Pituitary adenylate cyclase-activating polypeptide modulates plasminogen activator expression in rat granulosa cell

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a bioactive peptide isolated from ovine hypothalamus. It has been demonstrated to be transiently expressed in preovulatory follicles and to positively affect several parameters correlated with the ovulatory process. The aim of the present study was to investigate whether PACAP influences the plasminogen/plasmin system in rat ovary. Plasminogen activators (PAs) are serine proteases, modulated by gonadotropins and several peptides in preovulatory follicles, that appear to be involved in ovulation. Granulosa cells obtained from immature eCG-treated rats were cultured for 24 h in the presence of increasing concentrations of PACAP and vasoactive intestinal peptide (VIP). A significant, dose-dependent increase in tissue-type PA (tPA) activity and decrease in urokinase-type (uPA) PA activity were observed in PACAP-treated cells. These effects were exerted at the mRNA level. The use of cycloheximide, a protein synthesis inhibitor, suggested that PACAP requires an intermediary protein to decrease uPA-mRNA, but not to induce tPA-mRNA. However, no significant modulation of PAs was observed in the presence of VIP. When granulosa cells were stimulated within the intact follicle (i.e., maintaining the three-dimensional structure and in the presence of the theca cell layers), both PACAP and VIP dose-dependently stimulated tPA. These data suggest that, in addition to the PACAP type I receptor present on granulosa cells, different subtypes of PACAP receptors are present in the different ovarian compartments.     

Pituitary adenylate cyclase-activating polypeptide in intrinsic and extrinsic nerves of the rat pancreas

Pituitary adenylate cyclase-activating polypeptide (PACAP) is the latest member of the vasoactive intestinal polypeptide (VIP) family of neuropeptides present in nerve fibres in many peripheral organs. Using double immunohistochemistry, with VIP as a marker for intrinsic innervation and calcitonin-gene related peptide (CGRP) as a marker for mainly extrinsic innervation, the distribution and localization of PACAP were studied in the rat pancreas. PACAP was demonstrated in nerve fibres in all compartments of the pancreas and in a subpopulation of intrapancreatic VIP-containing ganglion cells. PACAP and VIP were co-stored in intra- and interlobular nerve fibres innervating acini, blood vessels, and in nerve fibres within the islets of Langerhans. No PACAP immunoreactivity was observed in the islet cells. Another population of PACAP-immunoreactive nerve fibres co-localized with CGRP innervated ducts, blood vessels and acini. PACAP/CGRP-positive nerve fibres were also demonstrated within the islets. Neonatal capsaicin reduced the PACAP-38 concentration by approximately 50%, and accordingly a marked reduction in PACAP/CGRP-immunoreactive nerve fibres in the exocrine and endocrine pancreas was observed. Bilateral subdiaphragmatic vagotomy caused a slight but significant decrease in the PACAP-38 concentration compared with controls. In conclusion, PACAP-immunoreactive nerve fibres in the rat pancreas seem to have dual origin: extrinsic, most probably sensory fibres co-storing CGRP; and intrinsic, constituting a subpopulation of VIP-containing nerve cell bodies and fibres innervating acinar cells and islet cells. Our data provide a morphological basis for the reported effects of PACAP in the pancreas and suggest that PACAP-containing nerves in the rat pancreas may have both efferent and sensory functions.     

