Tissue distribution of PACAP27 and -38 in oligochaeta: PACAP27 is the predominant form in the nervous system of Lumbricus polyphemus.

The levels of pituitary adenylate cyclase-activating polypeptide (PACAP)27 and -38 were measured in the nervous, intestinal, excretory, and reproductive systems of Lumbricus polyphemus by radioimmunoassay. Although both PACAP27 and -38 were significantly detectable in all of the examined tissues, the distribution of the peptides was very heterogeneous. Their highest concentrations were found in the cerebral ganglia and the ventral cord, followed by the alimentary tract and the nephridial system, respectively. Moreover, the reproductive system also contained a substantial amount of PACAP. The dominant form of the peptide discovered in the majority of tissues was PACAP27. Interestingly, about 10 times more PACAP27 than PACAP38 was found, with the latter representing only a fraction of PACAP-like immunoreactivity in the tissues of Lumbricus polyphemus.

Postischemic spontaneous hyperthermia and its effects in middle cerebral artery occlusion in the rat.

This study examined the time course and effects of postischemic spontaneous hyperthermia after transient and permanent focal ischemia. Rats underwent a 90-min, 120-min, or permanent middle cerebral artery occlusion (MCAO). Body temperatures started rising 15-20 min after MCAO and reached 39-40.5 degrees C during the first hour. Sustained hyperthermia was observed during the rest of the first 24 h. In another experiment, rats were subjected to the same interventions, but a normothermic body temperature was maintained. Spontaneous hyperthermia significantly increased the infarct volumes measured 48 h after MCAO in all groups. Reperfusion 2 h after the onset of ischemia was not beneficial in the hyperthermic animals in contrast to the normothermic group. We also examined the effect of spontaneous hyperthermia on the temporal progression of infarcted and penumbral areas 4, 12, or 48 h after MCAO. During spontaneous hyperthermia, penumbral areas became infarcted areas more rapidly, which was most expressed at 4 h. These findings demonstrate that severe spontaneous hyperthermia can occur in rats after MCAO and that it not only increases the infarct volumes in both transient and permanent ischemia, but also accelerates the incorporation of penumbral areas into necrotic areas, which significantly decreases the window of opportunity for therapeutic interventions.

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.

Distribution of PACAP-like immunoreactivity in the nervous system of oligochaeta.

The marked similarity between the primary structures of human, other vertebrate, and the invertebrate tunicate PACAP suggests that PACAP is one of the most highly conserved peptides during the phylogeny of the metazoans. We investigated the distribution of PACAP-like immunoreactivity in the nervous system of three oligochaete (Annelida) worms with immunocytochemistry. The distribution pattern of immunoreactivity was similar in all three species (Lumbricus terrestris, Eisenia fetida, and Lumbricus polyphemus). The cerebral ganglion contains numerous immunoreactive cells and fibers. A few cells and fibers were found in the medial and lateral parts of the subesophageal and ventral cord ganglia. In the peripheral nervous system, immunoreactivity was found in the enteric nervous system, in epidermal sensory cells, and in the clitellum.

Neuroprotective effects of PACAP38 in a rat model of transient focal ischemia under various experimental conditions.

In the study reported here, we investigated the neuroprotective effect of PACAP38 in a rat model of middle cerebral artery occlusion (MCAO) under various experimental conditions. Stroke patients often develop mild hyperthermia that increases the infarct size and worsens the outcome. We compared the neuroprotective effect of PACAP38 in normothermic and moderately hyperthermic animals. Brain damage was more extensive in the hyperthermic animals, but PACAP38 significantly reduced the infarct size, measured 48 h after MCAO, in both the normothermic and the moderately hyperthermic animals, by more than 50%. However, a significant neuroprotection with PACAP38 could only be demonstrated when animals with no definite circling behavior and/or convulsions were excluded from the evaluations. In conclusion, moderate hyperthermia did not influence the effect of PACAP38, but animals with questionable brain damage or convulsions must be excluded from the sample in order to demonstrate the neuroprotection with PACAP38.

The source of origin of PACAP- and VIP-immunoreactive fibers in the laterodorsal division of the bed nucleus of the stria terminalis in the rat.

