Effect Of Administration Of Luteinizing-Hormone (Lh) And Deprivation Of Lh On The Proteins Of Smooth Endoplasmic-Reticulum Of Immature And Adult-Rat Leydig-Cells

Analysis of proteins of smooth endoplasmic reticulum (SER) of Leydig cells from immature and admit rats by two-dimensional polyacrylamide gel electrophoresis (SDS-PAGE) revealed the presence of several new proteins in the adult rats. Administration of human chorionic gonadotropin to immature rats for ten days also resulted in a significant increase as well as the appearance of several new proteins. The general pattern of SDS-PAGE analysis of the SER proteins of Leydig cells resembled that of the adult rat. SDS-PAGE analysis of the SER proteins of Leydig cells from adult rats following deprivation of endogenous luteinizing hormone by administration of antiserum to ovine luteinizing hormone resulted in a pattern which to certain extent resembled that of an immature I at. Western Blot analysis of luteinizing hormone antiserum treated rat Leydig cell proteins revealed a decrease in the 17-alpha-hydroxylase compared to the control. These results provide biochemical evidence for the suggestion that one of the main functions of luteinizing hormone is the control of biogenesis and/or turnover SER of Leydig cells in the rat.

Effect of specific FSH or LH deprivation on testicular function of the adult rat

While the need for FSH in initiating spermatogenesis in the immature rat is well accepted, its requirement for maintenance of spermatogenesis in adulthood is questioned. In the current study, using gonadotropin antisera to neutralize specifically either endogenous FSH or LH, we have investigated the effect of either FSH or LH deprivation for a 10-day period on (i) testicular macromolecular synthesis in vitro, (ii) the activities of testicular germ cell specific LDH-X and hyaluronidase enzymes, and finally (iii) on the concentration of sulphated glycoprotein (SGP-2), one of the Sertoli cell marker proteins. Both immature (35-day-old) and adult (100-day-old) rats have been used in this study. Since LH deprivation leads to a near total blockade of testosterone production, the ability of exogenous testosterone supplementation to override the effects of LH deficiency has also been evaluated. Deprivation of either of the gonadotropins significantly affected in vitro RNA and protein synthesis by both testicular minces as well as single cell preparations. Fractionation of dispersed testicular cells preincubated with labelled precursors of RNA and protein on Percoll density gradient revealed that FSH deprivation affected specifically the rate of RNA and protein synthesis of germ cell and not Leydig cell fraction. LH but not FSH deprivation inhibited [3H]thymidine incorporation into DNA. The inhibitory effect of LH could mostly be overriden by testosterone supplementation. LDH-X and hyaluronidase activities of testicular homogenates of adult rats showed significant reduction (50%; P less than .05) following either FSH or LH deprivation. Again testosterone supplementation was able to reverse the LH inhibitory effect.

Hormonal modulation of secretion of immunoreactive riboflavin carrier protein by adult rat Leydig cells in vitro

Adult rat Leydig cells in culture synthesize and secrete riboflavin carrier protein (RCP) as demonstrated by [S-35]-methionine incorporation into newly synthesized proteins followed by immunoprecipitation as well as specific radioimmunoassay. LH stimulates the secretion of RCP 4-fold which could be inhibited upto 75% by an aromatase inhibitor. 8-bromo-cyclic AMP and cholera toxin could mimic the LH stimulated secretion of the carrier protein. The extent of stimulation of RCP secretion brought about by exogenous estradiol-17 beta is comparable to that of LH. The antiestrogen tamoxifen, when added along with either LH or estrogen, inhibited the stimulated levels significantly. These results show that the estrogen-inducible riboflavin carrier is secreted by Leydig cells under positive regulation of LH.

Adult rat retinal ganglion cells and glia can be printed by piezoelectric inkjet printing.

