Keith J. Lookingland

Gene Transfer of Human Guanosine 5′-Triphosphate Cyclohydrolase I Restores Vascular Tetrahydrobiopterin Level and Endothelial Function in Low Renin Hypertension

Background—We recently reported that arterial superoxide (O2−) is augmented by increased endothelin-1 (ET-1) in deoxycorticosterone acetate (DOCA)-salt hypertension, a model of low renin hypertension. Tetrahydrobiopterin (BH4), a potent reducing molecule with antioxidant properties and an essential cofactor for endothelial nitric oxide synthase, protects against O2−–induced vascular dysfunction. However, the interaction between O2− and BH4 on endothelial function and the underlying mechanisms are unknown. Methods and Results—The present study tested the hypothesis that BH4 deficiency due to ET-1–induced O2− leads to impaired endothelium-dependent relaxation and that gene transfer of human guanosine 5′-triphosphate (GTP) cyclohydrolase I (GTPCH I), the first and rate-limiting enzyme for BH4 biosynthesis, reverses such deficiency and endothelial dysfunction in carotid arteries of DOCA-salt rats. There were significantly increased arterial O2− levels and decreased GTPCH I activity and BH4 levels in DOCA-salt compared with sham rats. Treatment of arteries of DOCA-salt rats with the selective ETA receptor antagonist ABT-627, NADPH oxidase inhibitor apocynin, or superoxide dismutase (SOD) mimetic tempol abolished O2− and restored BH4 levels. Basal arterial NO release and endothelium-dependent relaxations were impaired in DOCA-salt rats, conditions that were improved by apocynin or tempol treatment. Gene transfer of GTPCH I restored arterial GTPCH I activity and BH4 levels, resulting in reduced O2− and improved endothelium-dependent relaxation and basal NO release in DOCA-salt rats. Conclusions—These results indicate that a BH4 deficiency resulting from ET-1–induced O2− via an ETA/NADPH oxidase pathway leads to endothelial dysfunction, and gene transfer of GTPCH I reverses the BH4 deficiency and endothelial dysfunction by reducing O2− in low renin mineralocorticoid hypertension.

Evidence That Hypothalamic Periventricular Dopamine Neurons Innervate the Intermediate Lobe of the Rat Pituitary

The purpose of the present study was to provide neurochemical and endocrinological evidence that dopamine (DA) neurons terminating in the intermediate lobe of the rat pituitary originate in the periventricular nucleus of the hypothalamus. One week following surgical separation of the periventricular nucleus from the mediobasal hypothalamus, DA and 3,4-dihydroxyphenyl-acetic acid (DOPAC) concentrations in the intermediate lobe were reduced by 50%, and this was accompanied by an increase in plasma alpha-melanocyte-stimulating hormone (alpha-MSH) concentrations. In contrast, this procedure had no effect on concentrations of prolactin in the plasma, or DA or DOPAC in the median eminence, the region of the mediobasal hypothalamus containing terminals of tuberoinfundibular DA neurons. Electrical stimulation of the periventricular nucleus increased the ratio of DOPAC/DA in the intermediate lobe and reduced the concentrations of alpha-MSH in the plasma, whereas in these same animals the DOPAC/DA ratio in the median eminence and concentrations of prolactin in the plasma were unaltered. These results indicate that approximately 50% of all the DA neurons terminating in the intermediate lobe of the rat pituitary originate in or project through the periventricular nucleus of the hypothalamus, and that these DA neurons regulate the secretion of alpha-MSH from intermediate lobe melanotrophs.

