Endocytosis and recycling of neurokinin 1 receptors in enteric neurons

Neurotransmission depends on the availability of transmitter and on the presence of functional, high-affinity receptors at the plasma membrane that are capable of binding ligand. The pathway, mechanism and function of endocytosis and recycling of the substance P or neurokinin 1 receptor in enteric neurons were studied using fluorescent substance P, receptor antibodies and confocal microscopy. In both the soma and neurites, substance P induced rapid, clathrin-mediated internalization of the neurokinin 1 receptor into early endosomes, which also contained the transferrin receptor. After 4-8 h, there was a return in surface neurokinin 1 receptor immunoreactivity in the soma, which was not prevented by cycloheximide, and was thus independent of new protein synthesis. This return was prevented by acidotropic agents, therefore required endosomal acidification. This suggests that the neurokinin 1 receptor recycles in the soma. In contrast, in neurites, substance P and the neurokinin 1 receptor remained in endosomes and recycling was not detected. Neurons of the myenteric plexus were heavily innervated by substance P-containing nerve fibers, and K(+)-stimulated release of endogenous substance P from cultured neurons induced internalization of the neurokinin 1-receptor. Therefore, endogenous substance P may induce endocytosis of the neurokinin 1 receptor. In the soma, endocytosis and recycling correlated with loss and recovery of functional binding sites for substance P. suggesting that this process contributes to the regulation of peptidergic neurotransmission. Thus, ligand-induced endocytosis of the neurokinin 1 receptor in myenteric neurons is associated with a loss of surface receptors and functional binding sites. Since release of endogenous substance P induces neurokinin 1 receptor internalization, and neurokinin 1 receptor neurons are innervated by substance P-containing fibers, endocytosis of neuropeptide receptors may regulate neurotransmission.     

Mechanisms of desensitization and resensitization of G protein-coupled neurokinin1 and neurokinin2 receptors

We compared the desensitization of neurokinin1 and neurokinin2 (NK1 and NK2) receptors expressed in Chinese hamster ovary cells to substance P and neurokinin A, respectively. Substance P and neurokinin A stimulated a rapid increase in intracellular Ca2+ concentration ([Ca2+]i) for both receptors, which was due to release of Ca2+ from intracellular stores. This was followed by a plateau in [Ca2+]i, which was due to influx of extracellular Ca2+, and was more sustained for the NK2 receptor. When Ca2+ was present in the extracellular solution, the Ca2+ response of the NK1 receptor, but not the NK2 receptor, rapidly desensitized and slowly resensitized to two exposures to agonist. In contrast, the [Ca2+]i response, measured in Ca2+-free solution, and inositol triphosphate generation desensitized and resensitized similarly for the NK1 and NK2 receptors. Thus, differences in desensitization between the NK1 receptor and the NK2 receptor may be related to differences in entry of extracellular Ca2+. We compared endocytosis of the NK1 and NK2 receptors to determine whether disparities could account for differences in desensitization. Fluorescent and radiolabeled substance P and neurokinin A were internalized similarly by cells expressing NK1 and NK2 receptors. Thus, disparities in internalization cannot account for differences in desensitization. We used inhibitors to examine the contribution of endocytosis, recycling, and phosphatases to desensitization and resensitization of the NK1 receptor. Desensitization did not require endocytosis. However, resensitization required endocytosis, recycling, and phosphatase activity. This suggests that the NK1 receptor desensitizes by phosphorylation and resensitizes by dephosphorylation in endosomes and recycling.     

Activation of spinal N-methyl-D-aspartate or neurokinin receptors induces long-term potentiation of spinal C-fibre-evoked potentials

The use-dependent increase in synaptic strength between primary afferent C-fibres and second-order neurons in superficial spinal dorsal horn may be an important cellular mechanism underlying central hyperalgesia. This long-term potentiation can be blocked by antagonists of the N-methyl-D-aspartate subtype of glutamate receptor, the neurokinin 1 or the neurokinin 2 receptor. We have tested here whether activation of these receptors by superfusion of the spinal cord with corresponding agonists in the absence of presynaptic activity is sufficient to induce long-term potentiation. In urethane anaesthetized rats C-fibre-evoked field potentials were elicited in superficial laminae of lumbar spinal cord by electrical stimulation of the sciatic nerve. In rats with intact spinal cord, controlled superfusion of the spinal cord at recording segments for 60 min with N-methyl-D-aspartate, substance P or neurokinin A never induced long-term potentiation. Spinal superfusion with a mixture of N-methyl-D-aspartate, substance P and neurokinin A also failed to induce long-term potentiation in four rats tested. In spinalized rats, however, long-term potentiation was induced by either N-methyl-D-aspartate (at 10 microM, to 173 +/- 16% of control) substance P (at 10 microM, to 176 +/- 13% of control) or by neurokinin A (at 1 microM, to 198 +/- .20% of control). The induction of long-term potentiation by N-methyl-D-aspartate, substance P or neurokinin A was blocked by intravenous application of the receptor antagonists dizocilpine maleate (0.5 mg/kg), RP67580 (2 mg/kg) or SR48968 (0.2 mg/kg), respectively. Thus, activation of N-methyl-D-aspartate or neurokinin receptors may induce long-lasting plastic changes in synaptic transmission in afferent C-fibres and this effect may be prevented by tonic descending inhibition.     

