Choline acetyltransferase activity in the neurointermediate lobe of the rat pituitary

Summary CAT activity was determined in the neurointermediate lobe of the rat's pituitary gland. The level of enzyme activity in the adult rat is of 386.2±39.1 picomoles/hour/neurointermediate lobe. CAT activity is present in the pituitary gland of the newborn animal and increases with age. In organotypic cultured neurointermediates lobes CAT decreases progressively and practically disappears after 10 days.     

Choline acetyltrasferase activity in the neurointermediate lobe of the rat pituitary

CAT activity was determined in the neurointermediate lobe of the rat's pituitary gland. The level of enzyme activity in the adult rat is of 386.2 +/- 39.1 picomoles/hour/neurointermediate lobe. CAT activity is present in the pituitary gland of the newborn animal and increases with age. In organo-typic cultured neurointermediates lobes CAT decreases progressively and practically disappears after 10 days.     

Cardiovascular effects of dynorphin A-(1-8), dynorphin A-(1-13) and dynorphin A-(1-17) microinjected into the preoptic medialis nucleus of the rat

Multiple forms of Dynorphin A (Dyn A) are present in cardiovascular nuclei in the hypothalamus, along with kappa-opiate receptors. To study the potential role of Dyn A in cardiovascular regulation, various Dyn A species [Dyn A-(1-8; 1-13; 1-17)] were microinjected into the preoptic nucleus (POM) of the anesthetized rat. Dyn A-(1-17) was tenfold more potent than Dyn A-(1-13), and over 100-fold more potent than Dyn A-(1-8) in reducing systemic blood pressure. High doses of Dyn A-(1-17) (6 nmoles) produced shock with severe bradycardia and bradypnea. These data suggest that Dyn A may be an endogenous modulator of cardiorespiratory variables in the anteroventral hypothalamus, and demonstrate that the larger fragments of the peptide are more active in eliciting central cardiorespiratory activity in this region.     

Rostral and caudal ventricular infusion of antibodies to dynorphin A(1-17) and dynorphin A(1-8): effects on electrically-elicited feeding in the rat

Lateral ventricular injection of antibodies to dynorphin A(1-13) was previously shown to elevate lateral hypothalamic stimulation frequency threshold for eliciting feeding behavior. The antibodies utilized in that study cross-react completely with dynorphin A(1-17) and, to a lesser extent, dynorphin A(1-8). In the present study, highly specific antibodies to dynorphin A(1-17) and dynorphin A(1-8) were infused into the lateral ventricle and mesopontine aqueduct to determine which biologically active dynorphin A fragment mediates feeding and at what level of the CNS this activity is likely to occur. Both antibodies were found to elevate the feeding threshold. Dynorphin A(1-8) antibodies were effective at both injection sites while dynorphin A(1-17) antibodies were only effective at the lateral ventricular site. These findings suggest that feeding-related dynorphin A(1-17) activity may occur predominantly within the forebrain, while dynorphin A(1-8) activity occurs within the brainstem. Only the dynorphin A(1-8) antibodies, infused into the aqueduct, produced a naloxone-like pattern of progressive elevation in serially determined thresholds; this pattern was previously interpreted to reflect a reduction in consummatory reward. Dynorphin A(1-8) activity within some brainstem structure(s) may therefore contribute prominently to the opioid mechanism whose mediation of the hedonic response to food was previously inferred from naloxone antagonism.     

Repeated electroconvulsive shocks alter the biosynthesis of enkephalin and concentration of dynorphin in the rat brain

Ten daily electroconvulsive shocks (ECSs) caused a two-fold increase in (Met5)-enkephalin-like immunoreactivity (ME-LI) and an 80% increase in the level of mRNA coding for preproenkephalin A in the hypothalamus. These observations suggest that repeated ECSs increase the biosynthesis of hypothalamic ME. Ten daily ECSs also increased dynorphin A (1-8)-like immunoreactivity (DN-LI) in hypothalamus (45%) but not in frontal cortex. Unlike other brain regions, a 64% decrease of DN-LI was found in the hippocampus after 10 daily ECSs whereas a significant increase of ME-LI (40%) was observed. Furthermore, immunocytochemical studies revealed an increase of (Leu5)-enkephalin-like immunoreactivity in the perforant pathway and a decrease of DN-LI in the mossy fiber system of the hippocampus after 10 daily ECSs. These studies suggest that alterations in enkephalin and dynorphin in the limbic system may contribute to the behavioral changes observed after repeated ECSs.     