The metabolism of dopamine in the median eminence reflects the activity of tuberoinfundibular neurons

The purpose of the present study was to characterize the metabolism of dopamine (DA) in tuberoinfundibular (TI) neurons terminating in the median eminence and to examine the effects of procedures that alter the synthesis and turnover of DA in these neurons on the concentrations of dihydroxyphenylacetic acid (DOPAC) in the median eminence. The DA uptake inhibitor nomifensine (25 mg/kg, i.p.; 30 min) failed to alter median eminence DOPAC concentrations indicating that very little released DA is recaptured and metabolized by TIDA neurons. Within 5 min following the administration of the monoamine oxidase inhibitor pargyline (50 mg/kg, i.v.) median eminence DOPAC concentrations declined to 15% of control demonstrating that this metabolite has a high turnover rate and is rapidly removed from the median eminence. Median eminence DOPAC concentrations in diestrous female rats, whose TIDA neuronal activity is higher than in the male, were two-fold greater than in male rats. Prolactin (10 micrograms/rat, i.c.v.; 12 h), which increases TIDA neuronal activity, produced a corresponding increase in median eminence DOPAC concentrations in male rats. Restraint stress (30 min), which decreases TIDA neuronal activity, produced a corresponding decrease in median eminence DOPAC concentrations in diestrous female rats. The results from the present study suggest that DOPAC concentrations in the median eminence can be used as an index of TIDA neuronal activity.

Efferent projections from the region of the medial zona incerta containing A13 dopaminergic neurons: a PHA-L anterograde tract-tracing study in the rat

Potential efferent projections of A13 dopaminergic (DA) neurons were identified in the present study by examining the distribution of labelled fibers following iontophoretic injection of the anterogradely transported lectin Phaseolus vulgaris leucoagglutinin (PHA-L) into the medial zona incerta (MZI), the region of the diencephalon containing A13 DA neuronal perikarya. One week after injection, PHA-L labelled fibers were found throughout the brain with the heaviest labelling occurring ipsilateral to the injection site in the anterior hypothalamic area, lateral hypothalamus, lateral preoptic area, horizontal diagonal band of Broca, and parvocellular region of the paraventricular nucleus. Moderate labelling was observed in the ipsilateral median preoptic nucleus, lateral septum, lateral aspect of the bed nucleus of the stria terminalis, and central nucleus of the amygdala. Moderate labelling was also found in the contralateral MZI and parvocellular region of the paraventricular nucleus. Light labelling was detected in the ipsilateral medial preoptic area, supraoptic nucleus, ventromedial nucleus, arcuate nucleus, vertical limb of the diagonal band of Broca, and in the contralateral lateral hypothalamus. Few immunopositive fibers were present in the dorsomedial nucleus of the hypothalamus or the magnocellular region of the paraventricular nucleus. These results reveal that neurons located in the MZI (possibly A13 DA neurons) have ipsilateral efferent axonal projections to a variety of brain regions including the lateral hypothalamus, lateral preoptic area, and the limbic structures at the diencephalic-telencephalic juncture.

Neuroregulation of growth hormone secretion in domestic animals

Growth hormone (GH) is essential for postnatal somatic growth, maintenance of lean tissue at maturity in domestic animals and milk production in cows. This review focuses on neuroregulation of GH secretion in domestic animals. Two hormones principally regulate the secretion of GH: growth hormone-releasing hormone (GHRH) stimulates, while somatostatin (SS) inhibits the secretion of GH. A long-standing hypothesis proposes that alternate secretion of GHRH and SS regulate episodic secretion of GH. However, measurement of GHRH and SS in hypophysial-portal blood of unanesthetized sheep and swine shows that episodic secretion of GHRH and SS do not account for all episodes of GH secreted. Furthermore, the activity of GHRH and SS neurons decreases after steers have eaten a meal offered for a 2-h period each day (meal-feeding) and this corresponds with reduced secretion of GH. Together, these data suggest that other factors also regulate the secretion of GH. Several neurotransmitters have been implicated in this regard. Thyrotropin-releasing hormone, serotonin and gamma-aminobutyric acid stimulate the secretion of GH at somatotropes. Growth hormone releasing peptide-6 overcomes feeding-induced refractoriness of somatotropes to GHRH and stimulates the secretion of GHRH. Norepinephrine reduces the activity of SS neurons and stimulates the secretion of GHRH via alpha(2)-adrenergic receptors. N-methyl-D,L-aspartate and leptin stimulate the secretion of GHRH, while neuropeptide Y stimulates the secretion of GHRH and SS. Activation of muscarinic receptors decreases the secretion of SS. Dopamine stimulates the secretion of SS via D1 receptors and inhibits the secretion of GH from somatotropes via D2 receptors. Thus, many neuroendocrine factors regulate the secretion of GH in livestock via altering secretion of GHRH and/or SS, communicating between GHRH and SS neurons, or acting independently at somatotropes to coordinate the secretion of GH.

