Pituitary adenylate cyclase activating polypeptide concentrations in the sheep mammary gland, milk, and in the lamb blood plasma after suckling.

Pituitary adenylate cyclase activating polypeptide (PACAP) is involved in development and reproduction. We previously described elevated PACAP levels in the milk compared to the plasma, and the presence of its specific PAC1 receptor in the mammary gland. This study aimed to determine PACAP and vasoactive intestinal peptide (VIP) levels in female suckling lambs compared to ewe plasma and mammary gland, as well as their age-dependent alterations. mRNA expressions of PACAP, VIP, PAC1 receptor and brain-derived neurotrophic factor (BDNF) were quantified in the milk whey and mammary gland. PACAP38-like immunoreactivity (PACAP38-LI) was measured in plasma, milk whey and mammary gland by radioimmunoassay, VIP-LI by enzyme-linked immunoassay. PACAP38-LI was 5, 6 times higher in the milk compared to the plasma of lactating sheep. It significantly increased in the lamb plasma 1 h, but returned to basal level 2 h after suckling. However, VIP mRNA was not present in the mammary gland, we detected the VIP protein in the milk whey. BDNF mRNA significantly decreased with age to approximately 60% and 25% in the 3- and 10-year-old sheep respectively, compared to the 3-month-old lambs. No differences were found between mammary and jugular vein plasma PACAP and VIP concentrations, or during the daily cycle. We propose a rapid absorption of PACAP38 from the milk and/or its release in suckling lambs. PACAP accumulated in the milk might be synthesized in the mammary gland or secreted from the plasma of the mothers. PACAP is suggested to have differentiation/proliferation promoting and immunomodulatory effects in the newborns and/or a local function in the mammary gland.

Prenatal cigarette smoke exposure slightly alters neurobehavioral development in neonatal rats: Implications for developmental origins of health and disease (DoHAD).

Numerous studies indicate that smoking during pregnancy exerts harmful effects on fetal brain development. The aim of this study was to determine the influence of maternal smoking during pregnancy on the early physical and neurobehavioral development of newborn rats. Wistar rats were subjected to whole-body smoke exposure for 2 × 40 min daily from the day of mating until day of delivery. For this treatment, a manual closed-chamber smoking system and 4 research cigarettes per occasion were used. After delivery the offspring were tested daily for somatic growth, maturation of facial characteristics and neurobehavioral development until three weeks of age. Motor coordination tests were performed at 3 and 4 weeks of age. We found that prenatal cigarette smoke exposure did not alter weight gain or motor coordination. Critical physical reflexes indicative of neurobehavioral development (eyelid reflex, ear unfolding) appeared significantly later in pups prenatally exposed to smoke as compared to the control group. Prenatal smoke exposure also resulted in a delayed appearance of reflexes indicating neural maturity, including hind limb grasping and forelimb placing reflexes. In conclusion, clinically relevant prenatal exposure to cigarette smoke results in slightly altered neurobehavioral development in rat pups. These findings suggest that chronic exposure of pregnant mothers to cigarette smoke (including passive smoking) results in persisting alterations in the developing brain, which may have long-lasting consequences supporting the concept of developmental origins of health and disease (DoHAD).

Chronic stress-induced mechanical hyperalgesia is controlled by capsaicin-sensitive neurones in the mouse.

