Contractile response to substance P in isolated smooth muscle strips from the intestinal bulb of the carp (Cyprinus carpio)

1. The effect of substance P on the mechanical activity of carp intestinal bulb smooth muscle was investigated in vitro. 2. Bath-applied substance P (1 nM-1 microM) caused concentration-dependent contraction of the smooth muscle. The EC50 value was 20 +/- 3 nM (N = 13). 3. Pretreatment with tetrodotoxin (780 nM) or atropine (500 nM) partially decreased the contractile response to substance P, while methysergide (3 microM) did not decrease the response. 4. The contractile response to substance P was not decreased by [D-Pro2, D-Trp7.9]-substance P or [D-Pro4, D-Trp7.9]-substance P (4-11) pretreatment (10 microM for 5 min). 5. Exposure of the intestinal bulb to substance P (100 nM and 1 microM for 15 min) decreased the response to subsequent application of substance P, physalaemin and eledoisin in a concentration dependent manner, while the contractile response to acetylcholine or methionine-enkephalin was not affected. 6. Exposure of the intestinal bulb to physalaemin and eledoisin (100 nM for 15 min) decreased the response to subsequent application of substance P. 7. The above results indicate that substance P causes the contraction of the carp intestinal bulb smooth muscle through its direct action on the smooth muscle and its indirect action through enteric cholinergic nerves. Long-term exposure to substance P causes desensitization of the preparation to substance P, physalaemin and eledoisin at the receptor level.     

Presence of four tachykinins in an acid extract of the carp intestinal bulb (Cyprinus carpio)

1. The pharmacological and chemical properties of substance P-like peptides isolated from an acid extract of the carp intestinal bulb were examined using guinea-pig ileum longitudinal smooth muscle.2. On a Sephadex G25 column (3 × 96 cm), smooth muscle contracting material was eluted as two peaks (fraction-1 and fraction-2). The molecular weight of the fraction-1 was estimated to be 2300 and that of the fraction-2 to be 1530.3. The pharmacological properties of the contracting materials in fraction-1 and fraction-2 resembled those of substance P and neurokinin A.4. The susceptibility of the contracting activity of fraction-1 to proteolytic enzymes resembled that of physalaemin but, on the other hand, the susceptibility of that of fraction-2 resembled those of eledoisin and neurokinin A.5. Ion-exchange chromatography on sulfopropyl-Sephadex C25 indicated the presence of one contracting material in fraction-1 and three contracting materials in fraction-2. The elution positions of four materials were different from that of substance P.6. These results indicate that four tachykinins different from substance P are present in an acid extract of the carp intestinal bulb.     

Presence of four tachykinins in an acid extract of the carp intestinal bulb (Cyprinus carpio).

1. The pharmacological and chemical properties of substance P-like peptides isolated from an acid extract of the carp intestinal bulb were examined using guinea-pig ileum longitudinal smooth muscle. 2. On a Sephadex G25 column (3 x 96 cm), smooth muscle contracting material was eluted as two peaks (fraction-1 and fraction-2). The molecular weight of the fraction-1 was estimated to be 2300 and that of the fraction-2 to be 1530. 3. The pharmacological properties of the contracting materials in fraction-1 and fraction-2 resembled those of substance P and neurokinin A. 4. The susceptibility of the contracting activity of fraction-1 to proteolytic enzymes resembled that of physalaemin but, on the other hand, the susceptibility of that of fraction-2 resembled those of eledoisin and neurokinin A. 5. Ion-exchange chromatography on sulfopropyl-Sephadex C25 indicated the presence of one contracting material in fraction-1 and three contracting materials in fraction-2. The elution positions of four materials were different from that of substance P. 6. These results indicate that four tachykinins different from substance P are present in an acid extract of the carp intestinal bulb.     

