system plays an important role in gastric carcinogenesis, probably associated with cell proliferation of antral epithelial cells.

Influence of endogenous opiates on the hypotensive action of taurine in DOCA-salt rats.

We studied the role of endogenous opiate activation in the hypotensive action of taurine, a sulphur amino acid, in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Previous work had shown that supplementation with 1% taurine reduced blood pressure when given after DOCA-salt hypertension had been established. In the present study, in conscious rats, intraperitoneal injection of naloxone, an opiate antagonist, increased blood pressure in taurine-supplemented DOCA-salt rats, but not in DOCA-salt rats or vehicle-treated control rats. These results suggest that activation of an endogenous opiate might contribute to the hypotensive action of taurine in DOCA-salt hypertensive rats.

Chronic dietary administration of tryptophan prevents the development of deoxycorticosterone acetate salt induced hypertension in rats.

Hypertension developed within 3 to 5 weeks in uninephrectomized rats administered deoxycorticosterone acetate (DOCA) at a dose of 850 micrograms X kg-1 X day-1 via Silastic tubes and given isotonic saline to drink. Chronic dietary administration of tryptophan (25 and 50 g/kg of food) to DOCA-treated rats reduced their exaggerated intake of NaCl solution and attenuated the elevation of blood pressure induced by treatment with DOCA alone. Treatment with tryptophan also protected against the reduction in urinary concentrating ability during a 24-h dehydration that is characteristic of DOCA-treated rats. Other tests assessed the responsiveness to the beta-adrenergic agonist, isoproterenol. These included measurement of drinking and heart rate following acute administration of isoproterenol. The characteristically depressed drinking and chronotropic responses of DOCA-treated rats to acute administration of isoproterenol were unaffected by tryptophan. Responsiveness to angiotensin II (AII) was also tested by assessment of dipsogenic and metabolic responses to acute administration of AII. The increased drinking and tail skin temperature responses to administration of AII, characteristic of DOCA-treated rats, were reduced in a graded fashion by treatment with graded doses of tryptophan. The specific binding of AII to its receptors in membranes form the diencephalon of the brain was increased by treatment with DOCA but was returned to control level by concomitant treatment with tryptophan. The content of serotonin in the mesencephalon of the brain was not changed significantly by treatment with tryptophan, but the content of 5-hydroxyindole acetic acid in the same region increased significantly, suggesting that turnover of serotonin was increased by chronic treatment with tryptophan. The cardiac hypertrophy characteristic of treatment with DOCA was attenuated significantly by chronic treatment with tryptophan, while the low, resting plasma renin activity of the DOCA-treated group was unchanged. These results suggest that tryptophan provides significant protection against the development of DOCA-induced hypertension, polydipsia, polyuria, and cardiac hypertrophy in rats. It also reduces the hyperresponsiveness to treatment with AII, possibly by decreasing the specific binding of AII to its receptors. It also appears to increase the turnover of serotonin in the brain. Whether either one or all of these is responsible for the antihypertensive effect of tryptophan remains for further study.

Pharmacological properties of besulpamide, a new diuretic, in rats and dogs.

Besulpamide, a newly synthesized compound, has demonstrated significant diuretic activity in rats and dogs, similar to that of chlorthalidone, clopamide and xipamide. Antihypertensive activity of besulpamide is similar to that of hydrochlorothiazide and was demonstrated in rat one-kidney desoxycorticosterone acetate (DOCA)-salt hypertension. In addition, besulpamide, like hydrochlorothiazide, potentiated the antihypertensive activity of captopril in spontaneously hypertensive rats (SHR). Doses of besulpamide exceeding those normally required for pharmacological activity did not evoke adverse reactions in rats and mice.

[Dietary magnesium and mineralocorticoid DOCA-salt hypertension in the rat. Effect on the metabolism of sodium and magnesium]. / Magnésium alimentaire et hypertension minéralocorticoïde DOCA et NaCl chez le rat. Influence sur le métabolisme du sodium et du magnésium.

