Water deprivation and rat adrenal mRNAs for tyrosine hydroxylase and the norepinephrine transporter

1997 ◽  
Vol 272 (6) ◽  
pp. R1897-R1903 ◽  
Author(s):  
V. L. Brooks ◽  
T. A. Huhtala ◽  
T. L. Silliman ◽  
W. C. Engeland
Keyword(s):  
Tyrosine Hydroxylase ◽  
Water Deprivation ◽  
Norepinephrine Transporter ◽  
Ribonuclease Protection Assay ◽  
Mrna Levels ◽  
Total Rna ◽  
Adrenocortical Activity ◽  
Corticosterone Response ◽  
Ribonuclease Protection ◽  
Th Mrna

Experiments were performed in rats to test the hypothesis that adrenal mRNA levels of tyrosine hydroxylase (TH) and the norepinephrine transporter (NET) would be modified by water deprivation via activation of the sympathetic nervous system. TH and NET mRNA levels were measured using the ribonuclease protection assay. Adrenal TH mRNA was higher (P < 0.001) in water-deprived (921 +/- 39 fg/microgram total RNA) compared with the water-replete rats (657 +/- 45 fg/microgram total RNA). In contrast, water deprivation decreased (P < 0.01) adrenal NET mRNA levels (275 +/- 66 vs. 433 +/- 63 fg/microgram total RNA). The dehydration-induced increase in TH mRNA was prevented by prior splanchnicectomy, but the decrease in NET mRNA was produced even in the absence of adrenal nerves. Water deprivation also increased (P < 0.05) plasma adrenocorticotropic hormone (84 +/- 16 vs. 42 +/- 14 pg/ml) and corticosterone (358 +/- 87 vs. 44 +/- 15 ng/ml) levels. Interestingly, the corticosterone response was reduced (P < 0.05) by unilateral adrenal denervation. These results suggest that water deprivation increases both adrenal medullary and adrenocortical activity at least in part by stimulation of sympathetic nerve activity.

Tyrosine hydroxylase and norepinephrine transporter mRNA levels increase in locus coeruleus after coitus in rabbits

1997 ◽  
Vol 19 (3) ◽  
pp. 311-319 ◽  
Author(s):  
SP Yang ◽  
KY Pau ◽  
HG Spies
Keyword(s):  
Tyrosine Hydroxylase ◽  
Locus Coeruleus ◽  
Time Interval ◽  
Ribonuclease Protection Assay ◽  
Mrna Levels ◽  
Lh Surge ◽  
Serum Lh ◽  
Ribonuclease Protection ◽  
Previous Demonstration ◽  
Internal Marker

Hypothalamic norepinephrine (NE) plays an important role in the control of sexual behavior and in the secretion of gonadotropin. Our previous study showed that coitus induced simultaneous increases in hypothalamic NE and GnRH releases in female but not in male rabbits. To investigate the activities in noradrenergic neurons during the coitus-induced process of an LH surge, we measured tyrosine hydroxylase (TH, the rate-limiting enzyme in NE synthesis) and NE transporter (NET, a key protein for NE cellular reuptake) mRNA levels in locus coeruleus (LC) noradrenergic cells in female New Zealand White rabbits. Changes in LC-TH and LC-NET mRNA levels were also measured in males as controls. Female rabbits were killed before coitus and at 15, 30, 60, 120, and 240 min after coitus (n = 6-7/time point); males were killed before and at 30, 60, and 120 min after coitus (n = 3/time). Individual brainstems were sectioned, the LC neurons punched, and TH and NET mRNAs were quantified by ribonuclease protection assay (RPA). Rabbit-specific TH (330 bp) and NET (503 bp) cDNAs were used as probes in the RPA for gene-specific signals. A rabbit 'house-keeping' cDNA (cyclophilin, 158 bp) was also cloned and used as an internal marker for tissue RNA content. Trunk blood was collected to determine serum LH levels. In female rabbits, serum LH levels rose by 15 min after coitus, reached peak concentrations at 1-2 h, and declined thereafter. The time interval for changes in TH and NET mRNA levels in females was similar to that in serum LH levels. Both TH and NET mRNAs increased significantly by 15 min (73% and 85% respectively) and were elevated for 2 h (87% and 111% respectively). TH mRNA levels returned to basal levels by 4 h after coitus, whereas NET mRNA values were elevated throughout the 4 h of observation. In contrast, LH, TH and NET mRNA levels did not change after coitus in males. The enhanced gene expression of both TH and NET in the LC in females, in accord with our previous demonstration of increased hypothalamic NE release, suggests that regulation of NE synthesis and reuptake is an integral part of the coitus-induced NE/GnRH/LH surge process that includes the initiation, sustenance or recovery of the release and/or storage of these neurochemicals.