We have investigated whether inkjet printing technology can be extended to print cells of the adult rat central nervous system (CNS), retinal ganglion cells (RGC) and glia, and the effects on survival and growth of these cells in culture, which is an important step in the development of tissue grafts for regenerative medicine, and may aid in the cure of blindness. We observed that RGC and glia can be successfully printed using a piezoelectric printer. Whilst inkjet printing reduced the cell population due to sedimentation within the printing system, imaging of the printhead nozzle, which is the area where the cells experience the greatest shear stress and rate, confirmed that there was no evidence of destruction or even significant distortion of the cells during jet ejection and drop formation. Importantly, the viability of the cells was not affected by the printing process. When we cultured the same number of printed and non-printed RGC/glial cells, there was no significant difference in cell survival and RGC neurite outgrowth. In addition, use of a glial substrate significantly increased RGC neurite outgrowth, and this effect was retained when the cells had been printed. In conclusion, printing of RGC and glia using a piezoelectric printhead does not adversely affect viability and survival/growth of the cells in culture. Importantly, printed glial cells retain their growth-promoting properties when used as a substrate, opening new avenues for printed CNS grafts in regenerative medicine.

Neonatal exposure to estradiol in female rats influences neuroactive steroid concentrations and behaviour in the adult rat

The gonadal steroids, in particular estradiol, exert an important action during perinatal period in the regulation of sexual dimorphism and neuronal plasticity, and in the growth and development of nervous system. Exposure of the developing female to estrogens during perinatal period may have long-lasting effects that are now regarded as “programming” the female neuroendocrine axis to malfunction in adulthood. The purpose of this study was to describe the effect of a single administration of a low dose (10 μg) of β-estradiol 3-benzoate (EB) to female rats on the day of birth on brain and plasma concentrations of the neuroactive steroid allopregnanolone, general behaviours and behavioral sensitivity to benzodiazepines. Neonatal administration of EB induces a dramatic reduction in the cerebrocortical and plasma levels of allopregnanolone and progesterone that were apparent in both juvenile (21 days) and adult (60 days). In contrast, this treatment did not affect 17β-estradiol levels. Female rats treated with β-estradiol 3-benzoate showed a delay in vaginal opening, aciclicity characterized by prolonged estrus, and ovarian failure. Given that allopregnanolone elicits anxiolytic, antidepressive, anticonvulsant, sedative-hypnotic effects and facilitates social behaviour, we assessed whether this treatment might modify different emotional, cognitive and social behaviours. This treatment did not affect locomotor activity, anxiety- and mood-related behaviours, seizures sensitivity and spatial memory. In contrast, neonatal β-estradiol 3-benzoate-treated rats showed a dominant, but not aggressive, behaviour and an increase in body investigation, especially anogenital investigation, characteristic of male appetitive behaviour. On the contrary, neonatal administration of β-estradiol 3-benzoate to female rats increases sensitivity to the anxiolytic, sedative, and amnesic effects of diazepam in adulthood. These results indicate that the marked and persistent reduction in the cerebrocortical and peripheral concentration of the neuroactive steroid allopregnanolone induced by neonatal treatment with β-estradiol 3-benzoate does not change baseline behaviours in adult rats. On the contrary, the low levels of allopregnanolone seems to be associated to changes in the behavioural sensitivity to the positive allosteric modulator of the GABAA receptor, diazepam. These effects of estradiol suggest that it plays a major role in pharmacological regulation both of GABAergic transmission and of the abundance of endogenous modulators of such transmission during development of the central nervous system.

IMMUNOCYTOCHEMICAL AND RADIOIMMUNOMETRICAL DEMONSTRATION OF SEROTONIN-IMMUNOREACTIVITIES AND NEUROPEPTIDE-IMMUNOREACTIVITIES IN THE ADULT-RAT TAPEWORM, HYMENOLEPIS-DIMINUTA (CESTODA, CYCLOPHYLLIDEA)