The bed nucleus of the stria terminalis (BSTL), which is known to be involved in the modulation of stress responses, exhibits a dense network of pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) immunoreactive (ir) fibers. The origin of the PACAP-ir fibers is unknown, and the origin of the VIP-ir fibers remains uncertain. The most important brain regions connected to the BSTL are the amygdaloid nuclei, the paraventricular and ventromedial hypothalamic nuclei, mesencephalic periaqueductal grey, the dorsal and linear raphe nuclei, the parabrachial nucleus, and the dorsal vagal complex. After microinjecting cholera toxin B subunit (CTB) in the BSTL as a retrograde tracer, neurons were double labeled for CTB and PACAP or VIP immunohistochemistry and the cells from which the PACAP- and VIP-ir fiber networks in the BSTL originated were identified. Cholera toxin B subunit labeled and VIP-ir cells were found in the mesencephalic periaqueductal grey and the dorsal and linear raphe nuclei, but no double labeled cells were seen in the amygdaloid nuclei or the hypothalamic region. CTB- and PACAP-ir neurons were observed in the paraventricular nucleus and the dorsal vagal complex. No double labeled perikarya were seen in the parabrachial nucleus or in the amygdaloid nuclei.

Immunohistochemical demonstration of the intracellular localization of pituitary adenylate cyclase activating polypeptide-like immunoreactivity in the rat testis using the stamp preparation.

Radioimmunoassay has revealed an abundance of PACAP in the rat testis. In the present study, a novel stamp preparation together with light microscopic immunohistochemistry were used to investigate in detail the intracellular localization of PACAP-like immunoreactivity (PACAP-LI) in rat germ cells. Samples were obtained by pressing the freshly cut surfaces of the testes against glass slides. PACAP-LI was clearly identified in developing acrosomes using five antisera which recognize PACAP 38, or both PACAP 27 and PACAP 38. Immunoreactivity with the antisera specific to PACAP38 was strictly localized in the developing acrosomes of the spermatids but vanished in the mature spermatids. Using an antiserum which detects PACAP 27 specifically, little staining was observed. Based on the specificities of antisera used, it was suggested that the PACAP-LI in the acrosomes represents mainly PACAP 38-LI. In addition, the present results supported the usefulness of the stamp preparation for immunohistochemical study of testicular tissues.

Axon terminals containing PACAP- and VIP-immunoreactivity form synapses with CRF-immunoreactive neurons in the dorsolateral division of the bed nucleus of the stria terminalis in the rat.

The bed nucleus of the stria terminalis (BST) is a highly heterogeneous forebrain structure, within which the median and lateral BST play distinct functional roles. The medial BST (BSTM) is thought to be related to sexual behavior, while the lateral BST (BSTL) may have a stress-related function. In the human brain, the BST shows marked sexual dimorphism in the distribution of vasoactive intestinal polypeptide (VIP) immunoreactive fibers and also contains a very high concentration of pituitary adenylate cyclase activating polypeptide (PACAP) immunoreactivity (ir). Using immunohistochemistry (IHC) to examine the rat brain, the present study found that both VIP and PACAP containing afferent fibers are abundant in the BSTLd (dorsolateral division of BST), but not in the BSTM. IHC did not reveal any apparent difference between the sexes in the size of distribution of either immunoreactivity. Double staining IHC showed that axonal terminals of both VIP and PACAP neurons were in close proximity to dendrites or perikarya of corticotropin releasing factor (CRF) neurons. At the electron microscopic level IHC revealed the presence of axodendritic or axosomatic synapses between VIP-ir and PACAP-ir axon terminals and CRF-ir neurons. Although the origin of PACAP-ir fibers in the BSTLd remains to be determined, these morphological findings suggest that PACAP and VIP regulate the activity of CRF neurons in the BSTLd as neurotransmitters or neuromodulators.

Localization of endomorphin-2-like immunoreactivity in the rat medulla and spinal cord.