The effects of a diet enriched in magnesium were studied in mineralocorticoid DOCA-salt hypertensive rats from 6 to 15 weeks old, during the development of hypertension. A standard normomagnesic diet with 0.21% Mg and 0.24% Na, equilibrated in minerals and vitamins is used as a control diet. The high magnesium diet is the same standard diet enriched in magnesium with a content of 0.75% Mg. That in magnesium enriched diet lessens the level of blood pressure. This effect appears within 2 weeks and is long lasting. Increase of urinary sodium is observed but without any modification of sodium balance. Sodium plasma is not changed. Plasma, urinary and balance of magnesium are increased. These results establish that in mineralocorticoid DOCA-salt hypertension, a high magnesium diet decreases hypertension. The observed metabolic variations may perhaps explain the protective effect of this in magnesium enriched diet.

[Effects of single and repeated oral administration of MK-421 and captopril on blood pressures in normotensive and experimental hypertensive rats].

In the single dose study, the aortic blood pressure in conscious normotensive rats, 2-kidney, 1-clip renal hypertensive rats (2K-RHR), 1-kidney, 1-clip renal hypertensive rats (1K-RHR) or DOCA hypertensive rats was measured for 24 hr after the oral administration of angiotensin converting enzyme (ACE) inhibitors such as MK-421 or captopril. MK-421 at 3 mg/kg and captopril at 10 mg/kg markedly lowered the blood pressure of 2K-RHR. MK-421 at 10 mg/kg and captopril at 30 mg/kg only modestly lowered the blood pressure of 1K-RHR. In contrast, both ACE inhibitors failed to reduce blood pressure in DOCA and normotensive rats. In the repeated dose study, the systolic blood pressures in normotensive rats, 2K-RHR or spontaneously hypertensive rats (SHR) were measured twice a week for 3 weeks treatment of either MK-421 at 3 mg/kg or captopril at 10 mg/kg. Both ACE inhibitors produced significant antihypertensive effects in these model rats, and the effects were sustained throughout the treatment period. The antihypertensive effects in 2K-RHR were greater than those in SHR and normotensive rats. These results indicate that MK-421 and captopril cause the most significant antihypertensive effect in 2K-RHR in which the renin-angiotensin system played a dominant role in blood pressure regulation. The antihypertensive effect of MK-421 was approximately 3 times as potent as that of captopril in these hypertensive models.

Prevention of DOCA-salt hypertension with the calcium blocker nitrendipine.

Mononephrectomized female rats on a high sodium intake developed hypertension, hypokalemia, enlarged hearts and kidneys and slight adrenal involution under deoxycorticosterone treatment. Simultaneous administration of nitrendipine (5 mg/kg twice daily) completely prevented hypertension and reduced but did not abolish cardiac enlargement. There was no effect of the calcium slow-channel inhibitor on kidney enlargement, adrenal atrophy or hypokalemia. The ability of the steroid to produce cardiomegaly in the absence of an elevated blood pressure to account for it, tends to confirm the suggestion of other investigators that the steroid may have that effect by a mechanism not involving blood pressure elevation.

[Influence of desoxycorticosterone on the reaction of isolated segments of coronary arteries to noradrenaline in the presence of pyrogallol]. / Influenza del desossicorticosterone sulle risposte di segmenti di arterie coronarie isolate alla noradrenalina, in presenza di pirogallolo.

The effect of the desoxycorticosterone on the noradrenaline-induced relaxation of coronary arteries waw studied in vitro, after a known inhibitor of COMT, pyrogallol. Relaxation induced by noradrenaline was enhanced by desoxycorticosterone. Relaxation in response to noradrenaline was increased by desoxycorticosterone. Pyrogallol potentiated the responses of coronary strips to noradrenaline and also reduced or abolished the enhancing effects of desoxycorticosterone. It is concluded that desoxycorticosterone enhances the reponse of coronary smooth muscle to noradrenaline by inhibiting and enzymatic pathway for the inactivation of catecolamines.

[Action of morphine and amidopyrine against a background of mineralocorticoid hormone and its antagonist]. / Deistvie morfina i amidopirina na fone mineralokortikoidnogo gormona i ego antagonista.

The effect of morphine and amidopyrine, used against the background of a mineralocorticoid hormone or its antagonist excess (multiple administration of desoxycorticosteron acetate or verospiron), is mitigated. The pain-allaying effect of analgesics and their influence on the behavioral reactions are less marked in both models than it is in intact animals and their toxocity is down.