Biosynthesis and release of beta-endorphin-, N-acetyl beta-endorphin-, beta-endorphin-(1-27)-, and N-acetyl beta-endorphin-(1-27)-like peptides by rat pituitary neurointermediate lobe: beta-endorphin is not further processed by anterior lobe

Continuous labeling and pulse-chase techniques were employed to study the synthesis and secretion of multiple forms of immunoreactive beta-endorphin by cultured dispersed rat anterior lobe cells and intact neurointermediate pituitary lobe. Cell and medium extract immunoreactive beta-endorphin (specific immunoprecipitation and radioimmunoassay) exhibiting a Kav similar to authentic beta-endorphin upon gel filtration was characterized further by nonequilibrium isoelectric focusing, cation exchange chromatography, reverse phase high pressure liquid chromatography, and partial tryptic and chymotryptic mapping. Intact neurointermediate lobes incorporated radiolabeled amino acids into four to six forms of immunoreactive beta-endorphin. Four of these forms were physicochemically similar to authentic beta-endorphin, N-acetylated beta-endorphin, beta-endorphin-(1-27), and N-acetylated beta-endorphin-(1-27). Pulse-chase studies indicated that a beta-lipotropin-like molecule served as a metabolic intermediate for a beta-endorphin-like molecule. As beta-endorphin-like material accumulated in the cell, some of it was N-acetylated (approximately 18% at 2 hr chase and approximately 65% at 18 hr chase). At later chase times, beta-endorphin-(1-27)- and N-acetylated beta-endorphin-(1-27)-like peptides were the predominant molecular species detected. All endorphin forms were detected in unlabeled tissue maintained in culture or tissue continuously labeled for 72 hr and were released into the medium under basal, stimulatory (10(-8) M norepinephrine), or inhibitory (10(-7) M dopamine) incubation conditions. In all cases, beta-endorphin-(1-27)-like species were the predominant forms (more than 70% of total) present in the cells and released into the medium. In contrast, approximately 90% of radiolabeled immunoreactive beta-endorphin extracted from anterior lobe cells and medium similarly incubated appeared to represent the authentic beta-endorphin molecule. Continuous labeling (72 hr) revealed the beta-lipotropin/beta-endorphin molar ratio to be approximately 4. We conclude that, in anterior lobe, most of the beta-endorphin is not processed further and is released intact, while in neurointermediate lobe, it serves as a biosynthetic intermediate.     

Chronic haloperidol and clozapine differentially affect dynorphin peptides and substance P in basal ganglia of the rat

The effect of chronic neuroleptic treatment, using haloperidol or clozapine, on immunoreactive dynorphin peptide and substance P levels in basal ganglia of rats was examined. The drugs were administered i.p. in daily doses for 10 days (haloperidol 1 mg/kg and clozapine 10 mg/kg). Dynorphin A, dynorphin B and substance P were measured in substantia nigra, striatum, globus pallidus and hypothalamus using specific radioimmunoassays. The most prominent effects were observed with with clozapine which increased levels of all measured peptides in substantia nigra. Haloperidol only affected nigral substance P levels which declined, while nigral dynorphin peptide levels remained unchanged. In striatum, haloperidol slightly reduced dynorphin peptides while substance P was unaffected. Clozapine increased striatal substance P but the dynorphin peptides were not affected. Minor changes in dynorphin peptides found in globus pallidus and hypothalamus were not statistically reliable. Substance P was not changed in these structures after either of the two drugs. High molecular weight fragments (greater than or equal to 5,000) from the dynorphin precursor, proenkephalin B, were measured in substantia nigra and striatum using trypsin digestion and subsequent analysis of generated Leu-enkephalin-Arg6. These high molecular weight fragments were found to be affected in the same manner as the dynorphin peptides. This study indicates that the two types of neuroleptic drugs have different modes of interaction on peptide systems in basal ganglia of rats. Dynorphin peptides and substance P were also differentially affected.     