Effects of morphine and methionine-enkephalin on the smooth muscle tonus and the contraction induced by transmural stimulation in the carp (Cyprinus carpio) intestinal bulb

The effects of morphine and methionine-enkephalin (met-enkephalin) on the smooth muscle tonus and the contraction induced by transmural stimulation were investigated in the isolated intestinal bulb of carp in vitro. Morphine (30 nM-3 microM) and met-enkephalin (3 nM-5 microM) caused dose-dependent non-sustained contraction. Naloxone (10 nM) inhibited the contraction induced by morphine or met-enkephalin in a competitive manner. Tetrodotoxin (400 nM) or atropine (500 nM) did not inhibit the contraction induced by morphine or met-enkephalin. Cooling of the bath fluid from 20 to 10 degrees C decreased nicotine- and transmural stimulation-induced contraction. But met-enkephalin-induced contraction was not affected. Transmural stimulation-induced contraction (3 Hz) was not affected by pretreatment with morphine, met-enkephalin or naloxone. The results demonstrated that morphine or met-enkephalin caused contraction of the smooth muscle directly through the activation of opiate receptors on the smooth muscle cells and neither morphine nor met-enkephalin regulated the cholinergic neurotransmission presynaptically.     

Effects of some autonomic drugs and neuropeptides on the mechanical activity of longitudinal and circular muscle strips isolated from the carp intestinal bulb (Cyprinus carpio)

1. The mechanical responses to some autonomic drugs and neuropeptides of longitudinal muscle (LM) and circular muscle (CM) strips isolated from the carp intestinal bulb were investigated in vitro. 2. Acetylcholine and carbamylcholine caused concentration-dependent transient contraction of both LM and CM strips. Tetrodotoxin had no effect, but atropine selectively decreased the contractile responses to acetylcholine and carbamylcholine. 3. Excitatory alpha-2 and inhibitory beta adrenoceptors were present in both LM and CM strips. 4. 5-Hydroxytryptamine (5-HT) caused concentration-dependent contraction of both LM and CM strips. Tetrodotoxin, atropine and methysergide decreased the contractile responses to 5-HT. 5. Some neuropeptides (angiotensin I, angiotensin II, bombesin, bradykinin, neurotensin, somatostatin and vasoactive intestinal polypeptide) did not cause any mechanical response (contraction or relaxation) in either smooth muscle strip. 6. Substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) caused contraction of both LM and CM strips. However, the time course of the contraction in LM was different from that in CM. The order of potency was NKA greater than SP greater than NKB in LM strips and NKA greater than SP much greater than NKB in CM strips. In LM strips, the contractile responses to tachykinins were unaffected by spantide and methysergide, but partly decreased by tetrodotoxin and atropine. On the other hand, the contractile responses of CM strips were unaffected by tetrodotoxin, atropine, methysergide and spantide. 7. Dynorphin (1-13) (DYN), leucine-enkephalin (L-Enk) and methionine-enkephalin (M-Enk) caused concentration-dependent contraction of both LM and CM strips. The order of potency was DYN greater than M-Enk greater than L-Enk. Naloxone selectively decreased the responses to opiate peptides. 8. The present results indicate that acetylcholine, carbamylcholine, catecholamines, 5-HT, tachykinins (SP, NKA and NKB) and opiate peptides (DYN, L-Enk and M-Enk) affect the mechanical activity of LM and CM strips isolated from the carp intestinal bulb through their specific receptors.     

The ultrastructure of leucocytes in carp (Cyprinus carpio)

Leucocytes from anterior kidney, middle kidney, spleen, thymus and peripheral blood of Common carp were examined. Lymphocytes were found to have a similar fine structure to that of mammals. Thrombocytes had a similar appearance to lymphocytes, but the former were sometimes distinguishable by vesicles in series and/or microtubules below the plasma membrane. Blast cells, monocytes and clearly identifiable plasma cells were also seen. Neutrophils had varying proportions of at least two types of granules, which often presented inclusions. Owing to their different granules, eosinophils and basophils were morphologically distinguishable, but differentiated cells with equal proportions of the two types of granules were also seen. In addition, cells of uncertain nature, possessing rod-shaped granules, were observed. The different leucocyte types showed the same morphology in the different lymphoid organs and in peripheral blood, although they were present in different proportions.     