BACKGROUND: Clinical studies demonstrated peripheral nociceptor deficit in stress-related chronic pain states, such as fibromyalgia. The interactions of stress and nociceptive systems have special relevance in chronic pain, but the underlying mechanisms including the role of specific nociceptor populations remain unknown. We investigated the role of capsaicin-sensitive neurones in chronic stress-related nociceptive changes. METHOD: Capsaicin-sensitive neurones were desensitized by the capsaicin analogue resiniferatoxin (RTX) in CD1 mice. The effects of desensitization on chronic restraint stress (CRS)-induced responses were analysed using behavioural tests, chronic neuronal activity assessment in the central nervous system with FosB immunohistochemistry and peripheral cytokine concentration measurements. RESULTS: Chronic restraint stress induced mechanical and cold hypersensitivity and increased light preference in the light-dark box test. Open-field and tail suspension test activities were not altered. Adrenal weight increased, whereas thymus and body weights decreased in response to CRS. FosB immunopositivity increased in the insular cortex, dorsomedial hypothalamic and dorsal raphe nuclei, but not in the spinal cord dorsal horn after the CRS. CRS did not affect the cytokine concentrations of hindpaw tissues. Surprisingly, RTX pretreatment augmented stress-induced mechanical hyperalgesia, abolished light preference and selectively decreased the CRS-induced neuronal activation in the insular cortex. RTX pretreatment alone increased the basal noxious heat threshold without influencing the CRS-evoked cold hyperalgesia and augmented neuronal activation in the somatosensory cortex and interleukin-1α and RANTES production. CONCLUSIONS: Chronic restraint stress induces hyperalgesia without major anxiety, depression-like behaviour or peripheral inflammatory changes. Increased stress-induced mechanical hypersensitivity in RTX-pretreated mice is presumably mediated by central mechanisms including cortical plastic changes. SIGNIFICANCE: These are the first data demonstrating the complex interactions between capsaicin-sensitive neurones and chronic stress and their impact on nociception. Capsaicin-sensitive neurones are protective against stress-induced mechanical hyperalgesia by influencing neuronal plasticity in the brain.

A novel 3-(4,5-diphenyl-1,3-oxazol-2-yl)propanal oxime compound is a potent Transient Receptor Potential Ankyrin 1 and Vanilloid 1 (TRPA1 and V1) receptor antagonist.

Transient Receptor Potential Ankyrin 1 and Vanilloid 1 (TRPA1, TRPV1) ion channels expressed on nociceptive primary sensory neurons are important regulators of pain and inflammation. TRPA1 is activated by several inflammatory mediators including formaldehyde and methylglyoxal that are products of the semicarbazide-sensitive amine-oxidase enzyme (SSAO). SZV-1287 is a new 3-(4,5-diphenyl-1,3-oxazol-2-yl)propanal oxime SSAO inhibitor, its chemical structure is similar to other oxime derivatives described as TRPA1 antagonists. Therefore, we investigated its effects on TRPA1 and TRPV1 receptor activation on the cell bodies and peripheral terminals of primary sensory neurons and TRPA1 or TRPV1 receptor-expressing cell lines. Calcium influx in response to the TRPA1 agonist allyl-isothiocyanate (AITC) (200 µM) and the TRPV1 stimulator capsaicin (330 nM) in rat trigeminal neurons or TRPA1 and TRPV1 receptor-expressing cell lines was measured by microfluorimetry or radioactive (45)Ca(2+) uptake experiments. Calcitonin gene-related peptide (CGRP) release as the indicator of 100 µM AITC - or 100 nM capsaicin-induced peripheral sensory nerve terminal activation was measured by radioimmunoassay. SZV-1287 (100, 500 and 1000 nM) exerted a concentration-dependent significant inhibition on both AITC- and capsaicin-evoked calcium influx in trigeminal neurons and TRPA1 or TRPV1 receptor-expressing cell lines. It also significantly inhibited the TRPA1, but not the TRPV1 activation-induced CGRP release from the peripheral sensory nerve endings in a concentration-dependent manner. In contrast, the reference SSAO inhibitor LJP 1207 with a different structure had no effect on TRPA1 or TRPV1 activation in either model system. This is the first evidence that our novel oxime compound SZV-1287 originally developed as a SSAO inhibitor has a potent dual antagonistic action on TRPA1 and TRPV1 ion channels on primary sensory neurons.

Stimulatory effect of pituitary adenylate cyclase-activating polypeptide 6-38, M65 and vasoactive intestinal polypeptide 6-28 on trigeminal sensory neurons.

