Absence of androgen-mediated transcriptional effects in osteoblastic cells despite presence of androgen receptors.

Androgen excess and deficiency affect skeletal maturation and bone cell function. Understanding the molecular basis for these androgen effects could improve therapy/prevention of short stature and osteoporosis. Androgens act through binding to androgen receptors (ARs), which modulate gene transcription via interactions with DNA response elements on target genes. Because osteoblasts contain ARs at levels just below certain androgen-sensitive tissues, we sought to define the function of AR in a number of commonly used osteoblastic cell lines. Presence and quantification of AR protein and mRNA were evaluated by ligand binding assay, western blotting, and RNAse protection assay. AR-containing osteoblastic cell lines were exposed to nonaromatizable androgens and effects on gene expression were assessed. We found no evidence for direct effects of androgen on endogenous genes nor was androgen involved in modulation of parathyroid hormone effects on early gene activation. Androgen-sensitive reporter gene constructs were stimulated by androgen only when AR cDNA expression vectors were introduced into cells by cotransfection. We conclude that, in commonly used osteoblastic cell lines, the presence of AR at the levels described here does not guarantee androgen transcriptional activity. The effects of androgen on bone in vivo may involve direct stimulation of osteoblastic cells in a different setting or stage of differentiation. Alternatively, androgen may act on bone cells other than osteoblasts, or through metabolic conversion to estrogens.

N-methyl-D,L-aspartic acid differentially affects LH release and LHRH mRNA levels in estrogen-treated ovariectomized control and androgen-sterilized rats.

Excitatory amino acids such as N-methyl-D,L-aspartic acid (NMDA) are thought to play an important role in the regulation of gonadotropin secretion. NMDA induces significant increases in plasma LH in a variety of animal models and these effects occur by activation of neural processes involved in excitation of LHRH neurons rather than by a direct action on the pituitary gland. We have taken advantage of this information to study the effects of NMDA on LH release and on changes in levels of LHRH mRNA in single neurons of adult rats treated neonatally with a high dosage of androgen. While iv NMDA evoked an increase in plasma LH in estrogen-treated ovariectomized control and androgen-sterilized rats (ASR), significantly less LH was released in ASR. LHRH mRNA levels in the organum vasculosum of the lamina terminalis (OVLT), the rostral (r), media (m) and caudal (c) preoptic (POA) regions were quantitated using in situ hybridization histochemistry and quantitative image analysis methods. LHRH mRNA levels in untreated controls and ASR did not differ in any of the brain regions examined. Within 1 h after NMDA, LHRH mRNA had increased significantly in OVLT and rPOA but not in mPOA and cPOA neurons of control rats and these mRNA levels remained elevated for 4 h. In contrast, NMDA treatment of ASR did not affect basal levels of LHRH mRNA in any region of the rostral hypothalamus. These observations suggest that neonatal androgen treatment of female rats either directly or indirectly affects the responsiveness of LHRH neurons to NMDA.(ABSTRACT TRUNCATED AT 250 WORDS)

Temporal changes in tyrosine hydroxylase mRNA levels in A1, A2 and locus ceruleus neurons following electrical stimulation of A1 noradrenergic neurons.

We examined the effects of electrical stimulation (ES) of right A1 noradrenergic cells on temporal changes in tyrosine hydroxylase (TH) mRNA levels in A1, A2 and locus ceruleus (LC) neurons by in situ hybridization histochemistry and quantitative image analysis methods. The stimulation parameters used previously have been shown to increase hypothalamic norepinephrine (NE) release. Within 1 h after beginning A1 stimulation, TH mRNA levels were significantly increased and they continued to rise to reach plateau by 6 h. TH message levels at 12 h were not difference from 6 h values. A1-ES did not affect TH mRNA levels in contralateral A1 or in A2 or locus ceruleus neurons. These data suggest that changes in TH mRNA levels may serve as an index of increased A1 neuronal activity in circumstances when increases in hypothalamic NE secretion occur.

