Hypovolemic hemorrhage induces Fos expression in the rat hypothalamus: Evidence for involvement of the lateral hypothalamus in the decompensatory phase of hemorrhage.

This study tested the hypothesis that the hypothalamus participates in the decompensatory phase of hemorrhage by measuring Fos immunoreactivity and by inhibiting neuronal activity in selected hypothalamic nuclei with lidocaine or cobalt chloride. Previously, we reported that inactivation of the arcuate nucleus inhibited, but did not fully prevent, the fall in arterial pressure evoked by hypotensive hemorrhage. Here, we report that hemorrhage (2.2 ml/100g body weight over 20 min) induced Fos expression in a high percentage of cells in the paraventricular, supraoptic and arcuate nuclei of the hypothalamus as shown previously. Lower densities of Fos immunoreactive cells were also found in the medial preoptic area (mPOA), anterior hypothalamus, lateral hypothalamus (LH), dorsomedial hypothalamus, ventromedial hypothalamus (VMH) and posterior hypothalamus. Bilateral injection of lidocaine (2%; 0.1 µl or 0.3 µl) or cobalt chloride (5mM; 0.3 µl) into the tuberal portion of the LH immediately before hemorrhage was initiated reduced the magnitude of hemorrhagic hypotension and bradycardia significantly. Lidocaine injection into the VMH also attenuated the fall in arterial pressure and heart rate evoked by hemorrhage although inactivation of the mPOA or rostral LH was ineffective. These findings indicate that hemorrhage activates neurons throughout much of the hypothalamus and that a relatively broad area of the hypothalamus, extending from the arcuate nucleus laterally through the caudal VMH and tuberal LH, plays an important role in the decompensatory phase of hemorrhage.

Localization of pro-opiomelanocortin mRNA transcripts and peptide immunoreactivity in rat heart.

OBJECTIVE: alpha-Melanocyte-stimulating hormone (alpha-MSH), beta-endorphin and other pro-opiomelanocortin-(POMC) derived peptides have been detected in the heart, but it is uncertain whether they are synthesized by cardiomyocytes or by cardiac nerves innervating the heart. The objective of this study was to determine whether POMC peptides are synthesized by cardiomyocytes. METHODS: Pro-opiomelanocortin peptides were localized in rat heart by immunohistochemistry using antisera against alpha-MSH, beta-endorphin and alpha N-acetyl-beta-endorphin, the predominant POMC peptides found in heart. Pro-opiomelanocortin mRNA was investigated by reverse transcription polymerase chain reaction (RT-PCR) using primers that discriminate between full-length POMC mRNA and a 5' truncated POMC transcript that is presumed to be non-functional. RESULTS: alpha-Melanocyte-stimulating hormone, beta-endorphin and alpha N-acetyl-beta-endorphin immunoreactivities were localized in atrial myocytes, particularly in the atrial appendages, but not to a significant extent in ventricular myocytes. Cardiac nerves were not immunostained. Atrial natriuretic peptide (ANP) immunoreactivity was similarly distributed in the adult heart. In neonatal heart, POMC-peptide and ANP immunoreactivities were present in both atrial and ventricular myocytes. RT-PCR amplification showed that full-length POMC mRNA transcripts were present in both atrial and ventricular tissue and provide evidence that 5' truncated POMC mRNA is expressed in heart. CONCLUSIONS: These results support the hypothesis that cardiomyocytes synthesize POMC peptides.

Dopaminergic regulation of the biosynthetic activity of individual melanotropes in the rat pituitary intermediate lobe: a morphometric analysis by light and electron microscopy and in situ hybridization.