Pituitary adenylate cyclase-activating polypeptide and islet amyloid polypeptide in primary sensory neurons

Primary sensory neurons serve a dual role as afferent neurons, conveying sensory information from the periphery to the central nervous system, and as efferent effectors mediating, e.g., neurogenic inflammation. Neuropeptides are crucial for both these mechanisms in primary sensory neurons. In afferent functions, they act as messengers and modulators in addition to a principal transmitter; by release from peripheral terminals, they induce an efferent response, “neurogenic inflammation,” which comprises vasodilatation, plasma extravasation, and recruitment of immune cells. In this article, we introduce two novel members of the sensory neuropeptide family: pituitary adenylate cyclase-activating polypeptide (PACAP) and islet amyloid polypeptide (IAPP). Whereas PACAP, a vasoactive intestinal polypeptide-resembling peptide, predominantly occurs in neuronal elements, IAPP, which is structurally related to calcitonin gene-related peptide, is most widely known as a pancreatic β-cell peptide; as such, it has been recognized as a constituent of amyloid deposits in type 2 diabetes. In primary sensory neurons, under normal conditions, both peptides are predominantly expressed in small-sized nerve cell bodies, suggesting a role in nociception. On axotomy, the expression of PACAP is rapidly induced, whereas that of IAPP is reduced. Such a regulation of PACAP suggests that it serves a protective role during nerve injury, but that of IAPP may indicate that it is an excitatory messenger under normal conditions. In contrast, in localized adjuvant-induced inflammation, expression of both peptides is rapidly induced. For IAPP, studies in IAPP-deficient mice support the notion that IAPP is a pronociceptive peptide, because these mutant mice display a reduced nociceptive response when challenged with formalin.     

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates the oxygen sensing type I (glomus) cells of rat carotid bodies via reduction of a background TASK-like K+ current

Pituitary adenylate cyclase-activating polypeptide (PACAP)-deficient mice are prone to sudden neonatal death and have reduced respiratory response to hypoxia. Here we found that PACAP-38 elevated cytosolic [Ca(2+)] ([Ca(2+)](i)) in the oxygen sensing type I cells but not the glial-like type II (sustentacular) cells of the rat carotid body. This action of PACAP could not be mimicked by vasoactive intestinal peptide but was abolished by PACAP 6-38, implicating the involvement of PAC(1) receptors. H89, a protein kinase A (PKA) inhibitor attenuated the PACAP response. Simultaneous measurement of membrane potential and [Ca(2+)](i) showed that the PACAP-mediated [Ca(2+)](i) rise was accompanied by depolarization and action potential firing. Ni(2+), a blocker of voltage-gated Ca(2+) channels (VGCC) or the removal of extracellular Ca(2+) reversibly inhibited the PACAP-mediated [Ca(2+)](i) rise. In the presence of tetraethylammonium (TEA) and 4-aminopyridine (4-AP), PACAP reduced a background K(+) current. Anandamide, a blocker of TWIK-related acid-sensitive K(+) (TASK)-like K(+) channel, occluded the inhibitory action of PACAP on K(+) current. We conclude that PACAP, acting via the PAC(1) receptors coupled PKA pathway inhibits a TASK-like K(+) current and causes depolarization and VGCC activation. This stimulatory action of PACAP in carotid type I cells can partly account for the role of PACAP in respiratory disorders.     

Pituitary adenylate cyclase-activating polypeptide (PACAP) regulates the expression of PACAP in cultured tilapia astrocytes

Neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic hormone that is involved in numerous physiologic functions. The present study examines the presence and the functional significance of PACAP and its receptor in the brain and astrocytes of tilapia (Oreochromis mossambicus). This is the first demonstration of the full-length nucleotide sequence of tPACAP gene in tilapia pituitary, brain, and cultured astrocytes. Two cDNA variants of the growth hormone-releasing hormone (GHRH)-PACAP gene were identified in tilapia pituitary, brain, and cultured astrocytes as a result of exon skipping with a long form (271 bp) encoding both tPACAP(38) and tGHRH and a short form (166 bp) encoding only tPACAP(38). The short form was found to be more abundant in astrocytes. Addition of ovine PACAP(38) (1 nM) to cultured astrocytes significantly stimulated the expression of tPACAP(38) at 4 hrs, but the effect dropped after 8 hrs of treatment. By contrast, the expression of PACAP type I receptor (PAC(1)-R) mRNA in the astrocytes was not responsive to PACAP(38) treatment. The tPACAP(38) expression also was activated by the cAMP analog, dibutyryl-cAMP, in a dose-dependent manner. Adding high salinity (170 mM NaCl, 500 mOsm/kg osmolarity) to cultured medium substantially increased astroglial tPACAP(38) expression over 4 hrs to a level that was maintained for 16 hrs. This observation was not found when mannitol (270 mM) was supplemented as an osmolarity-enhancing agent (500 mOsm/ kg). Taken together, tPACAP expression in tilapia astrocytes was well regulated by exogenous PACAP, cAMP, and salinity and might be involved in the adaptation to high salinity when the fish is in a seawater environment.     