Autophagic Death of Adult Hippocampal Neural Stem Cells Following Insulin Withdrawal

Novel therapeutic approaches using stem cell transplantation to treat neurodegenerative diseases have yielded promising results. However, survival of stem cells after transplantation has been very poor in animal models, and considerable efforts have been directed at increasing the viability of engrafted stem cells. Therefore, understanding the mechanisms that regulate survival and death of neural stem cells is critical to the development of stem cell‐based therapies. Hippocampal neural (HCN) stem cells derived from the adult rat brain undergo cell death following insulin withdrawal, which is associated with downregulation of antiapoptotic Bcl‐2 family members. To understand the type of cell death in HCN cells following insulin withdrawal, apoptosis markers were assessed. Of note, DNA fragmentation or caspase‐3 activation was not observed, but rather dying cells displayed features of autophagy, including increased expression of Beclin 1 and the type II form of light chain 3. Electron micrographs showed the dramatically increased formation of autophagic vacuoles with cytoplasmic contents. Staurosporine induced robust activation of caspase‐3 and nucleosomal DNA fragmentation, suggesting that the machinery of apoptosis is intact in HCN cells despite the apparent absence of apoptosis following insulin withdrawal. Autophagic cell death was suppressed by knockdown of autophagy‐related gene 7, whereas promotion of autophagy by rapamycin increased cell death. Taken together, these data demonstrate that HCN cells undergo a caspase‐independent, autophagic cell death following insulin withdrawal. Understanding the mechanisms governing autophagy of adult neural stem cells may provide novel strategies to improve the survival rate of transplanted stem cells for treatment of neurodegenerative diseases.

Periventricular-Hypophysial Dopaminergic Neurons Innervate the Intermediate but Not the Neural Lobe of the Rat Pituitary Gland

The purpose of the present study was to determine the relative distribution of axon terminals of A14 periventricular-hypophysial dopaminergic (PHDA) neurons in the neural and intermediate lobes of the rat pituitary gland. Discrete unilateral injections of the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) into the periventricular nucleus resulted in labelling of extensively branched terminal axonal arbors in the intermediate lobe, but not the neural lobe of the pituitary gland. In contrast, unilateral injections of PHA-L into the paraventricular nucleus revealed thick, varicose terminal arborizations containing PHA-L in the neural lobe, but not the intermediate lobe. Terminal axonal branches and varicosities containing PHA-L immunoreactivity in the intermediate lobe were also immunoreactive for tyrosine hydroxylase. These results reveal that A14 PHDA neurons originating in the periventricular nucleus of the hypothalamus project axons to the intermediate lobe of the rat pituitary gland.

Exogenous Tryptophan Increases Synthesis, Storage, and Intraneuronal Metabolism of 5-Hydroxytryptamine in the Rat Hypothalamus

The effects of tryptophan administration on neurochemical estimates of synthesis [5‐hydroxytryptophan (5‐HTP) accumulation following administration of a decarboxylase inhibitor], storage [5‐hydroxytryptamine (5‐HT) concentrations], and metabolism [5‐hydroxyin‐doleacetic acid (5‐HIAA) concentrations] of 5‐HT in selected regions of the hypothalamus were determined using HPLC coupled to an electrochemical detector. Tryptophan methyl ester HCl (30–300 mg/kg i.p.) produced a dose‐dependent increase in the rate of 5‐HTP accumulation throughout the hypothalamus but had no effect on the rate of accumulation of 3,4‐dihydroxyphen‐ylalanine. Peak 5‐HTP levels were attained by 30 min following administration of tryptophan (100 mg/kg i.p.) and were maintained for an additional 60 min. Tryptophan also produced concomitant dose‐dependent increases in 5‐HT and 5‐HIAA concentrations in these same regions without changes in the 5‐HIAA/5‐HT ratio. These results indicate that exogenous tryptophan administration selectively increases the synthesis, storage, and metabolism of 5‐HT in the hypothalamus without altering the synthesis of catecholamines. Inhibition of 5‐HT uptake with chlorimipramine or fluoxetine produced modest (10–40%) reductions in 5‐HIAA concentrations throughout the hypothalamus, revealing that only a minor portion of 5‐HIAA is derived from released and recaptured 5‐HT, whereas the major portion of this metabolite reflects intraneuronal metabolism of unreleased 5‐HT. In both chlorimipramine‐ and fluoxetine‐treated rats, 5‐HIAA concentrations were significantly increased by tryptophan administration, indicating that the increase in synthesis of 5‐HT following precursor loading is accompanied by an increase in the intraneuronal metabolism of 5‐HT.