The primary cell type of the intermediate lobe (IL) of the rat pituitary is a polyhedral secretory cell with a smooth ovoid nucleus. The results of this study demonstrate, however, that IL melanotropes are a heterogeneous cell population. Melanotropes differed in the tinctorial properties of their cytoplasm; some cells appeared distinctly darker, others lighter, and cells staining in intermediate shades were also found. Electron microscopical morphometry revealed that darkly staining melanotropes have a denser cytosol and contain a greater amount of rough endoplasmic reticulum, mitochondria, and secretory vesicles than light cells. In addition, in situ hybridization studies, using a POMC probe, showed that POMC mRNA was distributed unevenly among melanotropes in a pattern comparable to the distribution of light and dark cells. These studies further demonstrated that dopaminergic drug treatments, which are known to alter the secretion of POMC-related peptides from the IL, produced parallel changes in both the histological staining properties and the amount of POMC mRNA per cell. Haloperidol treatment dramatically increased the number of dark melanotropes and the amount of POMC mRNA in each cell and eliminated the cellular heterogeneity in both staining properties and the distribution of POMC mRNA. After bromocriptine treatment the number of light melanotropes increased, and each cell contained reduced levels of POMC mRNA. These findings indicate that individual melanotropes maintain different levels of biosynthetic activity and that treatments that alter the secretion of POMC peptides affect both the rate of POMC synthesis in individual melanotropes and the cellular heterogeneity of the IL.

Equine Cushing's disease: differential regulation of beta-endorphin processing in tumors of the intermediate pituitary.

Equine Cushing's disease is caused by an adenomatous hyperplasia of the intermediate pituitary which secretes high levels of beta-endorphin, ACTH, and other peptide derivatives of POMC. In the present study we found that plasma and cerebrospinal fluid immunoreactive beta-endorphin (i beta-endorphin) levels were 60- and 120-fold higher than control values in horses with Cushing's disease. There were no significant differences in intermediate lobe i beta-endorphin concentrations, although anterior lobe i beta-endorphin was significantly reduced in Cushing's horses, presumably because high levels of circulating glucocorticoids inhibit POMC biosynthesis in corticotrophs. Although the i beta-endorphin concentration of the tumors was not different from that in normal tissue, the posttranslational processing of beta-endorphin in the two tissues differed significantly. In controls, beta-endorphin-(1-31) was extensively processed to N-acetyl-beta-endorphin-(1-31), -(1-27), and -(1-26) and des-acetyl beta-endorphin-(1-27). N-Acetyl-beta-endorphin-(1-27) was the predominant form, constituting 57% of the total i beta-endorphin, whereas beta-endorphin-(1-31) was quantitatively minor (less than 7% of the total immunoreactivity. In adenomatous pituitaries, the processing of beta-endorphin was restricted, significantly increasing the proportions of beta-endorphin-(1-31) and N-acetyl-beta-endorphin-(1-31) and lowering the amounts of N-acetyl-beta-endorphin-(1-27) and -(1-26). These changes in peptide processing were associated with markedly reduced levels of dopamine, suggesting that the dopaminergic neurons that normally control intermediate lobe secretion no longer innervate the hyperplastic tissue. These findings are consistent with evidence that the dopaminergic innervation of the intermediate pituitary regulates the posttranslational processing and release of beta-endorphin.

Alpha-melanocyte-stimulating hormone and N-acetyl-beta-endorphin immunoreactivities are localized in the human pituitary but are not restricted to the zona intermedia.

The adult human pituitary lacks a well defined intermediate lobe, and it is uncertain whether the POMC cells that remain in the zona intermedia represent melanotropes or corticotropes. In the present study, we investigated whether the N-acetylated beta-endorphin- and alpha-MSH-related peptides that are characteristically produced by melanotropes in the rat and other species are localized in the human pituitary. Sequential gel filtration and ion exchange HPLC analysis revealed that small amounts of alpha-N-acetyl-beta-endorphin-(1-31), as well as beta-endorphin-(1-27) and beta-endorphin-(1-26), were detectable in human pituitary extracts, although beta-endorphin-(1-31) was clearly the major form. Consistent with this analysis, low levels of alpha-MSH, but not N,O-diacetyl-alpha-MSH, were identified by reverse-phase HPLC, although again, the desacetyl form of alpha-MSH predominated. Immunohistochemistry revealed that N-acetyl-beta-endorphin immunoreactivity was colocalized with ACTH and beta-endorphin in a subpopulation of zona intermedia cells. Unexpectedly, immunoreactive N-acetyl-beta-endorphin was also observed in a comparable proportion of corticotropes dispersed throughout the anterior lobe. alpha-MSH immunoreactivity was similarly distributed. These results indicate that N-acetylation is not restricted to the zona intermedia, suggesting that the strict dichotomy between corticotrope and melanotrope POMC processing observed in the rat and other species does not extend to the human pituitary.

