Evidence that a substance P-like peptide mediates the non-cholinergic excitatory response of the carp intestinal bulb (Cyprinus carpio)

Summary The participation of substance P in the noncholinergic contraction induced by transmural stimulation (TMS) of the carp intestinal bulb was examined. In the presence of atropine, substance P caused the contraction of carp intestinal bulb smooth muscle in a concentration dependent manner (1 nmol/1 – 1 mol/l). The EC50 value was 28 ± 7 nmol/l (n = 6). Substance P-induced desensitization (1 mol/l for 15 min), decreased the response to substance P and the atropine-resistant contraction induced by TMS (20 Hz) selectively. In contrast, in the absence of atropine, the contraction induced by TMS (20 Hz) was slightly attenuated with the substance P-induced desensitization. The acid extract obtained from the carp intestinal bulb contained a smooth muscle excitatory material whose pharmacological properties were consistent with those of substance P. The present results indicate that a substance P-like peptide is present in the carp intestinal bulb which is involved in the non-cholinergic contraction induced by TMS.Send offprint requests to T. Kitazawa at the above address     

Lateral actions at the inner plexiform layer of the carp retina: effects of turning windmill pattern stimulus

Lateral action from amacrine to ganglion cells was studied in the isolated carp retina by using a truncated windmill pattern (TWP). About 25% of ganglion cells of both "on" and "off" center types were suppressed or enhanced in firing activity in response to TWP turning. The suppressed cells were more sensitive to slow turning velocities of TWP than the enhanced cells. In the "on-off" type amacrine cells, a steady depolarizing or hyperpolarizing component (less than several mV) was maintained by stationary TWP, while the cells were exclusively depolarized by turning TWP at a wide range of velocities. These results suggest that individual responses of ganglion cells induced by both stationary and turning TWP are depending on a balance between two factors: the polarizing direction of steady components of the "on-off" amacrine cells and the polarizing direction of ganglion cells synaptically produced by the amacrine cells.     

Zn(2+) modulates light responses of color-opponent bipolar and amacrine cells in the carp retina

The effects of Zn(2+) on color-opponent bipolar cells (BCs) and amacrine cells (ACs) were studied in the isolated superfused carp retina using intracellular recording techniques. Bath-applied Zn(2+) (25 micro M) depolarized R(+)G(-)-type BCs and suppressed both depolarizing responses of these cells to red (680 nm) flashes and hyperpolarizing ones to green (500 nm) flashes. Following Zn(2+) application, G(+)R(-)-type BCs were hyperpolarized, which was accompanied by a potentiation of their depolarizing responses to green flashes and a suppression of hyperpolarizing ones to red flashes. Similar Zn(2+) effects were observed in R(+)G(-)- and G(+)R(-)-type ACs. The Zn(2+) effects persisted in the presence of picrotoxin and strychnine, suggesting that modulation by Zn(2+) of GABA and glycine receptors was unlikely involved. Using whole-cell recording techniques, it was found Ca(2+) currents in cone terminals were dose-dependently suppressed by Zn(2+), suggesting that Zn(2+) may reduce glutamate release from cone photoreceptors. Furthermore, lowering extracellular Ca(2+), a procedure that increases glutamate release from photoreceptors, exerted actions on R(+)G(-)- and G(+)R(-)-type BCs, almost opposite to the Zn(2+) effects on these two types of BCs. It is therefore postulated that the Zn(2+) effects reported in the present work may reflect a consequence of the changes in input resistances of color-opponent BCs and driving forces for their light responses resulted from the reduced glutamate release by Zn(2+).     

Clonal growth of carp (Cyprinus carpio) T cells in vitro

Carp kidney leukocytes co-cultured with a supporting cell layer resulted in the rapid proliferation of various types of leukocytes including immature leukocytes. Expressions of marker genes for multiple blood cell lineages were observed in the primary culture. However, after several passages, the proliferating cells expressed only T cell and macrophage marker genes.Further RT-PCR analysis revealed that the proliferating cells expressed TCR constant regions (Cα, Cβ, Cγ, Cδ), CD3γ/δ and CD4 (CD4L-1), but did not express CD8α and CD8β. Additionally, in situ hybridization analysis showed that the majority of proliferating cells expressed Cα, Cβ, Cγ, Cδ and CD4. Moreover, 5′-RACE sequences of TCR variable regions (Vα, Vβ, Vγ, Vδ) revealed that the proliferating cells contained a polyclonal T cell repertoire, and most of the Vα and Vβ sequences were functional, but the Vγ and Vδ sequences were non-functional with frame shifts and stop codons. Taken together, these results indicate that the proliferating cells after serial passages predominantly contained CD4+ CD8− αβT cells that simultaneously co-expressed non-functional γδTCR. To obtain CD4+ αβT cell (helper T cell) clones, single cells were picked up from the bulk culture, seeded into each well of 96-well plates and cultured in the presence of supporting cells and conditioned media. T cell colonies formed from single cells after 2-3 weeks. These colony cells expressed Cα, Cβ, Cδ and CD4, and weakly expressed Cγ, but did not express CD8α, CD8β and CD4L-2. Taken together, these results indicate that these clonal T cells resemble a subpopulation of mammalian CD4+ helper T cells.     