Soybean meal induces intestinal inflammation in common carp (Cyprinus carpio L.)

The development of soybean meal (SBM) induced enteritis in the hindgut of the omnivorous common carp (Cyprinus carpio L.). The developed condition was assessed when carp, continuously fed on animal protein, were transferred to a diet in which 20% of the protein was replaced by SBM. After week 1, most of the inflammation parameters were already present, but at week 3, a strong aggravation of the condition was observed which included a shortening of the mucosal folds, the disappearance of the supranuclear vacuoles, an increased number of goblet cells, a thickened lamina propria and sub-epithelial mucosa with increased numbers of basophilic granulocytes as well as a decreased uptake capacity of enterocytes (impaired endocytosis and microvilli). Contrary to previous observations made with respect to Atlantic salmon, common carp start to recover from the fourth to the fifth week after switching to SBM feeding. At this stage, the supranuclear vacuoles refill and most of the parameters revert to basal levels. During the enteritis process, a real-time quantitative PCR analysis was conducted to measure the expression of inflammatory and anti-inflammatory cytokine genes in the isolated intraepithelial lymphocytes (IEL). The pro-inflammatory interleukin 1 beta (IL-1 beta) and tumour necrosis factor alpha1 (TNF-alpha1) genes were up-regulated during the inflammation process while the anti-inflammatory interleukin 10 (IL-10) was down-regulated after an initial up-regulation at week 1. Transforming growth factor beta (TGF-beta) expression showed an up-regulation from week 3 onwards despite the high Ct value and the low primer efficiency shown. This study confirms the contribution of IEL (mainly T-like cells) and basophils in the enteritis process. In addition, the results show a clear involvement of up- and down-regulated cytokine genes in both the onset and recovery of the SBM-induced enteritis in the hindgut of carp.     

An in vitro study of long-term potentiation in the carp (Cyprinus carpio L.) olfactory bulb

Long-term potentiation (LTP) of synaptic transmission is considered a cellular mechanism for neural plasticity and memory formation. Previously, we showed that in the carp olfactory bulb, LTP occurs at the dendrodendritic mitral-to-granule cell synapse following tetanic electrical stimulation applied to the olfactory tract, and suggested that it is involved in the process of olfactory memory formation. As a first step towards understanding mechanisms underlying plasticity at this synapse, we examined the effects of various drugs (glutamate and GABA receptor agonists and antagonists, noradrenaline, and drugs affecting cAMP signaling) on dendrodendritic mitral-to-granule cell synaptic transmission in an in vitro preparation. Two forms of LTP are involved: a postsynaptic form (tetanus-evoked LTP) and a presynaptic form. The postsynaptic form is evoked at the granule cell dendrite following tetanic olfactory tract stimulation and is suppressed by the NMDA receptor antagonist, D-AP5, enhanced by noradrenaline, and occluded by the metabotropic glutamate receptor agonist, trans-ACPD. The presynaptic form occurs at the mitral cell dendrite following blockade of the GABA_A receptor by picrotoxin and bicuculline, or via activation of cAMP signaling by forskolin and 8-Br-cAMP.     

Adaptation of intestinal morphology in the temperature-acclimated carp,Cyprinus carpio L.

Summary The effects of temperature and photoperiod acclimation upon the morphology of carp intestinal mucosa have been studied using morphometric techniques. Carp intestine showed an absence of anatomical regionalisation. There was a gradual reduction in the dimensions of villi along the tract. The decrease in the dimensions of the villi was greatest in the anterior half. Temperature acclimation had no effect on intestinal-somatic indices. Acclimation to 10° C or 30° C resulted in large differences in the dimensions of villi. Cold acclimation produced significant increases in mean villus height and breadth along the entire intestine. These villus shape changes resulted in a 58% increase in total mucosal surface area and a 102% increase in total volume of villi in cold-acclimated fish relative to warm-acclimated fish. Surface area of the unmodified intestinal tube increased with cold acclimation by 28%. The total number of villi remained unchanged by thermal acclimation. Because normalisation to a nominal surface area does not take account of the possibility of differentially developed mucosal surfaces in differently acclimated animals, experiments comparing transepithelial transport rates of differently-acclimated fish, using unstripped preparations, overestimates the differences in area-specific transport capacity.     