Coordinate regulation of peptide acetyltransferase activity and proopiomelanocortin gene expression in the intermediate lobe of the rat pituitary.

Proopiomelanocortin (POMC) is posttranslationally processed in the intermediate lobe of the pituitary to both N-terminally acetylated and nonacetylated forms of alpha MSH and beta-endorphin (beta END). N-Acetylation substantially modifies the physiological responses produced by both peptides, suggesting that the activity of the peptide acetyltransferase, which posttranslationally acetylates beta END and des-acetyl-alpha MSH, may play an important role in defining the biological activity of the secretory products of the intermediate pituitary lobe. The present results demonstrate that peptide acetyltransferase activity is induced by treating rats chronically with the dopamine receptor antagonist haloperidol. Haloperidol administration produced parallel and essentially equivalent increases in acetyltransferase activity, POMC mRNA levels, and the content and secretion of POMC-derived peptides in the neurointermediate pituitary. Time-course and dose-response studies further demonstrated that acetyltransferase activity covaried with POMC mRNA and peptide levels. Chronic treatment with the dopamine receptor agonist bromocriptine had the opposite effects; it lowered acetyltransferase activity, POMC mRNA levels, and alpha MSH and beta END immunoreactivities. Subcellular fractionation showed that acetyltransferase activity was localized in three subcellular compartments corresponding in density to secretory vesicles, rough endoplasmic reticulum and Golgi apparatus, and cytosol. Haloperidol treatment significantly increased the specific activity of the secretory vesicle-associated acetyltransferase without affecting the specific activity of the enzymes present in the endoplasmic reticulum or cytosol. Together, these data indicate that peptide acetyltransferase activity and POMC biosynthesis are coregulated.

Histological evaluation of the dopaminergic regulation of proopiomelanocortin gene expression in the intermediate lobe of the rat pituitary, involving in situ hybridization and [3H]thymidine uptake measurement.

The melanotroph, a polyhedral secretory cell with an ovoid smooth nucleus, is the primary cell type of the intermediate lobe (IL) of the rat pituitary. The melanotrophs are not uniform, but differ in the tinctorial properties of their cytoplasm; some cells appear distinctly darker, others lighter, and cells staining in intermediate shades are also found. In addition, in situ hybridization using proopiomelanocortin (POMC) probes shows an uneven distribution of POMC mRNA among melanotrophs, indicating that different cells maintain different levels of biosynthetic activity. Dopaminergic drugs known to alter the secretion of POMC-related peptides from the IL produced parallel changes in histological staining properties and the amount of POMC mRNA per cell, as determined by in situ hybridization. Acute bromocriptine treatment (6 h) produced a dramatic reduction in grain counts over melanotroph cytoplasm (to 10% of the control levels). A similar reduction persisted after chronic treatment. Six hours after a single haloperidol injection, the grain counts were 180% of control levels. After chronic haloperidol treatment, they were further elevated to 300% of control levels. Chronic bromocriptine and haloperidol treatment also changed the thickness of the IL. Bromocriptine reduced and haloperidol treatment increased the number of cell layers in the IL by changing the rate of cell proliferation. Thus, haloperidol treatment significantly increased and bromocriptine treatment significantly decreased the number of melanotrophs labeled by [3H]thymidine. The mitotic index followed the same trend. These results suggest that dopamine regulation of the IL acts by two different mechanisms: POMC gene expression and cellular proliferation. The change in POMC gene expression is the cell's first rapid response. The influence on the cell cycle appears after subchronic and chronic treatment.

A diurnal rhythm in proopiomelanocortin messenger ribonucleic acid that varies concomitantly with the content and secretion of beta-endorphin in the intermediate lobe of the rat pituitary.