Pituitary adenylate cyclase-activating polypeptide protects astroglial cells against oxidative stress-induced apoptosis

Oxidative stress, associated with a variety of disorders including neurodegenerative diseases, results from accumulation of reactive oxygen species (ROS). Oxidative stress is not only responsible for neuron apoptosis, but can also provoke astroglial cell death. Numerous studies indicate that pituitary adenylate cyclase-activating polypeptide (PACAP) promotes neuron survival, but nothing is known regarding the action of PACAP on astroglial cell survival. Thus, the purpose of the present study was to investigate the potential glioprotective effect of PACAP on H(2)O(2)-induced astrocyte death. Pre-treatment of cultured rat astrocytes with nanomolar concentrations of PACAP prevented cell death provoked by H(2)O(2) (300 μM), whereas vasoactive intestinal polypeptide was devoid of protective activity. The effect of PACAP on astroglial cell survival was abolished by the type 1 PACAP receptor antagonist, PACAP6-38. The protective action of PACAP was blocked by the protein kinase A inhibitor H89, the protein kinase C inhibitor chelerythrine and the mitogen-activated protein (MAP)-kinase kinase (MEK) inhibitor U0126. PACAP stimulated glutathione formation, and blocked H(2)O(2)-evoked ROS accumulation and glutathione content reduction. In addition, PACAP prevented the decrease of mitochondrial activity and caspase 3 activation induced by H(2)O(2). Taken together, these data indicate for the first time that PACAP, acting through type 1 PACAP receptor, exerts a potent protective effect against oxidative stress-induced astrocyte death. The anti-apoptotic activity of PACAP on astrocytes is mediated through the protein kinase A, protein kinase C and MAPK transduction pathways, and can be accounted for by inhibition of ROS-induced mitochondrial dysfunctions and caspase 3 activation.     

Pituitary adenylate cyclase-activating peptide (PACAP) immunolocalization in lymphoid tissues of the rat

Pituitary adenylate cyclase activating peptide (PACAP) is a novel peptide isolated from the ovine hypothalamus. PACAP exists in 2 molecular forms with 27 (PACAP27) or 38 (PACAP38) amino acid residues. PACAP localization was studied by immunohistochemical methods in central (bone marrow and thymus) and peripheral (spleen, lymph nodes and duodenal mucosa) lymphoid tissues with antisera raised against PACAP27 or PACAP38. PACAP-positive cells were found in all lymphoid tissues examined. These cells were highly positive for PACAP38 but were negative for PACAP27. Morphologically, they were small mononuclear cells with relatively scarce cytoplasm and lymphocyte-like features. PACAP38-positive cells were abundant in peripheral lymphoid tissues (i.e., mesenteric lymph nodes). In the duodenal mucosa, PACAP38-positive cells were located either in the lamina propria or epithelium. These results suggest that PACAP38-positive cells are present within lymphoid tissues and may represent a lymphocyte-like cell subpopulation that has a potential role in cell-to-cell interactions in the immune system and in the integrated communication between neuroendocrine and immune systems.     

