Tonya C. Murphy

Cardiovascular regulatory actions of the hypocretins in brain

The hypocretins, also known as the orexins, are alternate translation products of a single gene. The recognition of their production in neurons of the rostral diencephalon, and their axonal localization in brain sites known to be important in the control of appetite, led to the demonstration of their orexogenic actions. However, these peptides are not as potent as other appetite stimulating neuropeptides and they have been localized in areas of brain more related to cardiovascular function. We verified the orexogenic actions of hypocretin-1 (Hcrt-1) and hypocretin-2 (Hcrt-2) in an ad libitum feeding model and identified the threshold dose to be 1 nmol when given into the lateral cerebroventricle (i.c. v.). Even at threshold doses for feeding, both Hcrt-1 and Hcrt-2 given i.c.v. into conscious, unrestrained rats stimulated significant elevations in mean arterial blood pressure, that appeared dose related. These elevations were relatively long lasting, mirroring the time course of a pressor dose of angiotensin II (0.1 nmol i.c.v.); however, the magnitude of blood pressure elevation to hypocretin did not equal that of A II. These data suggest an additional, non-appetitive action of the hypocretins and indicate that the peptide and receptor mapping studies may have predicted important roles for the peptides in the central nervous system control of cardiovascular function.

A novel action of the newly described prolactin-releasing peptides: cardiovascular regulation

The physiological relevance of the recently described prolactin-releasing peptides (PrRPs) has yet to be established. Here, we demonstrate the low potency of the PrRPs (minimum effective dose: 100 nM), compared to that observed for thyrotropin-releasing hormone (TRH, minimum effective dose: 1.0 nM), to stimulate prolactin (PRL) release from cultured pituitary cells harvested from lactating female rats. Anatomic studies question the role of these peptides in neuroendocrine control of lactotroph function. Instead, peptide and peptide receptor mapping studies suggest potential actions in hypothalamus and brainstem unrelated to the control of anterior pituitary hormone secretion. Intracerebroventricular (i.c.v. ) administration of both PrRP-20 and PrRP-31 (0.4 and 4.0 nmol) resulted in significantly increased mean arterial blood pressure in conscious, unrestrained rats [peak elevations vs. baseline: PrRP-20, 10% and 16%, low and high dose peptide; PrRP-31, 7% and 10%; compared to the response to 0.1 nmol angiotensin II (A II), 15-17%]. Similar doses of peptide did not significantly alter water drinking in response to overnight fluid deprivation, or thirst or salt appetite in response to an isotonic hypovolemic challenge. Thus, the effect on blood pressure appeared relatively specific. We suggest that these peptides, identified originally as ligands for a receptor found in abundance in pituitary gland, play a broader role in brain function and that the ability of them to stimulate PRL release may not represent their primary biologic function.

Central mechanisms for the hypertensive effects of preproadrenomedullin-derived peptides in conscious rats

Peptides derived from postranslational processing of preproadrenomedullin exert potent hypotensive effects in the periphery. One of those peptides, adrenomedullin (AM) also has been demonstrated to act centrally in conscious rats to inhibit water drinking and salt appetite and, in anesthetized rats, surprisingly to increase blood pressure. We examined the effects of AM and the other postranslational product, proadrenomedullin NH2-terminal 20 peptide (PAMP), on blood pressure in conscious rats. Both AM and PAMP elicited dose-related increases in mean arterial pressure after cerebroventricular administration. The hypertensive effects of both AM and PAMP and of ANG II were blocked by peripheral administration of phentolamine, indicating actions of the peptides in brain to stimulate sympathetic nervous system function. Blockade of central ANG II receptors with saralasin prevented the hypertensive effects of both ANG II and PAMP, suggesting recruitment of endogenous angiotensinergic systems by central PAMP. The structural homolog of AM, calcitonin gene-related peptide (CGRP), at similar doses did nto significantly affect blood pressure. Furthermore, the hypertensive effects of ANG II, AM, and PAMP were not abrogated by prior administration of the CGRP antagonist. We hypothesize that AM and PAMP exert cardioprotective effects in brain, which may counterbalance the volume-unloading actions of the peptides in the periphery.Peptides derived from postranslational processing of preproadrenomedullin exert potent hypotensive effects in the periphery. One of those peptides, adrenomedullin (AM) also has been demonstrated to act centrally in conscious rats to inhibit water drinking and salt appetite and, in anesthetized rats, surprisingly to increase blood pressure. We examined the effects of AM and the other postranslational product, proadrenomedullin NH2-terminal 20 peptide (PAMP), on blood pressure in conscious rats. Both AM and PAMP elicited dose-related increases in mean arterial pressure after cerebroventricular administration. The hypertensive effects of both AM and PAMP and of ANG II were blocked by peripheral administration of phentolamine, indicating actions of the peptides in brain to stimulate sympathetic nervous system function. Blockade of central ANG II receptors with saralasin prevented the hypertensive effects of both ANG II and PAMP, suggesting recruitment of endogenous angiotensinergic systems by central PAMP. The structural homolog of AM, calcitonin gene-related peptide (CGRP), at similar doses did not significantly affect blood pressure. Furthermore, the hypertensive effects of ANG II, AM, and PAMP were not abrogated by prior administration of the CGRP antagonist. We hypothesize that AM and PAMP exert cardioprotective effects in brain, which may counterbalance the volume-unloading actions of the peptides in the periphery.

