Corticosteroid receptor analyses in rat and hamster brains reveal species specificity in the type I and type II receptors

In vitro cytosol binding, receptor autoradiography with radiolabelled corticosteroid analogs, and immunocytochemistry with monoclonal antibodies have revealed the presence of two receptor systems for corticosteroids in rat and hamster brains. The type I receptor is found mainly in the hippocampal region, and in the hamster it binds cortisol (F) and corticosterone (B) with similar affinity while in the rat (a species which unlike the hamster secretes solely B) the type I receptor shows high affinity to B and not to F. The type II receptor is more widely distributed in the brain and it binds to F (hamster) or B (rat) with affinity 4-6-fold lower than to the type I. in vivo, the hamster type I and II retain F much more than B while those in the rat show the opposite. In conclusion, the present study clearly indicates species-specificity in type I and type II receptor systems in these animals. Furthermore, the type I receptor displays in vivo stringent preference for retention of the animal's predominantly circulating corticosteroid (F in hamster, in B in rat).     

An antibody that blocks insulin-like growth factor (IGF) binding to the type II IGF receptor is neither an agonist nor an inhibitor of IGF-stimulated biologic responses in L6 myoblasts

To better define the biologic function of the type II insulin-like growth factor (IGF) receptor, we raised a blocking antiserum in a rabbit by immunizing with highly purified rat type II IGF receptor. On immunoblots of crude type II receptor preparations, only bands corresponding to the type II IGF receptor were seen with IgG 3637, indicating that the antiserum was specific for the type II receptor. Competitive binding and chemical cross-linking experiments showed that IgG 3637 blocked binding of 125I-IGF-II to the rat type II IGF receptor, but did not block binding of 125I-IGF-I to the type I IGF receptor, nor did IgG 3637 block binding of 125I-insulin to the insulin receptor. In addition, IgG 3637 did not inhibit the binding of 125I-IGF-II to partially purified 150- and 40-kDa IGF carrier proteins from adult and fetal rat serum. L6 myoblasts have both type I and type II IGF receptors. IGF-I was more potent than IGF-II in stimulating N-methyl-alpha-[14C]aminoisobutyric acid uptake, 2-[3H]deoxyglucose uptake, and [3H]leucine incorporation into cellular proteins. IgG 3637 did not stimulate either 2-[3H]deoxyglucose uptake, N-methyl-alpha-[14C]aminoisobutyric acid uptake, or [3H]leucine incorporation into protein when tested alone. Furthermore, IgG 3637 at concentrations sufficient to block type II receptors under conditions of the uptake and incorporation experiments did not cause a shift to the right of the dose-response curve for stimulation of these biologic functions by IGF-II. We conclude that the type II IGF receptor does not mediate IGF stimulation of N-methyl-alpha-[14C]aminoisobutyric acid and 2-[3H]deoxyglucose uptake and protein synthesis in L6 myoblasts; presumably, the type I receptor mediates these biologic responses. The anti-type II receptor antibody inhibited IGF-II degradation in the media by greater than 90%, suggesting that the major degradative pathway for IGF-II in L6 myoblasts utilizes the type II IGF receptor.     

Investigation of the corticosteroid receptor system in rat hippocampus by ion exchange fast protein liquid chromatography

In order to study the receptor system for adrenocortical steroids, hippocampal cytosolic preparations--containing both type I and type II receptors--were subjected to anion exchange fast protein liquid chromatography (FPLC). With running buffer containing Tris, EDTA, and glycerol three peaks (1-3) were eluted from the column at 220, 400 and 560 mM NaCl respectively regardless of whether [3H]corticosterone or [3H]RU 28362 had been used as radiotracer. None of the peaks was caused by serum transcortin as revealed by control studies. However, the sequestering influence of transcortin on receptor binding of corticosterone could be demonstrated by the FPLC technique with mixtures containing serum and hippocampus cytosol. Competition experiments with cytosolic samples revealed that type I receptor was present only in peaks 2 and 3 while type II was found in all three peaks in variable amounts, depending on the presence of molybdate. When molybdate was added to the running buffer only two peaks (2 and 3) were eluted, both containing type I and type II receptors. Peak 1 was attributed to the activated type II receptor while peak 2 represented nonactivated receptors. The origin of peak 3 remains uncertain. The data indicate that molybdate must be present in the cytosolic preparation and in the running buffer to keep type II receptor in its nonactivated form. Type I receptor was probably not transformed into the activated form in the absence of molybdate but lost binding capacity and/or affinity for corticosterone.     

