Gregory P. Mueller

The distribution of corticotropin releasing factor-like immunoreactive neurons in rat brain

Using the indirect immunofluorescent technique, corticotropin releasing factor (CRF)-like immunoreactive nerve fibers and cell bodies were observed to be widely distributed in rat brain. A detailed stereotaxic atlas of CRF-like immunoreactive neurons was prepared. Large numbers of CRF-containing perikarya were observed in the nucleus paraventricularis, with scattered cells in the following nuclei: accumbens, interstitialis stria terminalis, preopticus medialis, supraopticus, periventricularis hypothalami, amygdaloideus centralis, dorsomedialis, substantia grisea centralis, parabrachialis dorsalis and ventralis, tegmenti dorsalis lateralis, vestibularis medialis, tractus solitarius and reticularis lateralis. The most intense staining of CRF-containing fibers was observed in the external lamina of the median eminence. Moderate numbers of CRF-like fibers were observed in the following nuclei: lateralis and medialis septi, tractus diagonalis, interstitialis stria terminalis, preopticus medialis, supraopticus, periventricularis thalami and hypothalami, paraventricularis, anterior ventralis and medialis thalami, rhomboideus, amygdaloideus centralis, habenulae lateralis, dorsomedialis, ventromedialis, substantia grisea centralis, cuneiformis, parabrachialis dorsalis and ventralis, tegmenti dorsalis lateralis, cerebellum, vestibularis medialis, reticularis lateralis, substantia gelatinosa trigemini and lamina I and II of the dorsal horn of the spinal cord. The present findings suggest that a CRF-like peptide may be involved in a neurotransmitter or neuromodulator role, as well as a hypophysiotropic role.

Hypothalamic and Extrahypothalamic Distribution of CRF-Like Immunoreactive Neurons in the Rat Brain

Using an antiserum to synthetic ovine corticotropin-releasing factor (CRF), CRF-like immunoreactive neurons were observed in the central nervous system of normal and colchicine-treated rats. CRF immunoreactivity was observed in the hypothalamus, thalamus, amygdala, cerebral cortex, midbrain, pons medulla, cerebellum and spinal cord. The significance of the extrahypothalamic distribution of CRF-like immunoreactivity is unknown.

Distribution of the pro-opiomelanocortin derived peptides, adrenocorticotrope hormone, α-melanocyte-stimulating hormone and β-endorphin (ACTH, α-MSH, β-END) in the rat hypothalamus

Abstract Rat hypothalamic nuclei were removed and assayed for adrenocorticotropic hormone (ACTH), β-endorphin (β-END) and α-melanocyte-stimulating hormone (α-MSH) content by radioimmunoassay, from the same samples. We also performed immunostaining for these 3 pro-opiomelanocortin (POMC) derived peptides on paraffin embedded serial sections of the hypothalamus. Areas known to project to the external zone of the median eminence receive a dense POMC innervation while those projecting to the posterior pituitary are not innervated. In addition, hypothalamic areas previously suggested to project to medullary autonomic centers are densely innervated. This innervation pattern may provide the morphological basis for the involvement of POMC peptides in neuroendocrine and autonomic functions. The biochemical data raise the possibility that the POMC precursor is processed differently in various brain regions.

Corticotropin-releasing factor (CRF) produces analgesia in humans and rats

The analgesic activity of corticotropin releasing factor (CRF) was determined in a clinical model and in the rat hot plate test. Patients administered CRF reported significantly less postoperative pain than patients pretreated with placebo. In rats, injection of CRF resulted in a significant analgesia which was comparable in both intensity and duration to a 300 times greater molar dose of morphine. These findings suggest that endogenous CRF may play a physiologic role in modulating pain when released under conditions of stress.

Beta-endorphin immunoreactivity in rat plasma: Variations in response to different physical stimuli

Abstract The effects of two nociceptive testing procedures and several physical stressors on plasma levels of beta-endorphin-like immunoreactivity (s-END-LI) were compared in male rats. Tail flick analgesia testing did not significantly alter plasma s-END-LI, whereas hot plate testing increased circulating s-END-LI 2.8 times control values (P