Trans‐4‐hydroxy‐2‐hexenal is a neurotoxic product of docosahexaenoic (22:6; n‐3) acid oxidation

Lipid peroxidation of docosahexaenoic (22:6; n‐3) acid (DHA) is elevated in the CNS in patients with Alzheimer’s disease and in animal models of seizure and ethanol withdrawal. One product of DHA oxidation is trans‐4‐hydroxy‐2‐hexenal (HHE), a six carbon analog of the n‐6 fatty acid derived trans‐4‐hydroxy‐2‐nonenal (HNE). In this work, we studied the neurotoxic potential of HHE. HHE and HNE were toxic to primary cultures of cerebral cortical neurons with LD50’s of 23 and 18 μmol/L, respectively. Toxicity was prevented by the addition of thiol scavengers. HHE and HNE depleted neuronal GSH content identically with depletion observed with 10 μmol/L of either compound. Using an antibody raised against HHE–protein adducts, we show that HHE modified specific proteins of 75, 50, and 45 kDa in concentration‐ and time‐dependent manners. The time‐dependent formation of HHE differed from that of F4‐neuroprostanes following in vitro DHA oxidation likely as a result of the different oxidation pathways involved. Using purified mitochondrial aldehyde dehydrogenase ALDH5A, we found that HHE was oxidized 6.5‐fold less efficiently than HNE. Our data demonstrate that HHE and HNE have similarities but also differences in their neurotoxic mechanisms and metabolism.

Gender-biased activity of the novel prolactin releasing peptides: comparison with thyrotropin releasing hormone reveals only pharmacologic effects.

The prolactin- (PRL) releasing activities of the newly described PRL-releasing peptides (PrRPs) were compared to that of thyrotropin-releasing hormone (TRH) in dispersed, rat anterior pituitary cell cultures. A dose-related stimulation of PRL release by TRH was observed in cells harvested from both intact male and random cycle female pituitary donors. The minimum effective dose of TRH ranged from 1 to 10 nM. Neither PrRP-20 nor PrRP-31 significantly altered PRL secretion in cells from male donors even at doses as high as 1 µM. In cells harvested from females, only the highest doses of PrRP-20 and PrRP-31 tested (0.1 and 1.0 µM) significantly stimulated PRL secretion. The PRL-releasing action of TRH was observed already at 15 min of incubation, whereas those of PrRP-20 and PrRP-31 appeared only after 1 and 2 h of incubation, and the magnitude of PRL release in the presence of 1 µM PrRPs was significantly less than that of a similar dose of TRH. These data do not suggest a physiologically relevant role for the PrRPs in the neuroendocrine regulation of PRL secretion in intact male and nonlactating, random-cycle female rats.

Oxidation of 4‐hydroxy‐2‐nonenal by succinic semialdehyde dehydrogenase (ALDH5A)

Elevated levels of 4‐hydroxy‐trans‐2‐nonenal (HNE) are implicated in the pathogenesis of numerous neurodegenerative disorders. Although well‐characterized in the periphery, the mechanisms of detoxification of HNE in the CNS are unclear. HNE is oxidized to a non‐toxic metabolite in the rat cerebral cortex by mitochondrial aldehyde dehydrogenases (ALDHs). Two possible ALDH enzymes which might oxidize HNE in CNS mitochondria are ALDH2 and succinic semialdehyde dehydrogenase (SSADH/ALDH5A). It was previously established that hepatic ALDH2 can oxidize HNE. In this work, we tested the hypothesis that SSADH oxidizes HNE. SSADH is critical in the detoxification of the GABA metabolite, succinic semialdehyde (SSA). Recombinant rat SSADH oxidized HNE and other α,β‐unsaturated aldehydes. Inhibition and competition studies in rat brain mitochondria showed that SSADH was the predominant oxidizing enzyme for HNE but only contributed a portion of the total oxidizing activity in liver mitochondria. In vivo administration of diethyldithiocarbamate (DEDC) effectively inhibited (86%) ALDH2 activity but not HNE oxidation in liver mitochondria. The data suggest that a relationship between the detoxification of SSA and the neurotoxic aldehyde HNE exists in the CNS. Furthermore, these studies show that multiple hepatic aldehyde dehydrogenases are able to oxidize HNE.