Melanotropins: aldosterone- and corticosterone-stimulating activity in isolated rat adrenal cells

Stimulation of corticosterone and aldosterone production in rat adrenal decapsular and capsular cells respectively by alpha- and beta-melanotropins (alpha- and beta-MSH) have been investigated. Although they are considerably less potent than corticotropin (ACTH), the dose-response curves are parallel to those for ACTH except in the case of aldosterone stimulation by beta-MSH where the dose-response curve is biphasic. The steroidogenic actions of ACTH, beta-MSH and alpha-MSH are antagonized by corticotropin-inhibiting peptide in both capsular and decapsular cells. A synthetic analog of beta-MSH ([Gly10]-betac1-MSH) inhibits both beta-MSH and ACTH in aldosterone output in capsular cells, but it does not inhibit their corticosteroidogenic actions in decapsular cells.     

Corticosterone regulation of brain and lymphoid corticosteroid receptors

Circulating lymphocytes are often used as a model for brain corticosteroid receptor regulation in clinical disease states, although it is not known if lymphoid receptors are regulated in a similar manner as brain receptors. In the present study the regulation of brain (hippocampus, frontal cortex, hypothalamus and striatum), lymphoid (circulating lymphocytes, spleen and thymus) and pituitary glucocorticoid receptors in response to alterations in circulating corticosterone levels was examined. Seven days following adrenalectomy, type II corticosteroid receptors (i.e. glucocorticoid receptors) were significantly increased in the hippocampus, frontal cortex and hypothalamus, but not in any other tissues. Administration of corticosterone (10 mg/kg) for 7 days significantly decreased type II as well as type I (i.e. mineralocorticoid receptors) receptors in the hippocampus. Type II receptors in the frontal cortex, circulating lymphocytes and spleen were also significantly decreased by chronic corticosterone treatment. Immobilization stress (2 h a day for 5 days) failed to alter receptor density in any of the tissues. These results demonstrate that homologous regulation of corticosteroid receptors by corticosterone does not invariably occur in all tissues and emphasize the complex degree of regulation of these receptors. However, the simultaneous downregulation of both hippocampal and lymphocyte glucocorticoid receptors by corticosterone provides support for the hypothesis that circulating lymphocytes do reflect some aspects of brain glucocorticoid receptor regulation.     

Corticosterone decreases the efficacy of adrenaline to affect passive avoidance retention of adrenalectomized rats

Short-term (48h) adrenalectomy (ADX) resulted in a deficit in the retention of a passive avoidance response. An inverted U-shaped dose-response relationship was found following immediate post-learning administration of adrenaline (A). A in a dose range of 0.005 - 5 micrograms/kg s.c. facilitated later retention. While corticosterone (CS) replacement alone had no effect, pretreatment with CS (300 micrograms/kg) was followed by a shift in the dose-response curve of A in ADX rats. Ten thousand times higher doses of A were required to improve retention behavior. Administration of the potent synthetic glucocorticoid dexamethasone failed to affect the responsiveness to A. It is concluded that corticosterone decreases the efficacy by which adrenaline affects later retention behavior of ADX rats. The specificity of corticosterone in this interaction suggests the involvement of the corticosterone receptor system which has its predominant localization in hippocampal neurons.     