A low hippocampal dynorphin A (1-8) immunoreactivity in spontaneously hypertensive rats

Dynorphin A (1-8)-like immunoreactivity (DN-LI A(1-8] was determined by radioimmunoassay in the brains of age matched spontaneously hypertensive rats (SHR) and two normotensive control groups consisting of Wistar-Kyoto (WKY) and Sprague-Dawley (SD) strain rats. A significantly lower DN-LI A(1-8) was found in the hippocampus and hypothalamus of the SHR compared with the WKY groups. DN-LI A(1-8) was 24% of control WKY levels in hippocampus and 79% of that in WKY hypothalamus at 16 weeks. Similar lower levels of DN-LI A(1-8) were also observed in SHR at 4, 8, and 12 weeks during the development of hypertension when compared with both WKY and SD groups. We failed to find significant differences in brain stem DN levels between the groups. The relationship between the low hippocampal dynorphin levels in SHR and the hypertension is problematical because the differences were present before (4 wks), during (8 and 12 wks) and after (16 wks) its development.     

Neurotensin-like immunoreactivity and neurotensin receptors in the rat hypothalamus and in the neurointermediate lobe of the pituitary gland

In the rat hypothalamus, cell bodies containing neurotensin-like immunoreactivity were mainly found in the medial preoptic area, the periventricular nucleus, the paraventricular nucleus, the supraoptic nucleus and the arcuate nucleus. [3H]neurotensin binding sites were observed throughout the hypothalamus with a dense accumulation of silver grains over the paraventricular nucleus, the arcuate nucleus and the median eminence region. By radioimmunoassay neurotensin-like immunoreactivity was also found in the neurointermediate lobe of the pituitary gland of various mammalian species and in human postmortem posterior pituitary glands. In the rat studies involving pituitary stalk transections and the neurotoxin monosodium glutamate indicated the presence of a neurotensinergic pathway from the arcuate nucleus to the neurointermediate lobe of the pituitary gland. [3H]neurotensin binding sites were found to be concentrated over the intermediate lobe of the pituitary gland and their presence was not affected by pituitary stalk transection, indicating their localization on endocrine cells of the intermediate lobe of the pituitary gland.     

Unmyelinated primary afferent fiber stimulation depletes dynorphin A (1-8) immunoreactivity in rat ventral horn.

The present study demonstrates many dynorphin (DYN)-immunoreactive fibers and presumed presynaptic terminals in rat lumbar ventral horn. The fibers and terminals seem to arise largely from DYN-containing intrinsic neurons in the dorsal horn. The majority of the presumed terminals closely surround a subpopulation of motoneurons that tend to be located in flexor motoneuron columns. Acute C fiber, but not A fiber, primary afferent stimulation depletes the ventral horn DYN immunostaining. We interpret these findings to indicate that the spinal DYN neurons are well positioned to serve both as modulators of nociceptive input and as interneurons in motor reflexes. We further hypothesize that the depletion of DYN-immunoreactivity that follows either acute C fiber stimulation or intense nociceptive stimuli may be the trigger for the upregulation in spinal cord DYN that occurs in models of chronic pain states.     

Distribution of dynorphin and enkephalin peptides in the rat brain

The neuroanatomical distribution of dynorphin B-like immunoreactivity (DYN-B) was studied in the adult male and female albino rat. The distribution of DYN B in colchicine- and noncolchicine-treated animals was also compared to that of another opioid peptide derived from the prodynorphin precursor dynorphin A (1-8) (DYN 1-8), and an opioid peptide derived from the proenkephalin precursor met-enkephalin-arg-gly-leu (MERGL). DYN B cell bodies were present in nonpyramidal cells of neo- and allocortices, medium-sized cells of the caudate-putamen, nucleus accumbens, lateral part of the central nucleus of the amygdala, bed nucleus of the stria terminalis, preoptic area, and in sectors of nearly every hypothalamic nucleus and area, medial pretectal area, and nucleus of the optic tract, periaqueductal gray, raphe nuclei, cuneiform nucleus, sagulum, retrorubral nucleus, peripeduncular nucleus, lateral terminal nucleus, pedunculopontine nucleus, mesencephalic trigeminal nucleus, parabigeminal nucleus, dorsal nucleus of the lateral lemniscus, lateral superior olivary nucleus, superior paraolivary nucleus, medial superior olivary nucleus, ventral nucleus of the trapezoid body, lateral dorsal tegmental nucleus, accessory trigeminal nucleus, solitary nucleus, nucleus ambiguus, paratrigeminal nucleus, area postrema, lateral reticular nucleus, and ventrolateral region of the reticular formation. Fiber systems are present that conform to many of the known output systems of these nuclei, including major descending pathways (e.g., striatonigral, striatopallidal, reticulospinal, hypothalamospinal pathways), short projection systems (e.g., mossy fibers in hippocampus, hypothalamo-hypophyseal pathways), and local circuit pathways (e.g., in cortex, hypothalamus). The distribution of MERGL was, with a few notable exceptions, in the same nuclei as DYN B. From these neuroanatomical data, it appears that the dynorphin and enkephalin peptides are strategically located in brain regions that regulate extrapyramidal motor function, cardiovascular and water balance systems, eating, sensory processing, and pain perception.     