Endomorphin-1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) are endogenous ligands that have greater affinity and selectivity for the mu-opiate receptor than any other known mammalian peptide. A polyclonal antiserum, screened for specificity to endomorphin-2 by immunodot-blot assay and preabsorption controls, was used for localization of this peptide. Immunocytochemistry performed on the brainstem, spinal cord, and sensory ganglia of rats by the avidin-biotin-peroxidase method revealed a continuous dense aggregation of endomorphin-2-like immunoreactive varicose fibers in the superficial laminae of the dorsal horn of the medulla and spinal cord. Immunoreactive fibers were detected in the dorsal root as well as within the dorsal root ganglia. The results suggest that endomorphin-2 is synthesized in primary sensory neurons in ganglia, transported to the superficial dorsal horn, and released near neurons expressing mu receptors. Its distribution appears to represent a functional unit likely to be associated with modulation of nociceptive stimuli.

Human immunodeficiency virus infection of T-lymphoblastoid cells reduces intracellular pH.

Alterations in plasma membrane function are induced by many cytopathic viruses, including human immunodeficiency virus type 1 (HIV-1). These alterations can result in changes in the intracellular content of ions and other small molecules and can contribute to cytolysis and death of the infected cell. The pH-sensitive fluorescent probe 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein-acetoxymethyl ester was used to quantitate intracellular pH (pHi) in HIV-1-infected T cells. Infection of cells from the CD4+ T-lymphoblastoid line HUT-78 (RH9 subclone) with HIV-1 strain LAI resulted in a significant decrease of pHi, from approximately 7.2 in mock-infected cells to below 6.7 by day 4 after infection, when cells were undergoing acute cytopathic effects. The pHi in persistently infected cells that survived the acute cytopathic effects of HIV-1 was approximately 6.8 to 7.0. Studies with amiloride, an inhibitor of the Na+/H+ exchange system, suggest that HIV-1-induced intracellular acidification in lymphocytes is due, in part, to dysfunction of this plasma membrane ion transport system. The alterations in pHi may mediate certain cytopathic effects of HIV-1, thereby contributing to depletion of CD4+ T lymphocytes in patients with AIDS.

Alteration of intracellular potassium and sodium concentrations correlates with induction of cytopathic effects by human immunodeficiency virus.

Increases in intracellular concentrations of potassium ([K+]i) and sodium ([Na+]i) occur concomitantly with cytopathic effects induced in a CD4+ T-lymphoblastoid cell line acutely infected by human immunodeficiency virus (HIV). This [K+]i increase was greater in cells infected by cytopathic HIV strains than in cells infected by less cytopathic strains. T cells persistently infected by HIV had an increased [K+]i but displayed an [Na+]i similar to that of mock-infected cells. HIV induced increases in [K+]i and [Na+]i after cytopathic infection of human peripheral blood mononuclear cells, but the magnitude of the Na+ changes did not correlate with the extent of the cytopathic effect. Enhanced movement of cations may osmotically drive water entry, resulting in balloon degeneration and lysis of HIV-infected cells. These observations offer potential approaches for antiviral therapies.

Immunohistochemical localization of PACAP in the ovine digestive system.

The localization of immunoreactive PACAP (PACAP-IR) in the entire length of the sheep gastrointestinal tract and the pancreas was studied by an immunohistochemical method. PACAP-IR-containing nerve fibers innervated the longitudinal smooth muscle layer of the mucosa in the esophagus, stomach, and small and large intestines, the muscular layer of the stomach and intestine, Brunner's gland of the duodenum, and the walls of small arteries. PACAP-IR fibers also innervated the exocrine acini, islets of Langerhans, and the small arteries in the connective tissue septa of the pancreas. These findings suggest a regulatory role of PACAP in the digestive system.