Nitric oxide hinders antibody clearance from the surface of Trypanoplasma borreli and increases susceptibility to complement-mediated lysis

Trypanoplasma borreli is an extracellular blood parasite of carp belonging to the same Order (Kinetoplastida) as African trypanosomes. These mammalian parasites have developed different strategies to evade the host immune system including antigenic variation, immunosuppression and clearance of surface-bound antibodies. The latter mechanism allows trypanosomes to use their swimming movement to cause surface-bound antibodies to ‘sail’ and accumulate at the posterior end of the parasite, to be internalized via the flagellar pocket and be degraded. There is no evidence that T. borreli shows antigenic variation, but during the late phases of infection NO-mediated immunosuppression is observed. High levels of nitric oxide (NO) lead to extensive tissue nitration whereas the parasite itself is not affected. Therefore, the induction of NO has thus far been considered a parasite-driven response with immunosuppressive effects. In the present study, we show that the induction of NO, particularly during the early phase of T. borreli infections, should be re-considered an effective part of the host immune response. We show that T. borreli rapidly removes surface-bound IgM. In addition, moderate concentrations of NO, by hindering surface antibody clearance, maintain high the concentrations of membrane-bound IgM, thereby favoring antibody-dependent complement-mediated parasite lysis. We performed a comprehensive quantitative gene expression analysis of in total seven different complement factors involved in all three activation pathways, differentiating between 1 and 4 isoforms for each complement gene. Our gene expression analysis supports an important role for antibody-dependent complement-mediated lysis of T. borreli in vivo. To our knowledge, NO-dependent inhibition of antibody clearance from the surface of kinetoplastid parasites has not been investigated. Our data support a role for NO as an important player in host–parasite interactions, not only as immune suppressor (late response) but also as immune effector (early response) in infections with bloodstream parasites such as T. borreli.     

A role for multiple estrogen receptors in immune regulation of common carp

Estrogens are important for bi-directional neuroendocrine-immune interaction. They act via nuclear estrogen receptors (ERα and ERβ) and/or G-protein coupled receptor - GPR30.We found expression of ERα, ERβ and GPR30 in carp lymphoid tissues and head kidney monocytes/macrophages, neutrophils and lymphocytes. Interestingly, ERβ is also expressed in some head kidney lymphocytes but not in naive PBLs. Immune stimulation altered the cell type specific profile of expression of these receptors, which depends on both activation and maturation stage.This implies direct leukocyte responsiveness to estrogen stimulation and therefore in vitro effects of 17β-estradiol (E2) on reactive oxygen species (ROS) production in monocytes/macrophages were determined. Short-time incubation with E2 increased ROS production in PMA-stimulated cells. Results comply with mediation by GPR30, partially functioning via phosphoinositide 3-kinase activation.These results furthermore demonstrate that neuroendocrine-immune communication via estrogen receptors is evolutionary conserved.     

GABA modulates color-opponent bipolar cells in carp retina

In the present work, the effects of γ-aminobutyric acid (GABA) were examined while recording intracellularly from color-opponent bipolar cells in the isolated, superfused carp retina. Bath-applied GABA differentially suppressed light responses of R/G cells, which depolarized and hyperpolarized, respectively, in response to red and green flashes, and G/R cells, which showed the opposite responses. In either R/G or G/R cells, the response driven by input from red cones was invariably suppressed to a greater extent. Such effects of GABA were completely blocked by picrotoxin, but only partially by bicuculline, suggesting that the GABA_C receptor, in addition to the GABA_A receptor, may also be involved. It is postulated that GABA modulates the color-opponent bipolar cells either through activating GABA receptors on these cells directly or those on cone terminals indirectly.     

5-hydroxytryptamine: its facilitative action on [3H]dopamine release from the retina

5-Hydroxytryptamine (5-HT) at 5 X 10(-4) M caused a two-fold increase in [3H]dopamine (DA) release from retinal particulate fractions of the carp (Cyprinus carpio). The 5-HT action was dose- and Ca2+-dependent. The three 5-HT agonists examined (5-methoxytryptamine, 5-methoxy-N,N-dimethyltryptamine and tryptamine) were more effective than 5-HT on [3H]DA release. 5,6-Dihydroxytryptamine was the strongest competitor among drugs tested for [3H ]DA uptake, but did not evoke any significant release of [3H]DA. Noradrenaline (or DA) at 5 X 10(-4) M produced a large increase in [3H]DA release from the particulate fractions, but its action was Ca2+-independent. The 5-HT-induced DA release could be seen in the frog retina but not in the rat retina, which does not contain indoleamine-accumulating cells. The results obtained strongly suggest that 5-HT stimulates [3H ]DA release through a receptor mechanism on DAergic terminals and modifies the DA-cell's function in the retina.     

Double color-opponent receptive fields of carp bipolar cells

Bipolar cells with double color-opponent receptive fields were found in the carp retina. They were classified into two types depending on their response polarity. In the receptive field center, type I cells showed hyperpolarization to a long wavelength spot (R-) and depolarization to a short wavelength spot (G+), while in the surround they showed R+, G- responses. Type II cells showed responses of opposite polarities to those of type I cells, namely R+ G- in the center, and R- G+ in the surround. From their spectral sensitivity, R components were identified as coming from red-sensitive cones, and G components from green-sensitive cones. These bipolar cells are thought to be the origin of double color-opponent receptive fields of retinal ganglion cells.