Pituitary adenylate cyclase-activating polypeptide (PACAP) acts on G protein-coupled receptors: the specific PAC1 and VPAC1/VPAC2 receptors. PACAP6-38 was described as a potent PAC1/VPAC2 antagonist in several models, but recent studies reported its agonistic behaviors proposing novel receptorial mechanisms. Since PACAP in migraine is an important research tool, we investigated the effect of PACAP and its peptide fragments on trigeminal primary sensory neurons. Effect of the peptides was studied with ratiometric Ca-imaging technique using the fluorescent indicator fura-2 AM on primary cultures of rat and mouse trigeminal ganglia (TRGs) neurons. Specificity testing was performed on PAC1, VPAC1 and VPAC2 receptor-expressing cell lines with both fluorescent and radioactive Ca-uptake methods. Slowly increasing intracellular free calcium concentration [Ca(2+)]i was detected after PACAP1-38, PACAP1-27, vasoactive intestinal polypeptide (VIP) and the selective PAC1 receptor agonist maxadilan administration on TRG neurons, but interestingly, PACAP6-38, VIP6-28 and the PAC1 receptor antagonist M65 also caused similar activation. The VPAC2 receptor agonist BAY 55-9837 induced similar activation, while the VPAC1 receptor agonist Ala(11,22,28)VIP had no significant effect on [Ca(2+)]i. It was proven that the Ca(2+)-influx originated from intracellular stores using radioactive calcium-45 uptake experiment and Ca-free solution. On the specific receptor-expressing cell lines the antagonists inhibited the stimulating actions of the respective agonists, but had no effects by themselves. PACAP6-38, M65 and VIP6-28, which were described as antagonists in numerous studies in several model systems, act as agonists on TRG primary sensory neurons. Currently unknown receptors or splice variants linked to distinct signal transduction pathways might explain these differences.

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.

Examination of PACAP38-like immunoreactivity in different milk and infant formula samples.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with special importance in reproductive and developmental processes. PACAP is found in two bioactive forms: PACAP27 and PACAP38. Recently, we have described that PACAP38 is present in high levels in the milk of human and ruminant animals. Breastfeeding is of utmost importance in proper nutrition of the newborn, but artificial nursing with infant formulas is necessary when breastfeeding is not available. Composition of the breast milk varies during the whole period of nursing and it shows differences at the beginning (foremilk) and the end of an actual suckling (hindmilk). The aim of this study was to investigate PACAP38-like immunoreactivity (PACAP38-LI) in different milk and infant formula samples by radioimmunoassay and to prove the presence of PACAP38 in the infant formula by mass spectrometry. We found similar PACAP38-LI in human mature foremilk and hindmilk samples, in the fresh and pasteurized cow milk and also in formulas. However, we found significantly higher PACAP38-LI in the hypoantigenic formula undergoing extensive hydrolysis compared to the non-hypoantigenic ones. Our results suggest that PACAP38 is relatively stable in the milk and it can withstand the manufacturing processes.

Effects of pituitary adenylate cyclase activating polypeptide on human sperm motility.

Pituitary adenylate cyclase activating polypeptide (PACAP), a neuropeptide with diverse effects, was originally isolated as a hypothalamo-hypophyseal peptide. Subsequent studies showed highest levels of PACAP in the testis after the brain, suggesting that it influences the development and functioning of spermatozoa. Indeed, it has been proven that PACAP has an effect on spermatogenesis, both locally and via influencing the hypothalamo-hypophyseal-gonadal axis. The aim of the present study was to determine whether PACAP has an effect on human sperm motility and whether it is present in the human seminal fluid. Furthermore, the sperm head morphology was studied in mice lacking endogenous PACAP. Human samples were obtained from healthy adult volunteers and andrological patients. The effects of PACAP on the motility of human sperm cells were investigated using a computer aided sperm analysis system. In cases where the motility was lower, addition of PACAP to the samples increased the motility and the ratio of rapid progressive and medium progressive sperm motility groups. The presence of PACAP could not be detected in human seminal fluid samples by means of mass spectrometry. Investigating sperm head morphology with routine histology in PACAP deficient mice revealed that both the longitudinal and transverse diameters were significantly lower in PACAP deficient mice, without marked difference in the shape, as revealed by scanning electron microscopy.