Types and activities of voltage-operated calcium channels change during development of rat pituitary neurointermediate lobe

Cultures of pituitary neurointermediate lobe cells were established from rats aged 1, 12, and 42 days to identify the types and assess the activities of Ca²⁺ channels present in melanotropes, glial-like cells, and fibroblasts during development. Day 12 represents the time at which dopaminergic axons have become distributed throughout the lobe, glial cells begin to lose their radial orientation, and melanotropes robustly express the short isoform of the dopamine D₂ receptor. Thus, we studied Ca²⁺ channels in relation to the event of innervation of melanotropes. Real-time fluorescence video microscopy, in the presence of pharmacological agents, which block L-, N-, P-, and T-type channels, was used as an indirect measurement of channel activity. Assessment of cell type was verified by triple-label fluorescence immunohistochemistry. In melanotropes, extracellular Ca²⁺ addition caused Ca²⁺ influx through ω-conotoxin GVIA-sensitive, N-type channels on days 1 and 12 but not on day 42. The K⁺ depolarization induced an increase in intracellular Ca²⁺ concentration in all age-groups. This effect was decreased by nifedipinc, an L-type channel blocker, at all ages, and by ω-agatoxin IVa, a P-type blocker, only on day 42. These results demonstrate that the predominance of N- or P-type channels on melanotropes is age-dependent and can be correlated with other developmental changes. The T-type blocker, NiSO₄, had no effect. In glial-like cells of all ages, extracellular Ca²⁺ addition resulted in an increase in intracellular Ca²⁺ concentration, which was inhibited only by NiSO₄. The percentage of responsive glial-like cells was equally high in days 1 and 12 cultures, then declined by day 42. The K⁺ depolarization had no effect on glial-like cells. Fibroblasts did not respond significantly to extracellular Ca²⁺ or K⁺ depolarization, indicating little detectable activity by this methodology from functional voltage-operated Ca²⁺ channels.     

Effects of immunoneutralization of dynorphin1-17 and dynorphin1-8 on the activity of central dopaminergic neurons in the male rat.

The effects of administration of antibodies against dynorphin1-17 (DYN1-17-AB) and dynorphin1-8 (DYN1-8-AB) were examined on the activity of dopaminergic (DA) neurons comprising the nigrostriatal, mesolimbic, tuberoinfundibular and periventricular-hypophysial systems in the male rat brain. DA neuronal activity was estimated by measuring the concentration of the dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in brain (striatum, nucleus accumbens, median eminence) and pituitary regions (intermediate lobe) containing terminals of these neurons. The intracerebroventricular administration of either DYN1-17-AB or DYN1-8-AB produced a time-related increase in the activity of tuberoinfundibular and periventricular-hypophysial DA neurons, but failed to alter the activity of nigrostriatal or mesolimbic DA neurons. The ability of both DYN1-17-AB and DYN1-8-AB to enhance the activity of tuberoinfundibular and periventricular-hypophysial DA neurons was reversed by the kappa opioid agonist U-50,488. These results indicate that DYN1-17-AB and DYN1-8-AB, presumably by binding endogenous dynorphins, remove a tonic inhibitory action of these opioid peptides on tuberoinfundibular and periventricular-hypophysial DA neurons.     

Analgesic actions of dynorphin A(1-13) antiserum in the rat brain stem.

This study was performed to evaluate the effects of dynorphin A(1-3) antiserum when microinjected into an active hyperalgesic region within the rat brain stem. When administered within the dorsal posterior mesencephalic tegmentum (DPMT) of intact conscious rats, dynorphin A(1-13) antiserum produced rapid onset and persistent prolongation of a low intensity thermally evoked tail avoidance response (LITETAR). These analgesic actions of the dynorphin A(1-13) antiserum appeared to be dose dependent. These studies support previous hypotheses about the existence of tonically active brain stem opioid hyperalgesic process. Further, the results provide indirect evidence for a potential role of brain stem dynorphin(s) in facilitating pain.     