Nerve growth factor processing and trafficking events following TrkA-mediated endocytosis

We expressed the high affinity nerve growth factor receptor TrkA in Chinese hamster ovary (CHO) fibroblasts to study nerve growth factor (NGF) trafficking and processing events following receptor-mediated ligand internalization in a nonneuronal and p75 minus cell line. These stable clonal cell lines express approximately 2.5 x 10(5) TrkA receptors and bind 125I-NGF with high affinity (Kd = 4 x 10(-10) M). The TrkA receptors are autophosphorylated on tyrosine residues upon NGF stimulation and are capable of tyrosine phosphorylating downstream signaling molecules. The t1/2 of 125I-NGF internalization is 5 min, and the probability of an occupied TrkA receptor internalizing within 1 min at 37 C is 9.8%. By 2 h following endocytosis, less than 10% of internalized 125I-NGF is degraded, as determined by TCA precipitation. Thirty minutes following ligand endocytosis, endocytosed 125I-NGF is delivered back to the cell surface and released by the cell (retroendocytosis), possibly by remaining associated with recycling TrkA receptors. We measured the effect of acidification on 125I-NGF-TrkA association and found that, at pH 6, 40% of 125I-NGF remains bound. Thus, NGF may remain associated with the TrkA receptor at low pH conditions in the endosome and can thereby be targeted back to the plasma membrane for release by the cell. In conclusion: 1) TrkA, in the absence of p75, is fully capable of mediating 125I-NGF endocytosis; 2) internalized 125I-NGF is slowly and inefficiently degraded; 3) following internalization, 125I-NGF is retroendocytosed; and 4) the ability of 125I-NGF to remain receptor-associated during acidic conditions may provide a mechanism for its retroendocytosis via recycling TrkA vesicles.     

Tachykinins alter inositol phosphate formation, but not cyclic AMP levels, in primary cultures of neonatal rat spinal neurons through activation of neurokinin receptors

The naturally occurring tachykinins, substance P, neurokinin A and neurokinin B, induce the formation of inositol phosphates or cAMP in a variety of tissues but their effects on neurons have not been resolved. We used primary cultures of neonatal rat spinal cord to determine whether neurokinin receptors mediate changes in these second messengers in spinal neurons. We found that substance P, neurokinin A and neurokinin B induced the formation of inositol phosphates in a concentration-dependent manner with similar potencies (EC50S: 3.6, 5.7 and 21.3 nM, respectively), but at concentrations tested (0.1-1.0 microM) these peptides had no effect on cAMP levels. All three tachykinins induced the formation of inositol phosphates predominately by activation of neurokinin1 receptors. CP-96,345 and WIN 51,708, neurokinin1 receptor antagonists, attenuated the response to substance P, neurokinin A and neurokinin B. GR 103,537, a neurokinin2 receptor antagonist, had no effect on the responses induced by any of the tachykinins. Furthermore, the selective neurokinin1 receptor agonist, GR-73632, induced the formation of inositol phosphates in a concentration-dependent manner, whereas the selective neurokinin2 receptor agonist, GR-64349, generated inositol phosphates only at the highest concentration tested (10 microM). Senktide, a neurokinin3 receptor agonist, did not induce the formation of inositol phosphates at any of the concentrations tested (0.01-10 microM). Inositol phosphate formation appeared to be due to a direct effect of the tachykinins on neuronal neurokinin1 receptors. These results suggest that biological responses in spinal neurons following activation of neurokinin1 receptors are mediated mainly by the hydrolysis of phosphoinositol 4,5-bisphosphate to form inositol 1,4,5-trisphosphate and diacylglycerol. It remains to be determined which of these second messengers mediates the increased neuronal excitability and depolarization that occurs in response to substance P.     