CaM kinase II and protein kinase C activations mediate enhancement of long-term potentiation by nefiracetam in the rat hippocampal CA1 region

Nefiracetam is a pyrrolidine-related nootropic drug exhibiting various pharmacological actions such as cognitive-enhancing effect. We previously showed that nefiracetam potentiates NMDA-induced currents in cultured rat cortical neurons. To address questions whether nefiracetam affects NMDA receptor-dependent synaptic plasticity in the hippocampus, we assessed effects of nefiracetam on NMDA receptor-dependent long-term potentiation (LTP) by electrophysiology and LTP-induced phosphorylation of synaptic proteins by immunoblotting analysis. Nefiracetam treatment at 1-1000 nM increased the slope of fEPSPs in a dose-dependent manner. The enhancement was associated with increased phosphorylation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor through activation of calcium/calmodulin-dependent protein kinase II (CaMKII) without affecting synapsin I phosphorylation. In addition, nefiracetam treatment increased PKCalpha activity in a bell-shaped dose-response curve which peaked at 10 nM, thereby increasing phosphorylation of myristoylated alanine-rich protein kinase C substrate and NMDA receptor. Nefiracetam treatment did not affect protein kinase A activity. Consistent with the bell-shaped PKCalpha activation, nefiracetam treatment enhanced LTP in the rat hippocampal CA1 region with the same bell-shaped dose-response curve. Furthermore, nefiracetam-induced LTP enhancement was closely associated with CaMKII and PKCalpha activation with concomitant increases in phosphorylation of their endogenous substrates except for synapsin I. These results suggest that nefiracetam potentiates AMPA receptor-mediated fEPSPs through CaMKII activation and enhances NMDA receptor-dependent LTP through potentiation of the post-synaptic CaMKII and protein kinase C activities. Together with potentiation of nicotinic acetylcholine receptor function, nefiracetam-enhanced AMPA and NMDA receptor functions likely contribute to improvement of cognitive function.     

Atrial natriuretic peptide inhibits the stimulation of aldosterone secretion by ACTH in vitro and in vivo

Previous studies have shown that atrial natriuretic peptide (ANP) inhibits the secretion of aldosterone by isolated adrenal glomerulosa cells stimulated by angiotensin II, ACTH and potassium in vitro and by angiotensin II in conscious unrestrained rats. In this study we investigated further the effects of synthetic ANP on the dose-response curve of aldosterone secretion stimulated by ACTH in vitro. ANP displaced the dose-response curve of aldosterone to ACTH to the right with a significant change in EC50. A similar effect of ANP was reproduced in vivo in conscious unrestrained rats. There was no significant effect of ANP on the corticosterone response to ACTH in vivo. ANP is a potent regulator of aldosterone secretion which may modulate the effects of ACTH on the adrenal in vitro and in vivo.     

Molecular cloning of a mineralocorticoid (type I) receptor complementary DNA from rat hippocampus

Rat brain expresses two types of corticosteroid-binding proteins. The type I receptor binds corticosterone with high affinity and is structurally related to the kidney mineralocorticoid receptor (MR), while the type II or classical glucocorticoid receptor binds corticosterone with lower affinity and displays an in vivo preference for dexamethasone. Here we describe the isolation and characterization of a cDNA coding for the MR, from a rat hippocampus cDNA library, by low stringency hybridization to radiolabeled human glucocorticoid receptor cDNA. The nucleotide and deduced amino acid sequence for rat hippocampal MR displays extensive homology to a MR cDNA isolated from human kidney, suggesting that they are orthologous genes. Southern analysis suggests that there is only one gene for the MR, and in vitro expression of the receptor generates a high affinity corticosterone-binding protein. These data provide evidence to support the contention that a single gene gives rise to the MR in renal tissues and type I receptors in the brain.     