Molecular cloning of the common carp (Cyprinus carpio) rhodopsin cDNA

A recombinant phage clone containing a 1584 nucleotides rhodopsin cDNA was screened from a carp retinal cDNA library. The inserted DNA consisting of a single open reading frame of 1062 nucleotides at positions 72 to 1133 encodes a 354 amino acid polypeptide. The deduced amino acid sequence of carp rhodopsin showed 95.7, 85.5 and 74.4% identity with that of goldfish, sand goby and lamprey, respectively. The sites of palmitoylation, glycosylation, disulfide bond formation and Schiff base formation in the putative rhodopsin are all conserved.     

Subcellular localization of muscle FBPase in carp (Cyprinus carpio) tissues

Subcellular localization of muscle FBPase-a regulatory enzyme of glyconeogenesis-was investigated in carp using immunohistochemistry and protein exchange method. Results of the experiments revealed that, in striated muscles, FBPase associates with alpha-actinin of the Z-line and co-localizes with aldolase. Additionally, in cardiac and smooth muscle cells FBPase is present inside the nuclei. In the light of findings on mammalian muscle FBPase, the data presented here indicates that interaction of the enzyme with specific cellular partners and nuclear presence of FBPase is a general phenomenon in contemporary vertebrates.     

Seasonal variations in the content of catecholamines in carp heart (Cyprinus carpio)

1. Seasonal changes in epinephrine and norepinephrine contents were demonstrated in ventricular muscle tissues of carp heart. 2. Patterns of changes in epinephrine and norepinephrine contents were similar; high during late spring-to-summer and low in winter; however, the fluctuation in epinephrine content was greater than that of norepinephrine. 3. Tyramine caused a positive inotropic effect in isolated electrically-stimulated ventricular muscles of carp heart. The inotropic effect was totally blocked by reserpine, reduced by propranolol, but not altered by atenolol. 4. These results suggest that the catecholamines released from sympathetic nerve terminals may contribute to the regulation of the seasonal change in the function of cardiac muscles in the carp.     

Monosaccharide uptake in common carp (Cyprinus carpio) EPC cells is mediated by a facilitative glucose carrier

In most animal cells, transport of monosaccharides across the plasma membrane is mediated by glucose transporters (GLUT). Mammals express at least five distinct transporters (GLUTs 1--5), which are well characterised both functionally and genetically. In contrast, the glucose transport system of fish remains poorly studied. Here we report studies of hexose uptake in carp EPC cells and cloning of a glucose transporter cDNA from these cells. Transport of radio-labelled methylglucose (3-OMG) followed Michaelis--Menten kinetics with a K(m) value (8.5 mM) similar to that of mammalian cells. The inhibition of transport by cytochalasin B and phloretin, but not by phloridzin or cyanide, strongly suggested the existence of a facilitative carrier. D-Glucose, 2-deoxyglucose, 3-OMG, D-mannose and D-xylose were competitive inhibitors of 3-OMG uptake, while L-glucose, mannitol, D-fructose, D-ribose and sucrose did not compete with 3-OMG. We cloned a carp glucose transporter (CyiGLUT1), using RT-PCR and RACE strategies. CyiGLUT1 was different from known carp and zebrafish EST sequences. The complete cDNA (3060 bp) contained one open reading frame encoding a predicted protein of 478 amino acids. The deduced amino acid sequence shared 78% identity with mammalian and avian GLUT1 proteins. Key amino acids involved in substrate selection and catalysis of mammalian GLUTs were conserved in the carp transporter.