Effects of pituitary adenylate cyclase-activating polypeptide on canine atrial electrophysiology

We hypothesized that pituitary adenylate cyclase-activating polypeptide (PACAP) activates intracardiac postganglionic parasympathetic nerves and has a different effect than cervical vagal stimulation. We measured effective refractory period (ERP) and conduction velocity at four atrial sites [high right atrium (HRA), low right atrium (LRA), high left atrium (HLA), and low left atrium (LLA)] and minimum atrial fibrillation (AF) cycle length at 12 atrial sites during cervical vagal stimulation and after PACAP in 26 autonomically decentralized, open-chest, anesthetized dogs. PACAP shortened ERP to a similar extent at all four sites (HRA, 58 +/- 2.0 ms; LRA, 60 +/- 6.3 ms; HLA, 68 +/- 11.5 ms; and LLA, 60 +/- 8.3 ms). Low- and high-intensity vagal stimulation shortened ERP at the HRA, but not in the other atrial sites (low-intensity stimulation: HRA, 64 +/- 4.0 ms; LRA, 126 +/- 5.1 ms; HLA, 110 +/- 9.5 ms; and LLA, 102 +/- 11.5 ms; high-intensity stimulation: HRA, 58 +/- 4.2 ms; and HLA, 101 +/- 4.0 ms). Conduction velocity was not altered by any intervention. Minimum AF cycle length after PACAP was similar in both atria but was shorter in the right atrium than in the left atrium during vagal stimulation. After atropine administration, no interventions changed ERP. These results suggest that PACAP shortens atrial refractoriness uniformly in both atria through activation of intrinsic cardiac nerves, not all of which are activated by cervical vagal stimulation.     

ATP consumption by uncoupled mitochondria activates sarcolemmal K(ATP) channels in cardiac myocytes

We tested whether close coupling exists between mitochondria and sarcolemma by monitoring whole cell ATP-sensitive K(+) (K(ATP)) current (I(K,ATP)) as an index of subsarcolemmal energy state during mitochondrial perturbation. In rabbit ventricular myocytes, either pinacidil or the mitochondrial uncoupler dinitrophenol (DNP), which rapidly switches mitochondria from net ATP synthesis to net ATP hydrolysis, had little immediate effect on I(K,ATP). In contrast, in the presence of pinacidil, exposure to 100 microM DNP rapidly activated I(K,ATP) with complex kinetics consisting of a quick rise [time constant of I(K,ATP) increase (tau) = 0.13 +/- 0.01 min], an early partial recovery (tau = 0.43 +/- 0.04 min), and then a more gradual increase. This DNP-induced activation of I(K,ATP) was reversible and accompanied by mitochondrial flavoprotein oxidation. The F(1)F(0)-ATPase inhibitor oligomycin abolished the DNP-induced activation of I(K,ATP). The initial rapid rise in I(K,ATP) was blunted by atractyloside (an adenine nucleotide translocator inhibitor), leaving only a slow increase (tau = 0.66 +/- 0.17 min, P < 0.01). 2,4-Dinitrofluorobenzene (a creatine kinase inhibitor) slowed both the rapid rise (tau = 0.20 +/- 0.01 min, P < 0.05) and the subsequent declining phase (tau = 0.88 +/- 0.19 min, P < 0.05). From single K(ATP) channel recordings, we excluded a direct effect of DNP on K(ATP) channels. Taken together, these results indicate that rapid changes in F(1)F(0)-ATPase function dramatically alter subsarcolemmal energy charge, as reported by pinacidil-primed K(ATP) channel activity, revealing cross-talk between mitochondria and sarcolemma. The effects of mitochondrial ATP hydrolysis on sarcolemmal K(ATP) channels can be rationalized by reversal of F(1)F(0)-ATPase and the facilitation of coupling by the creatine kinase system.     