Modulation of aldosterone receptors in rat kidney: effects of steroid treatment and potassium diet

The numbers of type I and type II aldosterone receptors in the kidney cytosol of adrenalectomized rats were estimated after animals were treated with various steroids, or fed with high or low potassium diets. Oestradiol and 5 beta-pregnane-3,20 dione, which exhibited no affinity for aldosterone receptors, did not modify the levels of type I or type II receptors. Cortisol, corticosterone, progesterone and spirolactones, which all competed with aldosterone for both types of receptor, reduced the number of type I sites, as does aldosterone itself. Steroid treatment has no appreciable effect on type II receptors. We conclude that type I receptors are modulated by steroids able to bind to aldosterone receptors and that steroid-receptor interaction is an essential step in the receptor modulation process. The effects of potassium on aldosterone receptor modulation were tested in adrenalectomized rats on hypo- or hyperkalaemic diets. No change in receptor levels was observed in the rats on a low potassium diet, but the number of type I receptors increased in animals on a high potassium diet. However, the effects of potassium on receptor modulation were of lesser magnitude than those of aldosterone agonists and antagonists.     

Blockade of the pre- and postjunctional effects of angiotensin in vivo with a non-peptide angiotensin receptor antagonist

Recent reports indicate that some imidazole-5-acetic acid derivatives are competitive antagonists of angiotensin II receptors. However, to our knowledge, there is no published information regarding: 1) what constant infusion rate of these non-peptide angiotensin receptor blockers is necessary to effectively antagonize angiotensin receptors in vivo, 2) whether imidazole-5-acetic acid derivatives antagonize both prejunctional and postjunctional angiotensin receptors, and 3) whether effective levels of these compounds exert non-specific actions and/or partial agonist activity. To address these issues, either vehicle, 2-butyl-4-chloro-1-(2-nitrobenzyl) imidazole-5-acetic acid (CV-2961; 30 and 100 micrograms/min) or a standard angiotensin receptor blocker, 1Sar8Ile-angiotensin II (100 ng/min), was infused intravenously into captopril-treated rats that were prepared for in situ perfusion of their mesenteric vascular beds. Infusion of CV-2961 for two and one-half hours did not alter arterial blood pressure, mesenteric perfusion pressure, plasma aldosterone level, or mesenteric vascular responses to sympathetic nerve stimulation or exogenous norepinephrine. The higher dose of CV-2961 (100 micrograms/min) completely blocked angiotensin II-induced enhancement of vascular responses to sympathetic nerve stimulation and shifted the angiotensin dose-response curve 10-fold to the right with respect to angiotensin II-induced increases in mesenteric perfusion pressure. The effects of the lower dose of CV-2961 (30 micrograms/min) on these actions of angiotensin II were not statistically significant. 1Sar8Ile-angiotensin II abolished both the prejunctional and postjunctional effects of angiotensin II. We conclude that in intact rats CV-2961, infused at 100 micrograms/min, antagonizes both prejunctional and postjunctional angiotensin II receptors, yet has a somewhat greater effect on the prejunctional actions of angiotensin II. CV-2961 is devoid of partial agonist activity, and no non-specific actions of CV-2961 are evident. Imidazole-5-acetic acid derivatives may find considerable utility as pharmacological probes and as therapeutic agents.     

Analysis of the dual mechanism of ACTH release by arginine vasopressin and its analogs in conscious rats

Plasma ACTH and/or corticosterone levels were measured in conscious rats 30 min after subcutaneous administration of arginine vasopressin (AVP), oxytocin (OT) and various analogs with a large range of activity on the vasopressor (V1), antidiuretic (V2) or oxytocic receptors. The comparison of their dose-response curves indicated that two different mechanisms are involved in the release of ACTH by neurohypophysial peptides and their analogs. AVP itself and a specific vasopressor agonist (Phe2, Orn8, OT) displayed a similar, high slope dose-response curve. Non-vasopressor analogs, such as dDAVP were characterized by a low slope dose-response curve. Furthermore, dDAVP potentiated CRF and neither its own ACTH-releasing action nor its potentiation of CRF were sensitive to previous VI- or V2-receptor blockade. These results, together with other available data, are interpreted as indicative of the existence of two mechanisms of action for ACTH release by AVP and its analogs in vivo: an indirect action via endogenous CRF release, mediated by a VI receptor mechanism, and a direct action on the pituitary, shared by dDAVP and other non-vasopressor analogs, with receptor characteristics different to both the V1 and the V2 classical types.     