Pituitary adenylate cyclase activating polypeptide (PACAP) increases cytosolic-free calcium concentration in folliculo-stellate cells and somatotropes of rat pituitary.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a new member of the secretin/VIP peptide family. In order to identify pituitary target cells for PACAP, we examined the effect of PACAP on the cytosolic-free Ca2+ concentrations, [Ca2+]i, in cells using Fura-2 and a digital imaging system. In a concentration-dependent manner, PACAP increased [Ca2+]i in pituitary cells, from 10(-11) to 10(-8) M. Some cells responded to PACAP at 10(-11) M, while others only at 10(-8) M, showing a diversity among cells in the sensitivity to PACAP. Both PACAP and growth hormone-releasing factor (GRF) at 10(-8) M increased [Ca2+]i in 25% and 33% of the total pituitary cells, respectively. About 38% of the GRF-responsive cells also responded to PACAP. Some of the cells that responded to PACAP were immunocytochemically identified as growth hormone (GH) cells or folliculo-stellate (FS) cells using antisera against GH or S-100 protein, respectively. The results indicate that both the folliculo-stellate cells and GH cells of the pituitary are the direct targets for PACAP, but the possibility that other cell types also respond to PACAP cannot be excluded.

Cytochemical characterization of anterior pituitary target cells for the neuropeptide, pituitary adenylate cyclase activating polypeptide (PACAP), using biotinylated ligands.

Two novel peptides, named PACAP (pituitary adenylate cyclase activating polypeptide) containing 38 (PACAP38) and 27 residues (PACAP27) were recently isolated from ovine hypothalami. In order to investigate the pituitary cell type(s) that bear a receptor for PACAP, PACAP38 was biotinylated and used for cytochemical examination of binding. The cells were also identified by immunocytochemical methods using the antisera against each of the rat anterior pituitary hormones or an antiserum against S-100 protein, a marker for pituitary folliculo-stellate (FS) cells. Biotinylated PACAP38 (biot-PACAP) exhibited adenylate cyclase stimulating activity (ACSA) comparable to PACAP38 in rat pituitary cell cultures, and displaced the bound 125I-PACAP27 to the rat pituitary membrane preparation to the same extent as PACAP38. After 2-4 days of culture, dispersed rat pituitary cells were incubated with varying concentrations of biot-PACAP at room temperature or 4 degrees C. The bound biot-PACAP38 was visualized by avidin-biotin-peroxidase complex (ABC) method with nickel intensification. Biot-PACAP-positive and pituitary hormone or S-100-positive cells were counted. More than 90% of S-100-positive cells bound biot-PACAP38. A considerable number of GH and PRL cells and a lesser number of ACTH cells also bound biot-PACAP38, whereas only a few identified LH, FSH, or TSH cells bound biot-PACAP38. These results suggest that FS cells are a major target cell type for PACAP. A recent study from our laboratory demonstrated that PACAP stimulated the release of interleukin (IL)-6 in rat pituitary cell cultures. FS cells are known to produce IL-6.

Pituitary adenylate cyclase activating polypeptide and vasoactive intestinal peptide increase cytosolic free calcium concentration in cultured rat hippocampal neurons.

Recently, pituitary adenylate cyclase activating polypeptide (PACAP) was isolated from ovine hypothalamus and it was shown to stimulate adenylate cyclase in rat pituitary cells, neurons, and astrocytes. PACAP exhibits a 68% amino acid sequence homology with vasoactive intestinal peptide (VIP); however, it is 1000 times more potent than VIP in stimulating adenylate cyclase. In view of the wide distribution of PACAP and its receptor in the central nervous system, PACAP is likely to act as a neurotransmitter or neuromodulator as well. In the present study, we investigated the effects of PACAP38 on cytosolic-free calcium concentrations ([Ca2+]i) and compared these effects with those of VIP in cultured rat hippocampal neurons. Calcium concentrations, at the single cell level, were measured using fura-2, a calcium sensitive fluorescent dye, and fura-2-loaded neurons were continuously superfused at 37 C and viewed under an inverted microscope. Images of these neurons were recorded at 10-sec intervals by a video camera equipped with an Argus-50/CA system which controls the image acquisition and display. [Ca2+]i was quantitated from the intensities of fluorescence of the cells at two excitation wavelengths of 340 and 380 nm. The ratio of the intensities of emitted fluorescence (340/380 nm) was calibrated to determine [Ca2+]i. PACAP38 (0.1 nM) increased [Ca2+]i in some hippocampal neurons. As the concentration of peptide was increased from 0.1 to 10 nM, the accumulated number of hippocampal neurons responding to PACAP38 progressively increased and reached a plateau at 10 nM. Total neurons (33.0 +/- 5.3%, n = 4; 502 neurons) were found to respond to 100 nM PACAP38. The half-maximal concentration (ED50) of PACAP38 was 2.60 +/- 0.77 nM. Typically, 60-90 sec after the addition of PACAP38 (10 nM), [Ca2+]i increased from basal levels of 50-100 to 150-300 nM. VIP also increased [Ca2+]i, but required 1 microM or higher concentration for a considerable number of cells to respond. The number of hippocampal neurons responding to VIP at 1 microM was 28.9 +/- 9.8% (n = 4; 442 cells) which was comparable to the population of neurons responding to 10 nM PACAP38. The ED50 for VIP was 0.68 +/- 0.38 microM which was approximately 260 times higher than the ED50 for PACAP38. Neither 1-10 microM nitrendipine, a L-type voltage-dependent Ca2+ channel blocker, or 1 microM omega-conotoxin GVIA, a N-type voltage-dependent Ca2+ channel blocker, altered the PACAP-induced Ca2+ increment. Removal of Ca2+ from the superfusion media did not influence the PACAP38-induced increase of [Ca2+]i.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunohistochemical localization of the neuropeptide, pituitary adenylate cyclase activating polypeptide (PACAP), in human and primate hypothalamus.