Pituitary adenylate cyclase activating polypeptide (PACAP) in the rat mammary gland

Pituitary adenylate cyclase activating polypeptide (PACAP), a member of the vasoactive intestinal polypeptide (VIP) family of peptides, is present in the brain and in neuronal elements of a number of peripheral organs. Since no information on PACAP in the mammary gland exists, we have investigated, by radioimmunoassay and immunohistochemistry, the occurrence and distribution of PACAP immunoreactivity in the mammary gland of lactating and non-lactating rats. A specific monoclonal mouse anti-PACAP antibody'has been used to show that the peptide is located in nerve fibres associated with bundles of circular and longitudinal smooth muscle surrounding the lactiferous duct of the nipple. PACAP-immunoreactive nerve fibres and nerve bundles are present in the subepidermal connective tissue of the nipple and in the mammary parenchyma, some of the fibres being in close contact with blood vessels. Occasionally, a few delicate varicose fibres are associated with secretory alveoli and lactiferous ducts. The majority of PACAP-positive nerve fibres are, however, located in the glabrous skin of the nipple and the hairy skin adjacent to the nipple forming a subepithelial plexus from which delicate varicose nerve fibres enter the overlying epithelium. Double immunostaining for PACAP and a marker for sensory neurons, calcitonin gene-related peptide, has disclosed that the two peptides are almost completely co-localized. A minor population of the PACAP-immunoreactive nerve fibres shows co-existence with VIP. Although no obvious changes at the immunohistochemical level could be observed during pregnancy or lactation, elevated concentrations of immunoreactive PACAP-38 in mammary extracts have been found during lactation. Our data suggest that PACAP is involved in the nervous control of mammary gland function, probably in the transmission of suckling stimuli.     

Pituitary adenylate cyclase-activating polypeptide inhibits collagen-induced arthritis: an experimental immunomodulatory therapy.

Rheumatoid arthritis is a chronic, systemic, autoimmune, and inflammatory disorder that affects the synovial lining of the joints. We describe the beneficial effects of the pituitary adenylate cyclase-activating polypeptide (PACAP) in the collagen-induced arthritis experimental murine model being proposed as a novel therapeutic approach in the treatment of rheumatoid arthritis. PACAP greatly decreases arthritis frequency and severity in the studied mice by improving clinical symptoms, ameliorating joint damage, and blocking both the inflammatory and autoimmune mediators which are the main keys of the pathogenesis of this disease. With this study, PACAP emerges as a promising candidate for the treatment of a pathology with a high world incidence but currently no effective treatment.     

Pituitary adenylate cyclase-activating polypeptide promotes differentiation of mouse neural stem cells into astrocytes

We have found that pituitary adenylate cyclase-activating polypeptide (PACAP) employed at the physiological concentrations induces the differentiation of mouse neural stem cells into astrocytes. The differentiation process was not affected by cAMP analogues such as dibutylic cAMP (db-cAMP) or 8Br-cAMP or by the specific competitive inhibitor of protein kinase A, Rp-adenosine-3',5'-cyclic monophosphothioate triethylamine salt (Rp-cAMP). Expression of the PACAP receptor (PAC1) in neural stem cells was detected by both RT-PCR and immunoblot using an affinity-purified antibody. The PACAP selective antagonist, PACAP(6-38), had an inhibitory effect on the PACAP-induced differentiation of neural stem cells into astrocytes. These results indicate that PACAP acts on the PAC1 receptor on the plasma membrane of mouse neural stem cells, with the signal then transmitted intracellularly via a PAC1-coupled G protein, does not involve Gs. This signaling mechanism may thus play a crucial role in the differentiation of neural stem cells into astrocytes.     

