Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Regulates the Hypothalamo-Pituitary-Thyroid (HPT) Axis via Type 2 Deiodinase in Male Mice.

The hypothalamic activation of thyroid hormones by type 2 deiodinase (D2), catalyzing the conversion of thyroxine to T3, is critical for the proper function of the hypothalamo-pituitary-thyroid (HPT) axis. Regulation of D2 expression in tanycytes alters the activity of the HPT axis. However, signals that regulate D2 expression in tanycytes are poorly understood. The pituitary adenylate cyclase-activating polypeptide (PACAP) increases intracellular cAMP level, a second messenger known to stimulate the DIO2 gene; however, its importance in tanycytes is not completely characterized. Therefore, we tested whether this ubiquitously expressed neuropeptide regulates the HPT axis through stimulation of D2 in tanycytes. PACAP increased the activity of human DIO2 promoter in luciferase reporter assay that was abolished by mutation of cAMP-response element. Furthermore, PAC1R receptor immunoreactivity was identified in hypothalamic tanycytes, suggesting that these D2-expressing cells could be regulated by PACAP. Intracerebroventricular PACAP administration resulted in increased D2 activity in the mediobasal hypothalamus, suppressed Trh expression in the hypothalamic paraventricular nucleus, and decreased Tshb expression in the pituitary demonstrating that PACAP affects the D2-mediated control of the HPT axis. To understand the role of endogenous PACAP in the regulation of HPT axis, the effect of decreased PACAP expression was studied in heterozygous Adcyap1 (PACAP) knockout mice. These animals were hypothyroid that may be the consequence of altered hypothalamic T3 degradation during set-point formation of the HPT axis. In conclusion, PACAP is an endogenous regulator of the HPT axis by affecting T3-mediated negative feedback via cAMP-induced D2 expression of tanycytes.

The selective PAC1 receptor agonist maxadilan inhibits neurogenic vasodilation and edema formation in the mouse skin.

We have earlier shown that PACAP-38 decreases neurogenic inflammation. However, there were no data on its receptorial mechanism and the involvement of its PAC1 and VPAC1/2 receptors (PAC1R, VPAC1/2R) in this inhibitory effect. Neurogenic inflammation in the mouse ear was induced by topical application of the Transient Receptor Potential Ankyrin 1 (TRPA1) receptor activator mustard oil (MO). Consequent neurogenic edema, vasodilation and plasma leakage were assessed by measuring ear thickness with engineer's micrometer, detecting tissue perfusion by laser Doppler scanning and Evans blue or indocyanine green extravasation by intravital videomicroscopy or fluorescence imaging, respectively. Myeloperoxidase activity, an indicator of neutrophil infiltration, was measured from the ear homogenates with spectrophotometry. The selective PAC1R agonist maxadilan, the VPAC1/2R agonist vasoactive intestinal polypeptide (VIP) or the vehicle were administered i.p. 15 min before MO. Substance P (SP) concentration of the ear was assessed by radioimmunoassay. Maxadilan significantly diminished MO-induced neurogenic edema, increase of vascular permeability and vasodilation. These inhibitory effects of maxadilan may be partially due to the decreased substance P (SP) levels. In contrast, inhibitory effect of VIP on ear swelling was moderate, without any effect on MO-induced plasma leakage or SP release, however, activation of VPAC1/2R inhibited the increased microcirculation caused by the early arteriolar vasodilation. Neither the PAC1R, nor the VPAC1/2R agonist influenced the MO-evoked increase in tissue myeloperoxidase activity. These results clearly show that PAC1R activation inhibits acute neurogenic arterial vasodilation and plasma protein leakage from the venules, while VPAC1/2R stimulation is only involved in the attenuation of vasodilation.

Protective effects of vasoactive intestinal peptide (VIP) in ischemic retinal degeneration.

Vasoactive intestinal peptide (VIP) is a pleiotropic neuropeptide, acting as a neuromodulator and neuroprotective peptide in the CNS after injuries. We have previously described that pituitary adenylate cyclase-activating polypeptide (PACAP), another member of the same peptide family, is retinoprotective in ischemic lesions. The aim of this study was to investigate the protective potential of VIP in bilateral common carotid artery occlusion (BCCAO)-induced ischemic retinal lesion. Two-month-old rats were subjected to BCCAO and treated with intravitreal VIP injection. Their retinas were processed for histology after 2 weeks of survival. We measured the number of the cells/100 µm of the ganglion cell layer and the thickness of each layer such as the outer nuclear, outer plexiform, inner nuclear, and inner plexiform layers as well as that of the whole retina. We found that treatment with 1,000 pmol VIP, but not 100 pmol VIP, had significant protective effects in BCCAO-injured retina, as shown by the morphometric analysis. Comparing the neuroprotective effects of VIP and PACAP in BCCAO-operated retinas, PACAP was more effective, already protective at 100-pmol doses. Similar to other studies, we found that VIP must be given at least in 10 times more concentration than PACAP to achieve a similar degree of neuroprotection in the retina.