The present study investigated whether diurnal variations in the secretion of alpha MSH and beta-endorphin from the intermediate lobe (IL) of the pituitary are associated with parallel changes in the synthesis of mRNA specifically encoding proopiomelanocortin (POMC). The results demonstrate that concomitant diurnal variations occur in both plasma and IL concentrations of immunoreactive beta-endorphin and alpha MSH. Plasma and IL peptide levels were relatively constant during daylight hours (0600-1800 h), but increased after the onset of darkness and reached maximal concentrations at 0200 h. To examine the possibility that this diurnal rhythm in the content and secretion of POMC-derived peptides resulted from diurnal changes in the biosynthesis of POMC, the concentration and rate of synthesis of POMC mRNA were examined. POMC mRNA levels were elevated during the dark period, reaching a maximum level at 0200 h that was 2-fold higher than that occurring during the light period. POMC mRNA synthesis, determined by measuring the number of RNA polymerase II complexes transcribing the POMC gene in isolated cell nuclei, also varied diurnally, with maximum transcription rates occurring at 1800 h, thus preceding maximal increases in POMC mRNA content and POMC peptide secretion by 8 h. Together, these data indicate that diurnal variations in the content and secretion of POMC-derived peptides are associated with parallel changes in POMC mRNA concentrations and are preceded by similar changes in POMC gene transcription.

Regional and laminar specificity of kainate-stimulated cobalt uptake in the rat hippocampal formation.

Kainate and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors were initially found to be impermeable to calcium, but cloning and expression studies subsequently revealed that certain kainate and AMPA receptor subunit combinations display substantial divalent cation permeability. The regional and cellular distribution of calcium-permeable kainate/AMPA receptors has not been extensively investigated, however. In this study, we used a histochemical technique, the kainate-stimulated cobalt uptake assay, to localize calcium-permeable kainate responsive receptors in the rat hippocampal formation. In the presence of cobalt, kainate produced a highly localized, dark granular precipitate in dendrites, perikarya, or both, of hippocampal neurons. Kainate-stimulated cobalt uptake was time- and concentration-dependent (1 microM-1 mM) and was blocked by the glutamate receptor antagonist, kynurenate. The specific cellular location of cobalt labeling varied regionally within the hippocampal formation, switching from selective labeling of only apical dendrites in Ammon's horn subregion la (CA1a) to a diffuse band of punctate labeling in CA1c to labeling of cell bodies along with their proximal dendrites in CA3. Furthermore, increasing the kainate concentration not only enhanced the intensity of cobalt labeling, but also changed the pattern of cellular staining from exclusively dendritic labeling to extensive staining of both cell bodies and dendrites in CA1a pyramidal neurons. High kainate concentrations or prolonged incubation times produced a diffuse cellular labeling suggestive of neurotoxicity. These data are consistent with evidence that specific kainate and AMPA receptor subunit combinations are targeted to specific synapses in hippocampal pyramidal neurons.

Choline as an agonist: determination of its agonistic potency on cholinergic receptors.

These experiments examined the potency of choline as a cholinergic agonist at both muscarinic and nicotinic receptors in rat brain and peripheral tissues. Choline stimulated the contraction of isolated smooth muscle preparations of the stomach fundus, urinary bladder and trachea and reduced the frequency of spontaneous contractions of the right atrium at high micromolar and low millimolar concentrations. The potency of choline to elicit a biological response varied markedly among these tissues; EC50 values ranged between 0.41 mM in the fundus to 14.45 mM in the atrium. Choline also displaced [3H]quinuclidinyl benzilate binding in a concentration-dependent manner although, again, its potency varied among different brain regions (Ki = 1.2 to 3.5 mM) and peripheral tissues (Ki = 0.28 to 3.00 mM). Choline exhibited a comparable affinity for nicotinic receptors. It stimulated catecholamine release from the vascularly perfused adrenal gland (EC50 = 1.3 mM) and displaced L-[3H]nicotine binding to membrane preparations of brain and peripheral tissues (Ki = 0.38 to 1.17 mM). However, the concentration of choline required to bind to cholinergic receptors in most tissues was considerably higher than serum levels either in controls (8-13 microM) or following the administration of choline chloride (200 microM). These results clearly demonstrate that choline is a weak cholinergic agonist. Its potency is too low to account for the central nervous system effects produced by choline administration, although the direct activation of cholinergic receptors in several peripheral tissues may explain some of its side effects.

Diethyldithiocarbamate depresses the acoustic startle response in rats.