Standard indirect immunocytochemical techniques have been interfaced with confocal scanning laser microscopy (for whole-mount preparations) and epifluorescence microscopy (for cryosections) to investigate the occurrence and distribution of serotoninergic and peptidergic nerve elements in adult H. diminuta. Serotonin (5-HT)-immunoreactivity (IR) was widespread throughout the worm, occurring in the paired cerebral ganglia, transverse commissure, the 10 longitudinal nerve cords and in a plethora of small nerve fibres of the peripheral nervous system. An abundance of serotoninergic nerve cell bodies was found in association with the lateral nerve cords. The genital atrium and accessory reproductive ducts were richly innervated with serotoninergic nerve fibres. Thirty-five antisera to 20 vertebrate regulatory peptides and 1 invertebrate peptide (FMRFamide) were used to screen the worm for neuropeptide IR. Immunostaining was obtained with antisera raised to pancreatic polypeptide (PP), peptide YY (PYY), neuropeptide Y (NPY), substance P (SP), peptide histidine isoleucine (PHI), xenopsin (XP) and FMRFamide. The most extensive pattern of IR occurred with antisera to PP and PYY, IR being evident in the cerebral ganglia, transverse commissure, longitudinal nerve cords and in small nerve fibres that ramified throughout the parenchyma. A series of bipolar nerve cell bodies between the median nerve cords displayed PP/PYY-IR. The distribution of FMRFamide-IR was reminiscent of the PP/PYY pattern but was less extensive. Comparison of the serotoninergic and peptidergic nervous systems has revealed general similarities and some distinct differences, especially with regard to the distribution of immunoreactive nerve cell bodies. Quantitative data are presented on the levels of PP-, SP-, PHI-, and gastrin-releasing peptide (GRP)-immunoreactivities demonstrable in acid-alcohol extracts of whole worms. The highest level of peptide IR determined was recorded for PP.

Temporal changes in mRNA expression of the brain nutrient transporters in the lithium-pilocarpine model of epilepsy in the immature and adult rat.

The lithium-pilocarpine model mimics most features of human temporal lobe epilepsy. Following our prior studies of cerebral metabolic changes, here we explored the expression of transporters for glucose (GLUT1 and GLUT3) and monocarboxylates (MCT1 and MCT2) during and after status epilepticus (SE) induced by lithium-pilocarpine in PN10, PN21, and adult rats. In situ hybridization was used to study the expression of transporter mRNAs during the acute phase (1, 4, 12 and 24h of SE), the latent phase, and the early and late chronic phases. During SE, GLUT1 expression was increased throughout the brain between 1 and 12h of SE, more strongly in adult rats; GLUT3 increased only transiently, at 1 and 4h of SE and mainly in PN10 rats; MCT1 was increased at all ages but 5-10-fold more in adult than in immature rats; MCT2 expression increased mainly in adult rats. At all ages, MCT1 and MCT2 up-regulation was limited to the circuit of seizures while GLUT1 and GLUT3 changes were more widespread. During the latent and chronic phases, the expression of nutrient transporters was normal in PN10 rats. In PN21 rats, GLUT1 was up-regulated in all brain regions. In contrast, in adult rats GLUT1 expression was down-regulated in the piriform cortex, hilus and CA1 as a result of extensive neuronal death. The changes in nutrient transporter expression reported here further support previous findings in other experimental models demonstrating rapid transcriptional responses to marked changes in cerebral energetic/glucose demand.

Sympathetic Stimulation and Hypertension in the Pyridoxine-Deficient Adult Rat

Pyridoxal phosphate is the coenzyme of various decarboxylases involved in the formation of monoamine neurotransmitters such as y-aminobutyric acid , serotonin , dopamine, and norepinephrine . Adult male Sprague-Dawley rats placed on a pyridoxine -deficient diet for 8 weeks showed significant hypertension compared with pyridoxine -supplemented controls . Hypothalamic contents of pyridoxal phosphate , y-aminobutyric acid, and serotonin in the pyridoxine - deficient rats were significantly lower than those in pyridoxine -supplemented controls . Hypertension was associated with sympathetic stimulation . Treatment of pyridoxine-deficient rats with a single dose of pyridoxine (10 mg/kg body weight) reversed the blood pressure to normal levels within 24 hours, with concomitant restorations of hypothalamic serotonin and y-aminobutyric acid as well as the return of plasma norepinephrine and epinephrine to normal levels . Also, pyridoxine treatment reversed the hypothalamic hypothyroidism observed in pyridoxine -deficient rats . These results indicate an association between pyridoxine deficiency and sympathetic stimulation leading to hypertension.