Attenuated Pituitary β-Endorphin Release in Estrogen-Treated Rats

Abstract Using a newly developed radioimmunoassay for β-endorphin (β-END) the influence of gonadal steroids and sex difference on basal and stimulated β-END secretion was investigated. Administration of estradiol benzoate (EB), but not testosterone proprionate, tended to decrease resting levels of plasma β-END and significantly attenuated the stress-induced increase in circulating β-END in male rats. Consistent with these findings is the observation that female rats had somewhat lower nonstress and stress-elevated levels of plasma β-END as compared to males. Over 95% of the total pituitary content of β-END (12.7 ± 1.7 μg) was found to be present in the neurointermediate lobe (NIL) and treatment with EB for 5 days significantly decreased (by up to 56%) stores of β-END in the NIL yet had no effect on the small amount of β-END present in the pars distalis (PD). Since daily administration of EB produced dose-related increases in plasma levels of corticosterone and dexamethasone treatment completely prevented the stress-induced release of β-END, a possible mechanism by which treatment with EB results in decreased β-END secretion may be related to the ability of estrogen to increase circulating levels of adrenal glucocorticoids. Together, these findings suggest that adrenal glucocorticoids may be responsible for the diminished release of β-END observed here in female rats and those treated with EB as compared to normal male rats. In addition, the very high concentration of β-END in the NIL as compared to the PD provides indirect evidence for the pars intermedia being the primary source of circulating β-END in the rat.

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 animals response to a new environment or to the stress induced by an unaccustomed experimental paradigm.

Dexamethasone alters plasma levels of beta‐endorphin and postoperative pain

Secretion of pituitary immunoreactive β‐endorphin is hypothesized to modulate the perception of pain. The present study examined this question by evaluating the effects of intravenous placebo or dexamethasone (0.1, 0.32, or 1.0 mg) on suppression of immunoreactive β‐endorphin secretion and development of postoperative pain after the surgical removal of impacted third molars in 48 patients. Compared with placebo, all doses of dexamethasone suppressed the postoperative increase in circulating levels of immunoreactive β‐endorphin. Patients administered 0.1 mg dexamethasone reported greater levels of pain, compared with those given placebo, from 60 through 120 minutes after surgery. Postoperative pain for the 0.32 and 1.0 mg doses did not differ from that for the placebo group. The increased pain after suppression of β‐endorphin release by the low dose of dexamethasone suggests that pituitary secretion of immunoreactive β‐endorphin alleviates postoperative pain under these conditions.

Protein microarray platforms for clinical proteomics

Proteomics for clinical applications is presently in a state of transition. It has become clear that the classical approaches based on 2‐DE and/or MS need to be complemented by different kinds of technologies. The well‐known problems include sample complexity, sensitivity, quantitation, reproducibility, and analysis time. We suggest that the new technologies for clinical proteomics can be supported by antibody‐centric protein microarray platforms. These platforms presently include antibody microarrays and lysate, or reverse capture/reverse phase protein microarrays. Other forms of these arrays are in less mature developmental stages, including ORF and self assembling protein microarrays. Bioinformatic support for interpreting these arrays is becoming more available as the whole field of systems biology begins to mature. The present set of applications for these platforms is profoundly focused on certain common cancers, immunology, and cystic fibrosis. However, we predict that many more disease entities will become studied as knowledge of the power and availability of these platforms becomes more widely established. We anticipate that these platforms will eventually evolve to accommodate label‐free detection technologies, human genome‐scale numbers of analytes, and increases in analytic and bioinformatic speeds.

Clonidine releases immunoreactive β-endorphin from rat pars distalis

Clonidine (10(-6), 10(-7) M) evokes the release of beta endorphin-like immunoreactivity (beta-END-LI) from cell cultures of anterior (pars distalis) but not neurointermediate (pars nervosa plus pars intermedia) lobe of the rat pituitary. This drug-induced secretion is blocked by alpha-adrenergic (phenoxybenzamine, yohimbine; 10(-5) M) but not beta-adrenergic (propranolol, 10(-5) M) antagonism. Gel filtration (Sephadex G-50) reveals that beta-END-LI released from anterior lobe cells consists of 2 major forms of immunoreactivity which coelute with beta-lipotropin or beta-endorphin standards. Conversely, beta-END-LI released spontaneously from neurointermediate lobe cells almost entirely corresponds to beta-endorphin. The data show that alpha-adrenergic stimulation by clonidine releases beta-END-LI selectively from cells of anterior but not neurointermediate lobe in vitro and suggests that the clonidine-induced release of pituitary beta-END-LI we have observed in vivo occurs in part by direct action on the corticotrophs of the pars distalis.