Comparative examination of inner ear in wild type and pituitary adenylate cyclase activating polypeptide (PACAP)-deficient mice.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a multifunctional neuropeptide with well-known neuroprotective and neurotrophic effects. The involvement of PACAP in sensory processing has also been documented, but little is known about its effects in the auditory system. PACAP and its specific receptor (PAC1) are present in the cochlea and in brain structures involved in auditory pathways. Recently, we have shown that PACAP protects cochlear cells against oxidative stress-induced apoptosis. The endolymphatic Ca(2+) concentration controlled by Ca(2+) buffers of the hair cells is essential for the normal hearing processes. In this study we examined the localization of PAC1 receptor and Ca(2+) buffering proteins (parvalbumin, calretinin, calbindin) in the inner ear of 5-day-old PACAP-deficient mice compared with wild-type mice in order to get a closer insight into the effect of endogenous PACAP in the cochlear function. We did not find differences in the distribution pattern of PAC1 receptors between the two groups, but wild-type animals showed significantly higher PAC1 receptor expression. In contrast, inner and outer hair cells of PACAP-deficient mice showed more pronounced parvalbumin, calbindin, and calretinin immunopositivity compared with wild-type mice. Elevated endolymphatic Ca(2+) is deleterious for cochlear function, while the high concentration of Ca(2+) buffers in hair cells may offer protection. The increased immunoreactivity of Ca(2+) binding proteins in the absence of PACAP provide further evidence the important role of PACAP in the hearing processes.

Mice deficient in pituitary adenylate cyclase activating polypeptide (PACAP) are more susceptible to retinal ischemic injury in vivo.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuroprotective peptide exerting protective effects in neuronal injuries. We have provided evidence that PACAP is neuroprotective in several models of retinal degeneration in vivo. Our previous studies showed that PACAP treatment ameliorated the damaging effects of chronic hypoperfusion modeled by permanent bilateral carotid artery occlusion. We have also demonstrated in earlier studies that treatment with PACAP antagonists further aggravates retinal lesions. It has been shown that PACAP deficient mice have larger infarct size in cerebral ischemia. The aim of this study was to compare the degree of retinal damage in wild type and PACAP deficient mice in ischemic retinal insult. Mice underwent 10 min of bilateral carotid artery occlusion followed by 2-week reperfusion period. Retinas were then processed for histological analysis. It was found that PACAP deficient mice had significantly greater retinal damage, as shown by the thickness of the whole retina, the morphometric analysis of the individual retinal layers, and the cell numbers in the inner nuclear and ganglion cell layers. Exogenous PACAP administration could partially protect against retinal degeneration in PACAP deficient mice. These results clearly show that endogenous PACAP reacts as a stress-response peptide that is necessary for endogenous protection against different retinal insults.

Involvement of transient receptor potential vanilloid 1 receptors in protease-activated receptor-2-induced joint inflammation and nociception.

Protease-activated receptor-2 (PAR-2) is a G-protein-coupled receptor activated through proteolytic cleavage. It is localized on epithelial, endothelial and inflammatory cells, as well as on transient receptor potential vanilloid 1 (TRPV1) receptor-expressing neurones. It plays an important role in inflammatory/nociceptive processes. Since there are few reports concerning PAR-2 function in joints, the effects of intraarticular PAR-2 activation on joint pain and inflammation were studied. Secondary hyperalgesia/allodynia, spontaneous weight distribution, swelling and inflammatory cytokine production were measured and the involvement of TRPV1 ion channels was investigated in rats and mice. Injection of the PAR-2 receptor agonist SLIGRL-NH(2) into the knee decreased touch sensitivity and weight bearing of the ipsilateral hindlimb in both species. Secondary mechanical allodynia/hyperalgesia and impaired weight distribution were significantly reduced by the TRPV1 antagonist SB366791 in rats and by the genetic deletion of this receptor in mice. PAR-2 activation did not cause significant joint swelling, but increased IL-1beta concentration which was not influenced by the lack of the TRPV1 channel. For comparison, intraplantar SLIGRL-NH(2) evoked similar primary mechanical hyperalgesia and impaired weight distribution in both WT and TRPV1 deficient mice, but oedema was smaller in the knockouts. The inactive peptide, LRGILS-NH(2), injected into either site did not induce any inflammatory or nociceptive changes. These data provide evidence for a significant role of TRPV1 receptors in secondary mechanical hyperalgesia/allodynia and spontaneous pain induced by PAR-2 receptor activation in the knee joint. Although intraplantar PAR-2 activation-induced oedema is also TRPV1 receptor-mediated, primary mechanical hyperalgesia, impaired weight distribution and IL-1beta production are independent of this channel.