Antisense oligonucleotide treatment reveals a physiologically relevant role for adrenomedullin gene products in sodium intake

Adrenomedullin (AM), a potent hypotensive peptide, is produced in numerous tissues including adrenal gland, kidney, brain and pituitary gland, where it acts to modify sodium homeostasis. Central AM administration dose-dependently inhibits sodium appetite. AM antisense oligonucleotide treatment significantly lowered peptide content in the hypothalamic paraventricular (PVN) nucleus and exaggerated the consumption of sodium. These results support a physiologic role for adrenomedullin gene products in the central regulation of sodium homeostasis.

Mitochondrial oxidation of 4‐hydroxy‐2‐nonenal in rat cerebral cortex

4‐Hydroxy‐trans‐2‐nonenal (HNE) is a neurotoxic product of lipid peroxidation whose levels are elevated in multiple neurodegenerative diseases and CNS trauma. The detoxification of HNE may take the route of glutathione conjugation to the C3 carbon and the oxidation or reduction of the C1 aldehyde. In this work, we examined whether the oxidation of HNE to its corresponding carboxylic acid, 4‐hydroxy‐trans‐2‐nonenoate (HNEAcid) was detoxifying event, if it occurred in rat cerebral cortex, and in which subcellular compartments. Our results show that HNEAcid did not form protein adducts and was non‐toxic to Neuro 2A cells. HNEAcid formation occurred in rat cerebral cortex slices following exposure to HNE in a time‐dependent and dose‐dependent fashion. Homogenate studies indicated that HNEAcid formation was NAD+ dependent. Subcellular fractionation demonstrated that mitochondria had the highest specific activity for HNEAcid formation with a KM of 21 µm HNE. These data indicate that oxidation of HNE to its corresponding acid is a major detoxification pathway of HNE in the CNS and that mitochondria play a role in this process.

Proadrenomedullin N-terminal 20 peptide inhibits adrenocorticotropin secretion from cultured pituitary cells, possibly via activation of a potassium channel

Preproadrenomedullin is processed into at least two biologically active peptides, adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP). Both peptides are hypotensive; however, they exert this action via differing mechanisms. In pituitary cells in culture, both basal and releasing factor-stimulated adrenocorticotropin (ACTH) secretion is inhibited by AM. Here we report that basal, but not stimulated, ACTH secretion from cultured rat pituitary cells is also inhibited by PAMP. The effect is dose-related, occurs in a physiologically relevant dose range that is similar to that of AM, and is blocked by the potassium channel blocker, glybenclamide. The failure of glybenclamide to inhibit AM’s effects on ACTH secretion indicates that in pituitary, as in other tissues, these two products of the same prohormone can exert similar biologic activity, although via differing mechanisms.

Nitrate-based vasodilators inhibit multiple vascular aldehyde dehydrogenases

Nitrate-based vasodilators (NBVs) are commonly used to treat multiple sequelae of atherosclerosis. A commonly used NBV, glyceryl trinitrate (GTN) is bioactivated by mitochondrial, class 2 aldehyde dehydrogenase (ALDH2). ALDH2 and other ALDHs are NAD(P)+-dependent enzymes critical to the detoxification of cytotoxic lipid-aldehydes elevated in atherosclerotic lesions, such as trans-4-hydroxy-2-nonenal (HNE). The GTN bioactivation step, however, inactivates ALDH2 and may alter the metabolism of these aldehydes. In this study, we tested the hypothesis that multiple ALDH enzymes are inhibited by different NBVs. ALDH2, ALDH3A, and ALDH5A were present in aorta with ALDH2 and ALDH3A localized to the smooth muscle layers. GTN (1 μM) inhibited ALDH2 activity (55±6% of control) and ablated ALDH3 activity. In contrast, isosorbide-2,5-dinitrate (ISDN, 1 μM) inhibited ALDH3 activity (1.1±0.4% of control) but did not inhibit ALDH2 activity even up to 50 μM ISDN. In homogenates of rat aorta, GTN (1 μM) inhibited the NAD+-dependent (41±5% of control) and NADP+-dependent (25±6% of control) detoxification of HNE. The inhibition of ALDH3A, but not ALDH2, could be prevented by the addition of dithiothreitol. These studies demonstrate that GTN and ISDN possess selectivity for ALDH inactivation with different mechanisms of inactivation.