Pituitary adenylate cyclase-activating polypeptide type 1 (PAC1) receptor is expressed during embryonic development of the earthworm

Pituitary adenylate cyclase activating polypeptide (PACAP)-like molecules have been shown to be present in cocoon albumin and in Eisenia fetida embryos at an early developmental stage (E1) by immunocytochemistry and radioimmunoassay. Here, we focus on detecting the stage at which PAC1 receptor (PAC1R)-like immunoreactivity first appears in germinal layers and structures, e.g., various parts of the central nervous system (CNS), in developing earthworm embryos. PAC1R-like immunoreactivity was revealed by Western blot and Far Western blot as early as the E2 developmental stage, occurring in the ectoderm and later in specific neurons of the developing CNS. Labeled CNS neurons were first seen in the supraesophageal ganglion (brain) and subsequently in the subesophageal and ventral nerve cord ganglia. Ultrastructurally, PAC1Rs were located mainly on plasma membranes and intracellular membranes, especially on cisternae of the endoplasmic reticulum. Therefore, PACAP-like compounds probably influence the differentiation of germinal layers (at least the ectoderm) and of some neurons and might act as signaling molecules during earthworm embryonic development.     

Pituitary adenylate cyclase-activating polypeptide induces testicular testosterone synthesis through PGE(2) mediation in crested newt,Triturus carnifex

The aim of the present work was to study the possible role of adenylate cyclase-activating polypeptide (PACAP) 38 in the testicular intracellular mechanism regulating steroidogenesis of crested newt, Triturus carnifex. Gonads were incubated in vitro with PACAP 38 and prostaglandin (PG) E(2) alone or with inhibitors of cyclooxygenase (COX), adenylate cyclase (AC), and phospholipase C (PLC) for 30 min and 60 min. PGE(2), PGF(2 alpha), testosterone, and estradiol-17 beta were measured in the culture medium; aromatase (AR) activity and cAMP were assessed in the tissue. PACAP 38 increased PGE(2) (30 min and 60 min), estradiol-17 beta (60 min), cAMP (60 min), and AR (60 min) but decreased testosterone (60 min). PGE(2) increased estradiol-17 beta, cAMP, and AR and decreased testosterone at 30 and 60 min.PLC inhibitor counteracted the effects of PACAP 38, while AC inhibitor counteracted these effects except for PGE(2) increase. AC inhibitor counteracted the effects of PGE(2), while PLC did not. COX inhibitor decreased PGF(2 alpha) (30 min and 60 min), PGE(2) (30 min and 60 min), estradiol-17 beta (60 min), cAMP (60 min), and AR (60 min), but increased testosterone (60 min). These in vitro results suggest that, in newt testis, PACAP 38 acts on PLC, inducing the increase of PGE(2) which, in turn, acting on AC, increases AR activity with the consequent estradiol-17 beta increase and testosterone decrease.     

Pituitary adenylate cyclase-activating polypeptide and vasoactive intestinal peptide attenuate glutamate-induced nNOS activation and cytotoxicity

Both vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) act as neurotransmitters in the central and peripheral nervous systems. Attention has been focused on these neuropeptides because among their numerous biological activities, they have been confirmed to show neuroprotective effects against ischemia and glutamate-induced cytotoxicity. It is well established that glutamate has excitatory effects on neuronal cells, and that excessive glutamate shows potent neurotoxicity, especially in neuronal nitric oxide synthase-containing neurons. Glutamate stimulates the production of nitric oxide (NO) in neurons, and the NO generated is tightly associated with the delayed death of neurons. We examined the effects of these neuropeptides on the glutamate-induced neural actions using PC12 cells, and we confirmed the important activities of PACAP/VIP on the production of NO as well as the delayed cell death stimulated by glutamate.     

Pituitary adenylate cyclase-activating polypeptide 27 is a functional ligand for formyl peptide receptor-like 1