scholarly journals Effects of Insulin Treatment without and with Recurrent Hypoglycemia on Hypoglycemic Counterregulation and Adrenal Catecholamine-Synthesizing Enzymes in Diabetic Rats

Endocrinology ◽  
2006 ◽  
Vol 147 (4) ◽  
pp. 1860-1870 ◽  
Author(s):  
Karen E. Inouye ◽  
Jessica T. Y. Yue ◽  
Owen Chan ◽  
Tony Kim ◽  
Eitan M. Akirav ◽  
...  
Keyword(s):  
Tyrosine Hydroxylase ◽  
Insulin Treatment ◽  
Diabetic Rats ◽  
Hyperinsulinemic Hypoglycemia ◽  
Mrna Levels ◽  
Relevant Model ◽  
Counterregulatory Hormones ◽  
Catecholamine Synthesizing Enzymes ◽  
Recurrent Hypoglycemia ◽  
Th Mrna

Untreated diabetic rats show impaired counterregulation against hypoglycemia. The blunted epinephrine responses are associated with reduced adrenomedullary tyrosine hydroxylase (TH) mRNA levels. Recurrent hypoglycemia further impairs epinephrine counterregulation and is also associated with reduced phenylethanolamine N-methyltransferase mRNA. This study investigated the adaptations underlying impaired counterregulation in insulin-treated diabetic rats, a more clinically relevant model. We studied the effects of insulin treatment on counterregulatory hormones and adrenal catecholamine-synthesizing enzymes and adaptations after recurrent hypoglycemia. Groups included: normal; diabetic, insulin-treated for 3 wk (DI); and insulin-treated diabetic exposed to seven episodes (over 4 d) of hyperinsulinemic-hypoglycemia (DI-hypo) or hyperinsulinemic-hyperglycemia (DI-hyper). DI-hyper rats differentiated the effects of hyperinsulinemia from those of hypoglycemia. On d 5, rats from all groups were assessed for adrenal catecholamine-synthesizing enzyme levels or underwent hypoglycemic clamps to examine counterregulatory responses. Despite insulin treatment, fasting corticosterone levels remained increased, and corticosterone responses to hypoglycemia were impaired in DI rats. However, glucagon, epinephrine, norepinephrine, and ACTH counterregulatory defects were prevented. Recurrent hypoglycemia in DI-hypo rats blunted corticosterone but, surprisingly, not epinephrine responses. Norepinephrine and ACTH responses also were not impaired, whereas glucagon counterregulation was reduced due to repeated hyperinsulinemia. Insulin treatment prevented decreases in basal TH protein and increased PNMT and dopamine β-hydroxylase protein. DI-hypo rats showed increases in TH, PNMT, and dopamine β-hydroxylase. We conclude that insulin treatment of diabetic rats protects against most counterregulatory defects but not elevated fasting corticosterone and decreased corticosterone counterregulation. Protection against epinephrine defects, both without and with antecedent hypoglycemia, is associated with enhancement of adrenal catecholamine-synthesizing enzyme levels.

PTH increases renal 25(OH)D3-1α-hydroxylase (CYP1α) mRNA but not renal 1,25(OH)2D3production in adult rats

2003 ◽  
Vol 284 (5) ◽  
pp. F1032-F1036 ◽  
Author(s):  
H. J. Armbrecht ◽  
M. A. Boltz ◽  
T. L. Hodam
Keyword(s):  
Ribonuclease Protection Assay ◽  
Mrna Levels ◽  
Fischer 344 Rats ◽  
Adult Rats ◽  
Dihydroxyvitamin D ◽  
Fischer 344 ◽  
Young Rats ◽  
Ribonuclease Protection ◽  
Cytochrome P 450