Cholinergic modulation of intrinsic fibre-evoked excitatory transmission contains a nicotinic component in immature but not adult rat piriform cortex, in vitro

The piriform cortex (PC) is highly prone to epileptogenesis, particularly in immature animals, where decreased muscarinic modulation of PC intrinsic fibre excitatory neurotransmission is implicated as a likely cause. However, whether higher levels of acetylcholine (ACh) release occur in immature vs. adult PC remains unclear. We investigated this using in vitro extracellular electrophysiological recording techniques. Intrinsic fibre-evoked extracellular field potentials (EFPs) were recorded from layers II to III in PC brain slices prepared from immature (P14-18) and adult (P>40) rats. Adult and immature PC EFPs were suppressed by eserine (1muM) or neostigmine (1muM) application, with a greater suppression in immature ( approximately 40%) than adult ( approximately 30%) slices. Subsequent application of atropine (1muM) reversed EFP suppression, producing supranormal ( approximately 12%) recovery in adult slices, suggesting that suppression was solely muscarinic ACh receptor-mediated and that some 'basal' cholinergic 'tone' was present. Conversely, atropine only partially reversed anticholinesterase effects in immature slices, suggesting the presence of additional non-muscarinic modulation. Accordingly, nicotine (50muM) caused immature field suppression ( approximately 30%) that was further enhanced by neostigmine, whereas it had no effect on adult EFPs. Unlike atropine, nicotinic antagonists, mecamylamine and methyllycaconitine, induced immature supranormal field recovery ( approximately 20%) following anticholinesterase-induced suppression (with no effect on adult slices), confirming that basal cholinergic 'tone' was also present. We suggest that nicotinic inhibitory cholinergic modulation occurs in the immature rat PC intrinsic excitatory fibre system, possibly to complement the existing, weak muscarinic modulation, and could be another important developmentally regulated system governing immature PC susceptibility towards epileptogenesis.

Δ9-tetrahydrocannabivarin suppresses in vitro epileptiform and in vivo seizure activity in adult rat

Purpose: We assessed the anticonvulsant potential of the phytocannabinoid Δ9-tetrahydrocannabivarin (Δ9-THCV) by investigating its effects in an in vitro piriform cortex (PC) brain slice model of epileptiform activity, on cannabinoid CB1 receptor radioligand-binding assays and in a generalized seizure model in rats. Methods: Δ9-THCV was applied before (10 μmΔ9-THCV) or during (10–50 μmΔ9-THCV) epileptiform activity induced by Mg2+-free extracellular media in adult rat PC slices and measured using multielectrode array (MEA) extracellular electrophysiologic techniques. The actions of Δ9-THCV on CB1 receptors were examined using [3H]SR141716A competition binding and [35S]GTPS assays in rat cortical membranes. Effects of Δ9-THCV (0.025–2.5 mg/kg) on pentylenetetrazole (PTZ)–induced seizures in adult rats were also assessed. Results: After induction of stable spontaneous epileptiform activity, acute Δ9-THCV application (≥20 μm) significantly reduced burst complex incidence and the amplitude and frequency of paroxysmal depolarizing shifts (PDSs). Furthermore, slices pretreated with 10 μmΔ9-THCV prior to induction of epileptiform activity exhibited significantly reduced burst complex incidence and PDS peak amplitude. In radioligand-binding experiments, Δ9-THCV acted as a CB1 receptor ligand, displacing 0.5 nm [3H]SR141716A with a Ki∼290 nm, but exerted no agonist stimulation of [35S]GTPS binding. In PTZ-induced seizures in vivo, 0.25 mg/kg Δ9-THCV significantly reduced seizure incidence. Discussion: These data demonstrate that Δ9-THCV exerts antiepileptiform and anticonvulsant properties, actions that are consistent with a CB1 receptor–mediated mechanism and suggest possible therapeutic application in the treatment of pathophysiologic hyperexcitability states.

Forced expression of the cyclin B1-CDC2 complex induces proliferation in adult rat cardiomyocytes

Repair of the mature mammalian myocardium following injury is impaired by the inability of the majority of cardiomyocytes to undergo cell division. We show that overexpression of the cyclin B1-CDC2 (cell division cycle 2 kinase) complex re-initiates cell division in adult cardiomyocytes. Thus strategies targeting the cyclin B1-CDC2 complex might re-initiate cell division in mature cardiomyocytes in vivo and facilitate myocardial regeneration following injury.