A 38 residue neuropeptide was recently isolated from ovine hypothalamus in our laboratory, and named pituitary adenylate cyclase activating polypeptide (PACAP38) based on its biological activity. Rabbit antisera against synthetic PACAP27 were characterized by ELISA for immunohistochemical use. PACAP-immunoreactive neuronal elements having similar distributions were demonstrated in both human and spider monkey hypothalami. Many PACAP-immunoreactive cell bodies were present in the supraoptic and paraventricular nuclei. Immunopositive nerve fiber networks were stained throughout the hypothalamus, including in both external and internal zones of the tuber cinereum, close to the transition of the pituitary stalk (median eminence). These results suggest that PACAP plays multifunctional roles as a hypophysiotropic hormone, neurohypophysial hormone, neurotransmitter or neuromodulator in higher vertebrate species including man.

Long-Term Dynamic in vitro System for Investigating Rat Pineal Melatonin Secretion.

Abstract Several details regarding the control of melatonin (MT) secretion from the pinealocytes are still to be clarified. To obtain more data on the mechanism and kinetics of MT secretion and on the interactions between bioactive materials affecting MT production, a perifusion system has been developed in our laboratory. In this dynamic in vitro system the surviving pinealocytes maintain their full responsiveness for at least 5 days. In order to determine the MT contents of large numbers of samples collected in the perifusion system, a sensitive MT radioimmunoassay was set up utilizing our specific MT antibody. In our perifusion system the basal MT release does not change significantly during the 5-day experiments. Norepinephrine (NE) was used at 1 muM concentration for 30 min as a marker of the responsiveness of pinealocytes, given at the beginning and at the end of the same experiments. No significant difference was found in the MT responses to NE stimulation over 5 days. The kinetics of MT response and the dose-response relationship were investigated after NE exposure at various concentrations (100 nM to 10 mM). NE at 100 nM was found to be ineffective. Between 1 muM and 1 mM concentrations NE increased the MT release in a dose-dependent manner. No significant difference was found between the responses above 1 mM concentration. NE seems to be a specific stimulator of pineal MT production. The MT production reached the maximum value after a relatively long lag (2 to 3 h) when NE application had been stopped, and returned to basal values after 5 to 6 h. This prolonged time-course of MT secretion, in contrast with the fast responses of pituitary cells to releasing hormones, suggests that NE stimulated the synthesis of MT rather than the release of stored hormone. The modulatory effects of light-dark cycle on basal and stimulated MT release of perifused pineals was also investigated: Neither basal nor NE-stimulated (100 nM to 10muM) MT release was influenced significantly by light-dark conditions showing that the light-dark cycle does not have a direct modulatory effect on MT secretion under in vitro circumstances. Based on our observations, this perifusion system should be a useful tool for investigating: 1) hormone interactions on the regulation of pineal MT release, and 2) accurate kinetics of MT response.