The relationship between norepinephrine (NE) content in cortex and spinal cord and acoustic startle amplitude was investigated in two experiments. Administration of diethyldithiocarbamate (DDC) depressed startle amplitude at the same time and dose that it most severely depleted NE content. These results support the conclusion that NE facilitates the normal elaboration of the acoustic startle reflex and also support evidence that NE activity in the spinal cord may be of particular importance in the maintenance of normal startle amplitude.

Glycyl-L-glutamine antagonizes alpha-MSH-elicited thermogenesis.

The objective of this study was to determine whether glycyl-L-glutamine [beta-endorphin(30-31)] modulates the thermoregulatory actions of alpha-MSH. Microinjection of alpha-MSH (0.06 nmol) into PGE2-responsive thermogenic sites in the medial preoptic area of rats generated a hyperthermic response, inducing a 0.85 +/- 0.19 degrees C rise in colonic temperature (Tc) within 45 min. Coadministration of glycyl-L-glutamine (3.0 nmol) completely blocked the response, maintaining Tc at baseline levels. This was not attributable to glycyl-L-glutamine hydrolysis because coadministration of glycine and glutamine had no effect on alpha-MSH-induced thermogenesis. Glycyl-L-glutamine, injected alone, was similarly without effect. These data indicate that glycyl-L-glutamine inhibits alpha-MSH-induced thermogenesis but is devoid of thermoregulatory activity itself.

POMC-derived peptide immunoreactivity in neural lobe axons of the human pituitary.

The efferent projections of proopiomelanocortin (POMC) neurons in the arcuate nucleus and nucleus of the solitary tract have been extensively characterized in the rat, but are less well understood in the human brain. We report here that ACTH, alpha-MSH, beta-endorphin, and N-acetyl-beta-endorphin immunoreactive axons are localized in the neural lobe of the human pituitary gland, in congruence with prior evidence that beta-endorphin and other POMC-derived peptides modulate vasopressin and oxytocin secretion.

Regional heterogeneity in the ratio of alpha-MSH: beta-endorphin in rat brain.

The molar ratio of alpha-MSH: beta-endorphin varies markedly among discrete microdissected regions of rat brain ranging from 0.57 in the median eminence to 2.74 in the lateral septum. This finding demonstrates that alpha-MSH and beta-endorphin (beta-END) are not uniformly distributed in a 1:1 molar ratio in rat brain as one might predict based on the consideration that the two peptides are synthesized in equimolar amounts as part of a common precursor molecule, pro-opiomelanocortin. The data indicate instead that the concentrations of alpha-MSH and beta-END, the two predominant peptides expressed by opiomelantropinergic neurons, are independently regulated in rat brain. The heterogeneity of alpha-MSH: beta-END ratios suggests that the regulation of alpha-MSH and beta-END is regionally specific and may impart functional selectivity to the multisecretory opiomelanotropinergic neuronal system.

Characterization of beta-endorphin- and alpha-MSH-related peptides in rat heart.

POMC-derived peptides and mRNA have been identified in heart tissue, although POMC processing has not been fully characterized. In the present study, we found that beta-lipotropin and ACTH were localized in rat heart, although they were almost entirely converted to beta-endorphin- and alpha-MSH-related peptides. Ion exchange HPLC analysis revealed that beta-endorphin(1-31) was further processed to alpha-N-acetyl-beta-endorphin(1-31), which comprised 35.9 +/- 0.1% of total immunoreactivity, and smaller amounts of beta-endorphin(1-27), beta-endorphin(1-26), and their alpha-N-acetylated derivates. The predominant alpha-MSH immunoreactive peptides coeluted with alpha-MSH and N,O-diacetyl-alpha-MSH by reverse-phase HPLC, although small amounts of ACTH(1-13)-NH2 were also present. Thus, multiple forms of beta-endorphin and alpha-MSH are localized in rat heart. beta-Endorphin(1-31) is a minor constituent, however, indicating that nonopioid beta-endorphin peptides predominate.

Localization of neuropeptide Y immunoreactivity and [125I]peptide YY binding sites in the human pituitary.