Effects of melatonin on the behavioral and hormonal responses of red-sided garter snakes (Thamnophis sirtalis parietalis) to exogenous corticosterone

We investigated possible interactions between melatonin and corticosterone in modulating the reproductive behavior of male red-sided garter snakes (Thamnophis sirtalis parietalis) following spring emergence. We also examined whether melatonin's modulatory actions could be explained by its potential properties as a serotonin receptor antagonist. Exogenous corticosterone significantly reduced courtship behavior of male snakes in a dose-dependent manner. Melatonin also significantly reduced courtship behavior of male garter snakes. Pretreatment with melatonin before administering corticosterone treatments further suppressed courtship behavior of red-sided garter snakes. These results indicate additive inhibitory effects of melatonin and corticosterone in modulating reproductive behavior. Snakes receiving ketanserin, a serotonergic type 2A receptor antagonist, followed by corticosterone also showed reduced courtship behavior; this serotonin receptor antagonist followed by treatment with vehicle did not significantly influence courtship behavior of male snakes. Neither melatonin nor corticosterone treatments significantly influenced testosterone + 5-alpha-dihydrotestosterone concentrations of male garter snakes, supporting a direct effect of melatonin and corticosterone on courtship behavior that is independent of any effect on androgen concentrations. We propose that a serotonin system is involved in the modulation of male courtship behavior by melatonin and corticosterone. In addition, our data support the hypothesis that melatonin may function as a serotonin receptor antagonist. Further research is necessary to discern whether the actions of melatonin and corticosterone are converging on the same pathway or if their effects on different pathways are having additive inhibitory effects on courtship behavior.     

Subcellular distribution and activation of rat submandibular cAMP-dependent protein kinase following beta-adrenergic receptor stimulation

The extent of activation of rat submandibular protein kinase A (EC 2.7.1.37) isozymes following beta-adrenergic receptor stimulation was determined in vitro using dispersed cells and an 8-N3-[32P]cAMP photoprobe. The half-maximal binding of the photoprobe for microsomal and cytosolic type I and cytosolic type II was 9 nM, 27 nM and 92 nM, respectively. 'Cold trap' studies indicated that 70% of type I protein kinase A was activated following maximal beta-adrenergic receptor stimulation, whereas type II activation was less than 40%. Both cytosolic and microsomal type I activation occurred rapidly following beta-adrenergic receptor stimulation and both remain activated throughout the entire secretory period. Type I inactivation occurred rapidly subsequent to beta-adrenergic receptor blockade. The dose-response relationship for the isotypes following beta-adrenergic receptor activation demonstrated a greater extent of type I activation at submaximal concentrations of agonist. Although protein kinase A may not be the only kinase involved in rat submandibular mucin release, these data add further support to a direct regulatory role for this kinase, with type I having potentially a greater role than type II.     

Cross-tolerance studies between caffeine and (-)-N6-(phenylisopropyl)-adenosine (PIA) in mice

Chronic administration of caffeine to mice (1 mg/ml in drinking water X 14 d) led to a downward shift in the dose-response curve for the locomotor effects of caffeine. Caffeine was also less effective as an antagonist against (-)-(N6-phenylisopropyl)-adenosine (PIA)-induced analgesia in the tail flick assay in these animals. The dose-response curves of PIA for both analgesia and locomotor depression were shifted to the left in animals chronically administered caffeine. In mice chronically administered PIA (1 mg/kg/d X 14 d), the dose-response curves of PIA for both analgesia and locomotor depression were shifted to the right. The dose-response curve for the locomotor effects of caffeine was shifted to the left, and caffeine exhibited greater antagonist activity against the analgesic action of PIA in these animals. There was no change in the Kd or Bmax values of either 3H-PIA or 3H-diethylphenylxanthine (DPX, a potent adenosine receptor antagonist) in mice chronically administered PIA. The Bmax values for both 3H-PIA and 3H-DPX were significantly increased, while the Kd values were not changed in mice chronically administered caffeine. There was no detectable change in the brain levels of either PIA or caffeine in animals chronically treated with either drug. The results demonstrate that chronic administration of caffeine increases the sensitivity of mice to the actions of PIA and vice versa, providing supportive evidence for the interaction of these drugs at the same receptor, which is probably an adenosine receptor.     