Effects of reserpine on tyrosine hydroxylase mRNA levels in locus coeruleus and medullary A1 and A2 neurons analyzed by in situ hybridization histochemistry and quantitative image analysis methods.

These studies examined the effects of reserpine on concentrations of norepinephrine (NE), dopamine (DA) and epinephrine (EPI) and on levels of tyrosine hydroxylase (TH) mRNA in locus coeruleus (LC) and medullary A1 and A2 neurons. Noradrenergic neurons in these regions first were identified by immunocytochemistry and, thereafter, by in situ hybridization histochemistry. Levels of TH mRNA were measured by quantitative image analysis methods. Changes in catecholamine concentrations in micropunches of these brain regions were analyzed by HPLC. Epinephrine was not detected in any of the nuclei examined. Twenty-four hours after reserpine treatment, NE concentrations declined in A1, A2 and LC neurons by 46, 69 and 34% respectively while DA declined only in the region of A2 neurons. This reserpine-induced depletion of NE was accompanied by a 2- to 3-fold increase in TH mRNA levels in LC and A1 neurons but no change in message levels occurred in A2 cells 24 h after reserpine. Forty eight hours later, message levels in A1 and LC neurons did not differ significantly from the elevated 24 h values but TH mRNA levels in A2 neurons now were significantly elevated compared to 24 h values. TH mRNA levels 72 h after reserpine did not differ from 48 h values in A1, A2 and LC neurons. Thus, TH gene expression in A1 neurons increases after reserpine treatment in a manner equivalent to that observed in LC, adrenal medulla and superior cervical ganglia. The reason why it required 48 h for TH mRNA to increase in A2 neurons remains unclear.

Changes in tyrosine hydroxylase mRNA levels in medullary A1 and A2 neurons and locus coeruleus following castration and estrogen replacement in rats.

Temporal changes in tyrosine hydroxylase (TH) mRNA levels in medullary A1 and A2 neurons and locus coeruleus (LC) cells were studied 6, 12 and 24 h following orchidectomy in rats. Brains from intact controls and sham castrated rats also were evaluated at these same time periods. In situ hybridization histochemistry and quantitative image analysis techniques were used to quantitate levels of cytoplasmic TH mRNA. Neither the time of day nor the stress of sham castration affected TH mRNA levels in A1, A2 and LC neurons. In contrast, 6 h following castration, TH mRNA levels in A1 neurons had declined significantly. Thereafter, there was a linear increase in A1 message levels such that, by 24 h, TH mRNA values were significantly higher than those obtained in intact controls. Placement of Silastic estrogen capsules immediately after castration prevented the 6 h decline in A1 message levels. At 12 h, TH mRNA levels in A1 neurons were significantly higher in estrogen-treated rats compared to those of the castrate or intact control groups. By 24 h, message levels in A1 neurons of steroid-treated rats were comparable to the intact control. Neither castration nor estrogen treatment altered TH mRNA levels in A2 neurons. TH mRNA levels in LC neurons increased significantly 6 h after castration and estrogen produced a further significant increase in message levels. Six hours later (12 h), TH mRNA values were still higher than controls but, in the estrogen-treated group, these levels had declined to those observed in the 12 h castrate group.(ABSTRACT TRUNCATED AT 250 WORDS)

Tyrosine Hydroxylase Messenger Ribonucleic Acid Levels Increase in A1 but not Locus Ceruleus Noradrenergic Neurons in Proestrous Rats but not in Diestrous or Androgen-Sterilized Animals.