High levels of neuropeptide Y (NPY) are found in the hypothalamus, median eminence, pituitary portal blood, and the pituitary in a number of species. Neuropeptide Y may influence the synthesis and secretion of a variety of hormones by interacting with specific receptors in the hypothalamus and/or the pituitary. To further define the function of NPY in the pituitary, we have examined the distribution of NPY immunoreactivity and NPY receptors in sections of human pituitary using immunohistochemical and autoradiographic techniques. Neuropeptide Y-immunoreactive varicose axons were seen throughout the neural lobe. A moderate number of NPY-immunoreactive cells were found in the anterior lobe. A very high level of [125I]PYY binding was seen in the neural lobe with low levels in the anterior lobe. The binding in the neural lobe was inhibited by NPY(13-36) at a Ki of 5.3 nM and [Leu31-Pro34]NPY at a Ki of 390 nM, indicating the receptor was the Y2 subtype. Therefore, neuronally released NPY may modulate human neural lobe function through a Y2 receptor.

Detection of pro-opiomelanocortin mRNA in human and rat caudal medulla by RT-PCR.

Pro-opiomelanocortin (POMC) mRNA has been localized in the NTS of the rat, but not in the human or other species. Here, we report that RT-PCR amplification of human caudal medulla RNA generated a distinct band on agarose gels corresponding in size and sequence to the predicted 742-bp POMC PCR product. The 742-bp signal was undetectable following amplification of cortex, amygdala or caudate nucleus RNA. An homologous, 678-bp band was amplified from rat caudal medulla and, unexpectedly, from other brain regions. Competitive RT-PCR demonstrated that POMC cDNA from rat cortex, striatum and cerebellum was 17%, 22% and 45% of caudal medulla levels. These data indicate that the POMC gene is expressed in human caudal medulla and suggest that small amounts of POMC mRNA are present in regions other than the hypothalamus and NTS of rat brain.

Dynorphin A(1-8) in human placenta: amino acid sequence determined by tandem mass spectrometry.

Presence of the kappa receptor-preferring neuropeptide dynorphin A(1-8) in human placenta has been demonstrated by mass spectrometry to establish rigorously the appropriate molecular weight and amino acid sequence. Liquid secondary ionization mass spectrometry produced the protonated molecule ion, (M + H)+, at m/z 981 of the endogenous peptide, and tandem mass spectrometry collected the product ion spectrum that contained the appropriate amino acid sequence-determining fragment ions produced from the precursor ion (M + H)+. The amino acid sequence of the peptide is YGGFLRRI.

The CCK-A and CCK-B receptor antagonists, devazepide and L-365,260, enhance morphine antinociception only in non-acclimated rats exposed to a novel environment.

Devazepide, a potent CCK-A receptor antagonist, and L-365,260, a selective CCK-B receptor antagonist, have been introduced as pharmacologic tools for differentiating the physiologic roles of CCK-A and CCK-B receptor subtypes. In the present study, we tested the effects of devazepide and L-365,260, on morphine antinociception in rats using the thermal sensorimotor tail flick test. Both devazepide and L-365,260 significantly enhanced the antinociceptive action of morphine, but only in rats that had not been acclimated to the laboratory environment or habituated to investigator handling. When tested with fully acclimated animals, devazepide and L-365,260 had no effect whatsoever; they neither enhanced nor attenuated morphine-induced antinociception. These observations indicate that the effects of devazepide and L-365,260, CCK antagonists, on morphine antinociception appear to be dependent on the animal's response to a new environment or to the stress induced by an unaccustomed experimental paradigm.

Long-term haloperidol treatment elevates beta-endorphin levels in the intermediate pituitary but not in rat brain.

Long-term treatment of rats with haloperidol, a dopamine receptor antagonist, produced a dose-dependent increase in immunoreactive beta-endorphin (i beta-END) concentrations in the neurointermediate lobe of the pituitary (NIL). In contrast, chronic haloperidol treatment had no significant effect on i beta-END levels in the hypothalamus, the midbrain or in discrete, microdissected brain nuclei even when administered at a dose (5 mg/kg) ten-fold higher than that which elevated i beta-END levels in the NIL. Chronic treatment with bromocriptine, a dopamine receptor agonist, had the opposite effect on the NIL, lowering i beta-END levels to approximately one-third of control values, but it did not affect hypothalamic i beta-END concentrations. These data are consistent with prior evidence that the synthesis of beta-END by IL melanotrophs is reciprocally modulated by dopaminergic ligands. The results indicate, however, that beta-END-releasing neurons are not similarly regulated.