The capacity of parathyroid hormone (PTH) to stimulate renal 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] production declines with age in the rat. The purpose of these studies was to determine whether this decline is due to a decreased capacity of PTH to increase the mRNA levels of CYP1α, the cytochrome P-450 component of the 25(OH)D3-1α-hydroxylase. Young (2 mo) and adult (12 mo) male Fischer 344 rats were parathyroidectomized (PTX). After 72 h, PTX rats were injected with PTH or vehicle at 24, 6, and 3 h before death, and renal CYP1α mRNA levels were measured by ribonuclease protection assay. In young rats, PTH markedly increased plasma 1,25(OH)2D3 and renal 1,25(OH)2D3 production. However, in adult rats, the response to PTH was less than 30% of that seen in young rats. Renal CYP1α mRNA levels, on the other hand, were increased over fivefold by PTH in both young and adult rats. In in vitro studies, PTH/forskolin increased CYP1α mRNA levels over twofold in renal slices from both young and adult PTX rats. These studies demonstrate that the decreased capacity of PTH to increase 1,25(OH)2D3 production in adult rats is not due to decreased induction of CYP1α mRNA.

scholarly journals DNase I Treatment of Total RNA Improves the Accuracy of Ribonuclease Protection Assay

BioTechniques ◽  
1998 ◽  
Vol 24 (5) ◽  
pp. 732-734 ◽  
Author(s):  
Dan A. Dixon ◽  
Darius L. Vaitkus ◽  
Stephen M. Prescott
Keyword(s):  
Dnase I ◽  
Ribonuclease Protection Assay ◽  
Protection Assay ◽  
Total Rna ◽  
Ribonuclease Protection

Renal and extra-renal levels of renin mRNA in experimental hypertension

1991 ◽  
Vol 80 (4) ◽  
pp. 339-344 ◽  
Author(s):  
Nilesh J. Samani ◽  
William J. Brammar ◽  
John D. Swales
Keyword(s):  
Adrenal Glands ◽  
Matched Control ◽  
Experimental Hypertension ◽  
Ribonuclease Protection Assay ◽  
Mrna Levels ◽  
Renin Gene ◽  
Salt Hypertension ◽  
Renin Mrna ◽  
Ribonuclease Protection ◽  
And Control

1. Using a ribonuclease-protection assay, renin mRNA levels were compared in the kidneys, livers, brains, hearts and adrenal glands of two-kidney, one-clip Goldblatt hypertensive rats with those of age-matched control rats at 4 weeks (‘early’) and 20 weeks (‘chronic’) after clipping, and in the kidneys and adrenal glands of rats treated for 3 weeks with deoxycorticosterone and salt (deoxycorticosterone-salt hypertension) with those of control rats. 2. While marked changes were observed in kidney renin mRNA levels in all three experimental groups compared with their respective controls, in most of the extrarenal tissue studied minimal, if any, difference was seen in renin mRNA levels between the hypertensive and control rats. 3. The findings suggest that in these extra-renal tissues renin gene expression is differently regulated from that in the kidney, and particularly that it is not profoundly affected by changes in the level of circulating angiotensin II. 4. An increase in renin mRNA was observed in the adrenal glands of the ‘chronic’ Goldblatt rats, which may be of relevance to the maintenance of hypertension in this model.

scholarly journals Regulation of tyrosine hydroxylase mRNA in catecholaminergic cells of embryonic rat: analysis by in situ hybridization.

1989 ◽  
Vol 37 (1) ◽  
pp. 1-5 ◽  
Author(s):  
G M Jonakait ◽  
M Rosenthal ◽  
J I Morrell
Keyword(s):  
In Situ Hybridization ◽  
Tyrosine Hydroxylase ◽  
Messenger Rna ◽  
Sympathetic Ganglia ◽  
Mrna Levels ◽  
Pregnant Rats ◽  
Reserpine Treatment ◽  
Catecholaminergic Cells ◽  
Th Mrna

In situ hybridization was used to examine the appearance of mRNA specific for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine (CA) biosynthesis, in neural crest derivatives of the rat embryo. These derivatives include sympathetic ganglia and transient catecholaminergic cells of embryonic intestine. Messenger RNA is first detected in sympathetic ganglia at E11.5, the age corresponding to the initial immunocytochemical expression of TH protein. In older embryos increased accumulation of TH-specific mRNA in sympathetic ganglia parallels the increase in TH immunoreactivity. By contrast, mRNA for TH is difficult to detect in embryonic intestines at E11.5 but is found instead in cells clustered at the dorsal boundaries of the pharynx and foregut. Cells expressing TH mRNA are infrequently found in embryonic intestines at any age, even though TH protein is immunohistochemically apparent. Treatment of pregnant rats with doses of reserpine, known to increase circulating levels of glucocorticoid hormones and prolong the expression of TH protein in embryonic gut cells, dramatically but transiently increases the number of gut cells at E12.5 with detectable TH mRNA. After E13.5 TH mRNA is undetectable even in reserpine-treated guts. Reserpine treatment also increases the labeling density in sympathetic ganglia. Taken together, these data are consistent with the hypothesis that the microenvironment of the embryonic intestine affects gene expression directly to alter phenotype. Moreover, although reserpine administration briefly increases TH mRNA levels, the effect is short-lived and does not alter neurotransmitter phenotypic conversion.