Role of G1 phase cyclins and cyclin dependent kinases during hypertrophic growth of adult rat cardiomyocytes

Cell cycle regulatory molecules are implicated in cardiomyocyte hypertrophy. We have investigated protein expression of cyclins A, D1–3, and E and cyclin-dependent kinases (CDKs) 2, 4, 5, and 6 in left ventricular (LV) tissues during the development of LV hypertrophy in rats following aortic constriction (AC). Compared with their expression in sham-operated controls (SH), expression of cyclins D2 and D3 and of CDK4 and CDK6 increased significantly fromday 3 to day 21 after AC concomitant with increased LV mass. However, no significant difference was observed for CDK2 or CDK5. Cyclins A, D1, and E were undetectable. In vitro kinase activities of CDK4 and CDK6 increased ∼70% from day 7 to day 14 in AC myocytes compared with SH myocytes (P< 0.03). Fluorescence-activated cell sorter analysis revealed a G0/G1to G2/M phase progression in AC myocyte nuclei (22.0 ± 1.1% in G2/M) by day 7 postoperation compared with progression in SH myocyte nuclei (14.0 ± 0.8% in G2/M;P < 0.01). Thus an upregulation of certain cell cycle regulators is associated with cardiomyocyte hypertrophy.

Regulation of mitogen-activated protein kinase cascade in adult rat heart preparations in vitro.

The regulation of mitogen-activated protein kinase (MAPK) and MAPK kinase (MEK) was studied in freshly isolated adult rat heart preparations. In contrast to the situation in ventricular myocytes cultured from neonatal rat hearts, stimulation of MAPK activity by 1 mumol/L phorbol 12-myristate 13-acetate (PMA) was not consistently detectable in crude extracts. After fast protein liquid chromatography, MAPK isoforms p42MAPK and p44MAPK and two peaks of MEK were shown to be activated > 10-fold in perfused hearts or ventricular myocytes exposed to 1 mumol/L PMA for 5 minutes. The identities of MAPK or MEK were confirmed by immunoblotting and, for MAPK, by the "in-gel" myelin basic protein phosphorylation assay. In retrogradely perfused hearts, high coronary perfusion pressure (120 mm Hg for 5 minutes), norepinephrine (50 mumol/L for 5 minutes), or isoproterenol (50 mumol/L for 5 minutes) stimulated MAPK and MEK approximately 2- to 5-fold. In isolated myocytes, endothelin 1 (100 nmol/L for 5 minutes) also stimulated MAPK, but stimulation by norepinephrine or isoproterenol was difficult to detect. Immunoblotting showed that the relative abundances of MAPK and MEK protein in ventricles declined to < 20% of their postpartal abundances after 50 days. This may explain the difficulties encountered in assaying the activity of MAPK in crude extracts from adult hearts. We conclude that potentially hypertrophic agonists and interventions stimulate the MAPK cascade in adult rats and suggest that the MAPK cascade may be an important intracellular signaling pathway in this response.

Thyroid hormones alter the adenine nucleotide hydrolysis in adult rat blood serum

The effects of ATP, ADP, and adenosine in the processes of platelet aggregation, vasodilatation, and coronary flow have been known for many years. The sequential hydrolysis of ATP to adenosine by soluble nucleotidases constitutes the main system for rapid inactivation of circulating adenine nucleotides. Thyroid disorders affect a number of biological factors including adenosine levels in different fractions. Then, we intend to investigate if the soluble nucleotidases responsible for the ATP, ADP, and AMP hydrolysis are affected by variations in the thyroid hormone levels in blood serum from adult rats. Hyperthyroidism was induced by daily intraperitoneal injections of L-thyroxine (T4) (2.5 and 10.0 mu g/100 g body weight, respectively) for 7 or 14 days. Hypothyroidism was induced by thyroidectomy and methimazole (0.05%) added to their drinking water during 7 or 14 days. The treatments efficacy was confirmed by determination of hemodynamic parameters and cardiac hypertrophy evaluation. T4 treatment predominantly inhibited, and hypothyroidism (14 days after thyroidectomy) predominantly increased the ATP, ADP, and AMP hydrolysis in rat blood serum. These results suggest that both excess and deficiency of thyroid hormones can modulate the ATP diphosphohydrolase and 5`-nucleotidase activities in rat blood serum and consequently modulate the effects mediated by these enzymes and their products in vascular system. (C) 2010 International Union of Biochemistry and Molecular Biology, Inc.