Norepinephrine (NE) turnovers (an index of secretion) increase in the hypothalamus of proestrous rats concomitant with luteinizing hormone surges, whereas, neither of these events are observed in diestrous nor in androgen-sterilized rats. Increased hypothalamic NE release may occur as a consequence of the withdrawal of local inhibitory γ-aminobutyric acid and opiate controls on specific presynaptic NE terminals and/or as a result of an increase in activity within noradrenergic neurons. Tyrosine hydroxylase (TH) is the rate-limiting enzyme for the synthesis of NE and our earlier studies revealed that increases in TH mRNA in A1 and locus ceruleus (LC) neurons can serve as an index of increased activity within these cells. In the present study, we evaluated whether TH message levels change in A1 and LC neurons prior to and during the hours when luteinizing hormone surges and increased NE turnovers are observed. As controls, TH mRNA levels in A1 and LC neurons were evaluated at the same hours of day in diestrous day 2 and in androgen-sterilized rats. In situ hybridization histochemistry and quantitative image analysis methods were used to measure changes in TH mRNA levels. Luteinizing hormone surges in proestrous rats began at 1500 h, peaked between 1600 and 1700 h and declined, thereafter, to 2000 h. In contrast, plasma luteinizing hormone remained basal throughout the day in diestrous and androgen-sterilized rats. While A1 neuronal TH mRNA levels did not differ in the three groups of rats during the morning (0930 to 1030 h), these message levels were significantly elevated in proestrous rats during the afternoon (1645 to 1715 h) and remained high at 2000 to 2030 h. In contrast, no changes in TH mRNA levels were observed in A1 neurons throughout the afternoon in diestrous animals or androgen-sterilized rats. TH mRNA levels in the LC did not differ in the three groups of rats and they remained unchanged throughout the afternoon hours we examined. From these observations we conclude that concomitant with afternoon proestrous luteinizing hormone surges and the accompanying increase in hypothalamic NE secretion, there is an increase in activity within A1 but not LC neurons. These data suggest that the proestrous increase in hypothalamic NE turnover we previously observed is not due solely to withdrawal of local inhibitory controls of presynaptic NE release but it also involves an increase in activity within A1 but not LC neurons.

Pituitary gland responsiveness to LHRH and LHRH neuronal responsiveness to excitatory stimuli are severely impaired in female rats treated neonatally with high doses of androgen.

Treatment of neonatal female rats with androgen renders these animals permanently sterile as adults. Previously, we reported that these androgen-sterilized rats (ASR) do not respond to the positive feedback effects of estrogen by having LH surges. We also reported that this defect might be due to the failure of these animals to show increased hypothalamic norepinephrine turnovers (an index of secretion) in response to steroid treatment. Although LHRH-catecholamine synapses are established before or at birth, whether such synapses are functional remains to be resolved. Accordingly, in the present studies, female rats were given 1.25 mg of testosterone propionate at 5 days of age and, at 100 days of age, these ASR and controls were ovariectomized and treated with estradiol. In these animals, we examined whether activation of medullary A1 noradrenergic neurons would amplify LH release following preliminary depolarization of LHRH neurons with an electrochemical stimulus (ECS). As well, we reexamined whether LHRH neuronal responsiveness to exogenous NE and pituitary responsiveness to LHRH differ in controls versus ASR. In controls, two pulses of LHRH given 60 min apart elicited increases in plasma LH with the second pulse inducing greater LH release than the first pulse. In ASR, significantly less LH was released after either LHRH pulse and particularly after the second pulse. When the spacing between the two LHRH pulses was reduced to 25 min, equivalent levels of LH release occurred in controls and ASR after the second pulse.(ABSTRACT TRUNCATED AT 250 WORDS)

Tactile stimulation and preterm infants.

A critical challenge for care providers is improving the outcomes for premature infants. The issues of how to control various kinds of stimulation, provide appropriate sensory stimulation, and maintain the quality of life of premature infants becomes the central focus of care given in neonatal intensive care units. Therefore, intervention research studies that improve the development and quality of life for premature infants are vitally important. This article comparatively analyzes and critiques five intervention studies of premature infants using tactile stimulation and provides future research directions in this area. By examining the effectiveness of the tactile stimulation studies, some evidence and guidance can be provided for researchers generating knowledge in this area as well as nurses involved in clinical care.