Developmental and tissue-regulated expression of IGF-I and IGF-II mRNAs in Sparus aurata

1996 ◽  
Vol 16 (2) ◽  
pp. 123-132 ◽  
Author(s):  
S J Duguay ◽  
J Lai-Zhang ◽  
D F Steiner ◽  
B Funkenstein ◽  
S J Chan
Keyword(s):  
Sparus Aurata ◽  
Mrna Level ◽  
Gilthead Seabream ◽  
Ribonuclease Protection Assay ◽  
Mrna Levels ◽  
Amino Acid Residues ◽  
Sequence Comparisons ◽  
Regulated Expression ◽  
Igf I ◽  
Ribonuclease Protection

ABSTRACT Recent studies have shown that homologues of the mammalian IGF-I and -II genes are also found in teleosts. We report here the cDNAs coding for IGF-I and IGF-II cloned from the gilthead seabream, Sparus aurata. Sequence comparisons revealed that both IGFs have been well conserved among teleosts, although Sparus IGF-I is shorter by three amino acid residues due to truncated B-and C-domains. Using the cloned cDNAs as probes, the relative expression of IGF-I and IGF-II mRNAs were assayed in different Sparus tissues. Sparus liver clearly contained the highest level of IGF-I mRNA while relatively high levels of IGF-II mRNA were found in liver, heart and gill using the ribonuclease protection assay. After GH administration the amount of IGF-I mRNA was increased by 220% in liver but no changes in IGF-II mRNA levels were detected in any tissue. We also assayed the expression of IGF-I and IGF-II in Sparus during early development. The IGF-II mRNA level was highest in larva 1 day after hatching and decreased thereafter. In contrast, IGF-I mRNA was detected in 1-day-old larva but there was an increase in expression in 12- and 16-day-old larva. These results demonstrated that the expression of IGF-I and IGF-II is highly regulated in teleosts and suggest that they play distinct roles during growth and development.

scholarly journals Estradiol Alters Only GAD67 mRNA Levels in Ischemic Rat Brain with No Consequent Effects on GABA

2005 ◽  
Vol 26 (4) ◽  
pp. 518-526 ◽  
Author(s):  
Hung-Dong Joh ◽  
Robin V Searles ◽  
Michael Selmanoff ◽  
Nabil J Alkayed ◽  
Raymond C Koehler ◽  
...  
Keyword(s):  
Glutamic Acid ◽  
Rat Brain ◽  
Glutamic Acid Decarboxylase ◽  
Brain Regions ◽  
Artery Occlusion ◽  
Acid Decarboxylase ◽  
Ribonuclease Protection Assay ◽  
Mrna Levels ◽  
Gaba Transporters ◽  
Ribonuclease Protection

The present study tested the hypothesis that estradiol reduces tissue infarction after middle cerebral artery occlusion (MCAO) in estradiol-deficient females by augmenting glutamic acid decarboxylase (GAD) expression and thus activity, leading to increases in γ-amino-butyric acid (GABA) tissue levels. Glutamic acid decarboxylase is the principal enzyme for GABA synthesis and has two isoforms, GAD65 and GAD67, which differ in size and cellular distribution. Rats were ovariectomized 7 to 8 days before receiving no hormone, placebo, or 25 μg estradiol via subcutaneous implant 7 to 10 days before harvesting tissue in either ischemic cohorts after 2 h of MCAO (end-ischemia) or in nonischemic cohorts. Selected cortical and striatal regions were microdissected from harvested brains. GAD65/67 mRNA levels were determined by microlysate ribonuclease protection assay. End-ischemic GABA concentrations were determined by HPLC. Steroid treatment selectively decreased ischemic cortical GAD67 mRNA levels. In most brain regions evaluated, regional GABA concentrations increased with ischemia regardless of treatment. Estradiol blocked MCAO-induced increases in GABA concentration only in dorsomedial cortex. These data suggest that estradiol repletion in ischemic rat brain selectively decreases GAD67 mRNA levels but does not alter steady-state GABA concentrations. It may be that estradiol under ischemic conditions is attenuating GABA metabolism rather than enhancing synthesis or is augmenting other aspects of GABAergic transmission such as GABA transporters and receptors.

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