Dopamine receptor-mediated regulation of incertohypothalamic dopaminergic neurons in the male rat.

The incertohypothalamic dopaminergic (DA) neuronal system has been divided into a rostral component of neurons originating in the rostral periventricular nucleus and projecting to the preopticosuprachiasmatic and medial preoptic nuclei and a caudal component originating in the medial zona incerta and projecting to the dorsomedial and anterior hypothalamic nuclei. The purpose of the present study was to determine if the activity of these intrahypothalmic DA neurons is regulated by DA receptor-mediated mechanisms, as are those in the major ascending nigrostriatal and mesolimbic neurons, or if they resemble another group of intrahypothalamic DA neurons, those that comprise the tuberoinfundibular system, which are not responsive to the acute actions of DA agonists or antagonists. The rate of DA turnover (decline after alpha-methyltyrosine) in micropunched regions of the striatum (ST), nucleus accumbens (NA) and hypothalamic regions which contain cell bodies or terminals of incertohypothalamic DA neurons was increased after administration of a DA antagonist (haloperidol) and decreased after administration of a DA agonist (bromocriptine). gamma-Butyrolactone increased DA concentrations in the ST, NA and hypothalamic brain regions containing incertohypothalamic DA neurons, and this effect was blocked by the DA agonist apomorphine. In contrast, none of these treatments affected the concentration or rate of turnover of DA in the median eminence (terminal region of tuberoinfundibular neurons). Injections of either gamma-hydroxybutyric acid or baclofen into the substantia nigra/ventral tegmental region of the midbrain increased DA concentrations in the NA and/or ST but failed to alter DA concentrations in any hypothalamic region. These results suggest that the incertohypothalamic DA system is composed of neurons whose activity can be rapidly modulated by DA receptor-mediated mechanisms and thus resemble the DA neurons in the major ascending nigrostriatal and mesolimbic systems rather than the hypothalamic neurons which comprise the tuberoinfundibular DA system.

Regulation of Male Rat Copulatory Behavior by Preoptic Incertohypothalamic Dopamine Neurons

The role of dopaminergic terminals in the medial preoptic area (MPO) in the regulation of male rat copulatory behavior was investigated. A 6-hydroxydopamine (6-OHDA) injection into the MPO of animals pretreated with desipramine resulted in a small (23%) depletion of DA, and no impairment of copulatory activity. Further depletion of catecholamines with alpha-methyl p-tyrosine (AMPT) produced several deficits in the copulatory behavior of 6-OHDA-treated males, at a dose of AMPT that did not adversely affect copulation prior to 6-OHDA administration. The dose-related effects of intracranial apomorphine (APO) injections were also altered by 6-OHDA injections into the MPO. The inhibition previously found with 0.2 microgram of APO into the lateral ventricle of normal males was abolished by 6-OHDA treatment. A facilitation of copulatory behavior was observed following the injection of 0.2 microgram of APO into the MPO of 6-OHDA-treated animals, whereas this treatment did not affect the copulatory behavior of intact animals. Finally, inhibitory effects observed following an injection of 0.1 microgram of APO into the MPO of normal males were blocked by 6-OHDA administration. The relative roles of presynaptic autoreceptors and postsynaptic DA receptors in the MPO in mediating the dose-related effects of APO on copulatory behavior are discussed.