(Thr-59)-insulin-like growth factor I stimulates 2-deoxyglucose transport in BC3H1 myocytes through the insulin-like growth factor receptor, not the insulin receptor

The murine non-fusing muscle cell line contains distinct receptors for insulin and insulin-like growth factors. Pretreatment of myocytes with insulin for 20 h at 37 degrees C inhibits the binding of [125I]iodoinsulin by 60% without affecting the binding of [125I]iodoinsulin-like growth factor I. The ED50 values for down-regulation of the insulin and insulin-like growth factor receptor by their respective ligands are 1 nM and 3 nM, respectively. Insulin, (Thr-59)-insulin-like growth factor I and multiplication-stimulating activity stimulate 2-[3H]deoxyglucose transport in myocytes with ED50 values of 5 nM, 5.6 nM and 33 nM, respectively. In order to determine whether (Thr-59)-insulin-like growth factor I stimulates 2-[3H]deoxyglucose transport in myocytes via its own receptor or the insulin receptor, we determined the activity of these peptides after down-regulation of the insulin receptor. The rate of 2-[3H]deoxyglucose transport in myocytes pretreated with insulin (5 nM) is elevated but returns to control levels by 1 h after the washout of insulin. The dose-response curve for insulin-stimulated 2-[3H]deoxyglucose transport is shifted to the right (ED50 greater than 100 nM) immediately after insulin washout but is normal by 1 h after insulin washout. In contrast, the dose-response curve for (Thr-59)-insulin-like growth factor I is unchanged in insulin-pretreated cells immediately after insulin washout. These data show that (Thr-59)-insulin-like growth factor I stimulates 2-[3H]deoxyglucose transport in myocytes by acting through an insulin-like growth factor receptor and not through the insulin receptor. Since multiplication-stimulating activity is 6-fold less active than (Thr-59)-insulin-like growth factor, they both may be acting through a type 1 insulin-like growth factor receptor.     

Receptors for insulin-like growth factors I and II in developing embryonic mouse limb bud

Insulin-like growth factors (IGF) or somatomedins (SM) have been classically defined as promoting the actions of growth hormone in skeletal growth. IGF is divided into two groups, IGF-I and II, and are presumed to act via IGF type I (higher affinity for IGF-I and II and very low affinity for insulin) and II (higher affinity for IGF-II than I and no affinity for insulin) receptors, respectively. Recently, a switchover role of IGF-II to I during fetal to adult growth has been suggested. We have investigated the possible transitional role of IGF-II to I in a developing mouse embryonic limb bud organ culture model. In this in vitro system, limb bud develops from the blastoma stage to a well-differentiated cartilage tissue. Both IGF type I and II receptors were found to be present in limb buds at all stages of differentiation. Type I receptor decreased with differentiation while Type II receptor increased. The effect of IGF-I on [3H]thymidine and [35S]sulfate uptake by the tissue increased with differentiation while the effect of IGF-II on [3H]thymidine uptake of the undifferentiated tissue was abolished with differentiation of the tissue. The increase of the IGF-I response with decreased type I receptor may reflect an altered receptor sensitivity (occupancy) during differentiation. The decrease of the IGF-II response with increased type II receptor with differentiation may on the other hand suggest that IGF-II in differentiated tissue no longer acts as a classical growth factor. These results tend to support the hypothesis of the switchover role of IGF-I and II during fetal and adult growth, however, confirmation of the precise role of IGF-I and II in biological growth may have to wait until further studies clarifying the significance of the increased IGF type II receptor in differentiated tissue are made.