Although the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) has been implicated in the regulation of several immune responses, its target receptors and signaling mechanisms have yet to be fully elucidated in immune cells. In this study, we found that PACAP27, but not PACAP38, specifically stimulated intracellular calcium mobilization and ERK phosphorylation in human neutrophils. Moreover, formyl peptide receptor-like 1 (FPRL1) was identified as a PACAP27 receptor, and PACAP27 was found to selectively stimulate intracellular calcium increase in FPRL1-transfected rat basophil leukocytes-2H3 cell lines. In addition, PACAP27-induced calcium increase and ERK phosphorylation were specifically inhibited by an FPRL1 antagonist, Trp-Arg-Trp-Trp-Trp-Trp (WRW4), thus supporting the notion that PACAP27 acts on FPRL1. In terms of the functional role of PACAP27, we found that the peptide stimulated CD11b surface up-regulation and neutrophil chemotactic migration, and that these responses were completely inhibited by WRW4. The interaction between PACAP27 and FPRL1 was analyzed further using truncated PACAPs and chimeric PACAPs using vasoactive intestinal peptide, and the C-terminal region of PACAP27 was found to perform a vital function in the activation of FPRL1. Taken together, our study suggests that PACAP27 activates phagocytes via FPRL1 activation, and that this results in proinflammatory behavior, involving chemotaxis and the up-regulation of CD11b.     

Modeling transmural heterogeneity of K(ATP) current in rabbit ventricular myocytes

To investigate the mechanisms regulating excitation-metabolic coupling in rabbit epicardial, midmyocardial, and endocardial ventricular myocytes we extended the LabHEART model (Puglisi JL and Bers DM. Am J Physiol Cell Physiol 281: C2049–C2060, 2001). We incorporated equations for Ca²⁺ and Mg²⁺ buffering by ATP and ADP, equations for nucleotide regulation of ATP-sensitive K⁺ channel and L-type Ca²⁺ channel, Na⁺-K⁺-ATPase, and sarcolemmal and sarcoplasmic Ca²⁺-ATPases, and equations describing the basic pathways (creatine and adenylate kinase reactions) known to communicate the flux changes generated by intracellular ATPases. Under normal conditions and during 20 min of ischemia, the three regions were characterized by different I_Na, I_to, I_Kr, I_Ks, and I_Kp channel properties. The results indicate that the ATP-sensitive K⁺ channel is activated by the smallest reduction in ATP in epicardial cells and largest in endocardial cells when cytosolic ADP, AMP, PCr, Cr, P_i, total Mg²⁺, Na⁺, K⁺, Ca²⁺, and pH diastolic levels are normal. The model predicts that only K_ATP ionophore (Kir6.2 subunit) and not the regulatory subunit (SUR2A) might differ from endocardium to epicardium. The analysis suggests that during ischemia, the inhomogeneous accumulation of the metabolites in the tissue sublayers may alter in a very irregular manner the K_ATP channel opening through metabolic interactions with the endogenous PI cascade (PIP₂, PIP) that in turn may cause differential action potential shortening among the ventricular myocyte subtypes. The model predictions are in qualitative agreement with experimental data measured under normal and ischemic conditions in rabbit ventricular myocytes. ATP-sensitive K⁺ channel; creatine and adenylate kinase reactions; phosphatidylinositol phosphates; heart; mathematical model     

Electron microscopic observation of pituitary adenylate cyclase-activating polypeptide (PACAP)-containing neurons in the rat retina

The distribution and localization of pituitary adenylate cyclase-activating polypeptide (PACAP) in the rat retina were studied by immunocytochemistry with both light and electron microscopy. PACAP-like immunoreactivity (PACAP-LI) was detected in the amacrine and horizontal cells as well as in the inner plexiform layer, the ganglion cell layer and the nerve fiber layer. PACAP-LI seemed to be concentrated predominantly in the neuronal perikarya and their processes, but not in other cells in the retina. At the ultrastructural level, PACAP-LI was visible in the plasma membranes, rough endoplasmic reticulum, and cytoplasmic matrix in the PACAP-positive neurons in the inner nuclear layer. In the inner plexiform layer, PACAP-positive amacrine cell processes made synaptic contact with immunonegative amacrine cell processes, bipolar cell processes, and ganglion cell terminals. These findings suggest that PACAP may function as a neurotransmitter and/or neuromodulator.