Dopamine release and synthesis in the neurointermediate lobe of the rat hypophysis in vitro after electrical stimulation of the pituitary stalk

The isolated neurointermediate lobe of the rat hypophysis was incubated in Krebs solution and the stalk was electrically stimulated. The endogenous dopamine (DA) released into the medium was estimated by HPLC with electrochemical detection. Stimulation with biphasic pulses (1 ms, 10 Hz) in the presence of pargyline elicited a mean DA output of 200 fg X pulse-1. This release was calcium-dependent but was only partially inhibited by tetrodotoxin (TTX) (1 microM), effects typical for direct electrical depolarization of the nerve endings. Reducing the duration of the electric pulses to 0.2 ms (15 Hz) caused a reduction in DA output to about 40 fg X pulse-1 which was completely blocked by TTX indicating that it was evoked by propagated action potentials. DA overflow was enhanced when the action potential was prolonged with tetraethylammonium (TEA) or when DA uptake was inhibited with GBR 12921. Evidence for a calcium-dependent increase in DA synthesis in electrically stimulated NILs has been obtained when monoamine oxidase was inhibited and TEA or GBR 12921 was present in the Krebs solution. The present results complete the requirements for the DA in the NIL to be classified as a neurotransmitter substance. The NIL-pituitary stalk preparation is a useful model for studying regulatory mechanisms in dopaminergic nerve terminals.     

Identification of MSH release-inhibiting elements in the neurointermediate lobe of the rat

Neurointermediate lobes of rats comprise elements which, when excited in vitro, bring about an inhibition of the release of melanocyte stimulating hormone (MSH). Superfusion of neurointermediate lobes of intact donor rats with medium containing 45 mM K+ induced a stimulation of the release of oxytocin, arginine-vasopressin and dopamine (DA) and inhibited the release of MSH. Fluorescence histochemical observations and the results of release studies indicate that electrothermic lesions in the mediobasal hypothalamus induced a more rapid degeneration of dopaminergic than of peptidergic terminals in the neurointermediate lobe. Dopaminergic nerve terminals and the stimulated release of DA had vanished completely on the second day after these lesions, which coincided with the disappearance of K+-induced inhibition of MSH release. Frontal hypothalamic deafferentations resulted in disappearance of peptidergic nerve terminals as evidenced by the development of diabetes insipidus and the strong decline of depolarization-induced release of oxytocin and vasopressin from neurointermediate lobes in vitro. In contrast, the dopaminergic plexus was left intact, as was the K+-induced stimulation of DA release and inhibition of MSH release. We conclude that the K+-induced inhibition of MSH release is mediated by DA rather than by neuropeptides from terminals in the neurointermediate lobe. The results are in agreement with the proposed MSH release-inhibiting role of the dopaminergic tuberoinfundibular neurones.     

Effects of dynorphin A (1-17), dynorphin A (1-13) and D-ala2-D-leu5-enkephalin on the excitability of pyramidal cells in CA1 and CA2 of the rat hippocampus in vitro

The effects of D-ala2-D-leu5-enkephalin (DADL), dynorphin (DYN) A (1-17), and DYN A (1-13) on the excitability of hippocampal (HC) pyramidal cells in CA1 and CA2 were compared in an in vitro preparation of the rat. DADL and DYN A (1-13) increased the size of the population spike (PS) evoked in CA1 and CA2 by stimulation of the stratum radiatum. Paired-pulse stimulation showed that both peptides decreased the inhibition produced by the conditioning stimulus. These effects were naloxone-reversible. DYN A (1-17) decreased the PS in CA2 and increased the inhibition evoked by the conditioning stimulus. When applied in the same concentration-range, the depressant effects of DYN A (1-17) were more pronounced in CA1 than in CA2 and not naloxone-reversible. DYN A (1-17) reduced the excitatory effects of DADL. Low concentrations of DYN A (1-17) (0.1 nM) produced, like DYN A (1-13) and DADL, an increase in the PS in CA1 in the majority of tests. The present data suggest that DYN A (1-17) shows the excitatory action of other opioids HC pyramidal cells only at low concentration.