Changes of PACAP levels in the brain show gender differences following short-term water and food deprivation.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a pleiotropic neuropeptide exerting diverse actions in the central and peripheral nervous systems. A few studies indicate that PACAP is involved in the regulation of feeding and water homeostasis. The aim of the present study was to investigate changes in PACAP38 concentrations in different brain areas following food or water deprivation in male and female rats. Rats were sacrificed 12, 36 and 84h after water or food removal. PACAP levels were determined by radioimmunoassay. Our results show that levels of PACAP decreased in the hypothalamus in both sexes after water deprivation, with a more marked, significant decrease in females at 12h. A decrease was observed also in the telencephalon, with a similar pattern in both genders: levels were lowest after 12h, and showed a gradual increase at the other two time-points. PACAP levels increased in the brainstem of male rats, while females had a decrease 12h after water deprivation. The pattern of changes in PACAP levels was very different after food deprivation. In male rats, PACAP levels showed a significant increase in the hypothalamus, telencephalon and brainstem 12h after the beginning of starvation. In females, a less marked increase was observed only in the hypothalamus while no changes were found in the other brain areas. Our results show a sensitive reaction in changes of endogenous PACAP levels to water and food deprivation in most brain areas, but they are differentially regulated in male and female rats.

Effect of pituitary adenylate cyclase activating polypeptide-38 on sensory neuropeptide release and neurogenic inflammation in rats and mice.

Substance P (SP) and calcitonin gene-related peptide (CGRP), released from capsaicin-sensitive sensory nerves induce local neurogenic inflammation, while somatostatin exerts systemic anti-inflammatory actions. The aim of the present study was to investigate the release of pituitary adenylate cyclase activating polypeptide-38 (PACAP-38) and its effects on sensory neuropeptide release in vitro and acute neurogenic ear swelling in vivo. Capsaicin (10(-6) M) or electrical field stimulation (EFS; 40 V, 0.1 ms, 10 Hz, 120 s; 1200 impulses)-induced release of PACAP-38, SP, CGRP and somatostatin from isolated rat tracheae was measured with radioimmunoassay. Mustard oil-induced neurogenic inflammation in the mouse ear was determined with a micrometer and in the rat hind paw skin by the Evans Blue leakage technique. Capsaicin and EFS evoked 27% and more than twofold elevation of PACAP-38 release respectively, compared with the prestimulated basal values from isolated trachea preparation. Exogenously administered PACAP-38 (20-2000 nM) diminished both capsaicin- and EFS-evoked sensory neuropeptide release in a concentration-dependent manner. The maximal inhibitory effects of PACAP on capsaicin-induced substance P, CGRP and somatostatin release amounted to 75.4%, 73.3% and 90.0%, while EFS-evoked release of these peptides was 80.03%, 87.7% and 67.7%. In case of capsaicin stimulation the EC50 values for substance P, CGRP and somatostatin were 82.9 nM, 60.1 nM and 66.9 nM, respectively. When EFS was performed, these corresponding EC50 data were 92.1 nM, 67.8 nM and 20.9 nM. PACAP-38 (10, 100 and 1000 microg/kg i.p. in 200 microl volume) inhibited neurogenic ear swelling in the mouse. Furthermore, 100 microg/kg i.p. PACAP also significantly diminished mustard oil-evoked plasma protein extravasation in the rat skin. These results suggest that PACAP-38 is released from the stimulated peripheral terminals of capsaicin-sensitive afferents and it is able to inhibit the outflow of sensory neuropeptides. Based on this mechanism of action PACAP is also able to effectively diminish/abolish neurogenic inflammatory response in vivo after systemic administration.

Delayed systemic administration of PACAP38 is neuroprotective in transient middle cerebral artery occlusion in the rat.

BACKGROUND AND PURPOSE: Many substances have been shown to reduce brain damage in models of stroke, but mainly when given either before or shortly after the onset of ischemia. Delayed systemic administration of pituitary adenylate cyclase-activating polypeptide (PACAP) has been shown to attenuate the neuronal damage in the hippocampus in a model of global ischemia in rats. The present study examined the neuroprotective action of delayed systemic administration of PACAP38 in a model of transient focal ischemia produced by middle cerebral artery occlusion (MCAO) in rats. METHODS: We administered PACAP38 as an intravenous bolus (20 nmol/kg body wt) followed by an intravenous infusion for 48 hours using a micro-osmotic pump at a rate of 160 pmol/microL per hour, beginning 4, 8, or 12 hours after a 2-hour transient MCAO using a filament model. The size of the infarct was determined by examining 2-mm-thick brain sections stained with triphenyltetrazolium chloride, followed by image analysis. Control animals received intravenously 0.1% bovine serum albumin in 0.9% saline as a bolus and infusion at the same time intervals. RESULTS: The administration of PACAP38 beginning 4 hours after MCAO significantly reduced the infarct size by 50.88%. Treatment with PACAP38 starting 8 or 12 hours after the onset of ischemia did not result in a significant reduction of the infarct size, although infarct volumes tended to be smaller than in the control groups. CONCLUSIONS: Systemic administration of PACAP38 should be clinically useful for reducing brain damage resulting from stroke even when administration is delayed for several hours.