Genetic basis of disease: studies of eicosanoids, and some complex disease traits

The three main tachykinins, substance P, neurokinin A (NKA), and neurokinin B, are believed to be selective ligands for respectively the NK-1, NK-2 and NK-3 receptors. However, NKA also has actions which cannot be mediated through its normal NK-2 receptor and the synthetic peptide [pGlu6,Pro9]-Substance P9-11--called septide--is known to have tachykinin-like actions despite its apparent lack of binding to any known tachykinin receptor. In the cloned NK-1 receptor expressed in COS-7 cells NKA and septide as expected were poor competitors for radiolabeled substance P. However, by using radiolabeled NKA and septide directly, it was found that both peptides in homologous binding assays as well as in competition against each other in fact bound to the NK-1 receptor with high affinity: Kd values of 0.51 +/- 0.15 nM (NKA) and 0.55 +/- 0.03 nM (septide). It is concluded that NKA and septide are high-affinity ligands for the NK-1 receptor but that they are poor competitors for substance P, which in contrast competes very well for binding with both NKA and septide.     

The rate of internalization of the mannose 6-phosphate/insulin-like growth factor II receptor is enhanced by multivalent ligand binding

The cation-independent mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF-II receptor) undergoes constitutive endocytosis, mediating the internalization of two unrelated classes of ligands, mannose 6-phosphate (Man-6-P)-containing acid hydrolases and insulin-like growth factor II (IGF-II). To determine the role of ligand valency in M6P/IGF-II receptor-mediated endocytosis, we measured the internalization rates of two ligands, beta-glucuronidase (a homotetramer bearing multiple Man-6-P moieties) and IGF-II. We found that beta-glucuronidase entered the cell approximately 3-4-fold faster than IGF-II. Unlabeled beta-glucuronidase stimulated the rate of internalization of 125I-IGF-II to equal that of 125I-beta-glucuronidase, but a bivalent synthetic tripeptide capable of occupying both Man-6-P-binding sites on the M6P/IGF-II receptor simultaneously did not. A mutant receptor with one of the two Man-6-P-binding sites inactivated retained the ability to internalize beta-glucuronidase faster than IGF-II. Thus, the increased rate of internalization required a multivalent ligand and a single Man-6-P-binding site on the receptor. M6P/IGF-II receptor solubilized and purified in Triton X-100 was present as a monomer, but association with beta-glucuronidase generated a complex composed of two receptors and one beta-glucuronidase. Neither IGF-II nor the synthetic peptide induced receptor dimerization. These results indicate that intermolecular cross-linking of the M6P/IGF-II receptor occurs upon binding of a multivalent ligand, resulting in an increased rate of internalization.     

Potencies of agonists acting at tachykinin receptors in the oestrogen-primed rat uterus: effects of peptidase inhibitors

The uterotonic potencies of the naturally occurring mammalian tachykinins and the synthetic subtype-selective agonist analogues of these agents [Lys5,MeLeu9,Nlel0]neurokinin A-(4-10) and [Nle10]neurokinin A-(4-10) (tachykinin NK2 receptor-selective), [Sar9,Met(O2)11]substance P (tachykinin NK1 receptor-selective) and senktide (tachykinin NK3 receptor-selective) were determined using preparations from oestradiol-treated rats. The endopeptidase 24.11 inhibitor, N-[N-[1-(S)-carboxyl-3-phenylpropyl]-(S)-phenyl-alanyl-(S)-isoserine+ ++ (SCH 39370), potentiated responses to neurokinin A, neurokinin B and substance P, but not to [Lys5,MeLeu9,Nle10)]neurokinin A-(4-10) or senktide. [Nle10]neurokinin A-(4-10) effects were potentiated by SCH 39370 with amastatin and those to [Sar9,Met(O2)11]substance P were potentiated by SCH 39370 and captopril in combination. In the presence of optimal concentrations of peptidase inhibitors the relative order of agonist potency was: neurokinin A > substance P > neurokinin B for the naturally occurring mammalian tachykinins and [Lys5,MeLeu9,Nle10]neurokinin A-(4-10) > [Nle10]neurokinin A-(4-10) > [Sar9,Met(O2)11]substance P > senktide for the synthetic tachykinin analogues. Thus, while a tachykinin NK2 receptor predominates in the oestrogen-primed uterus, a tachykinin NK1 receptor may also be present. The non-peptide tachykinin NK3 receptor antagonist, SR 142801, did not antagonise the effects of senktide suggesting that tachykinin NK3 receptors do not mediate its relatively minor effect on the uterus of the oestrogen-primed rat.     

Role of endothelin receptors, calcium and nitric oxide in the potentiation by endothelin-1 of the sympathetic contraction of rabbit ear artery during cooling

1. To examine further the potentiation by endothelin-1 on the vascular response to sympathetic stimulation, we studied the isometric response of isolated segments, 2 mm long, from the rabbit central ear artery to electrical field stimulation (1-8 Hz), under different conditions, at 37 degrees C and during cooling (30 degrees C). 2. Electrical stimulation produced frequency-dependent contraction, which was reduced (about 63% for 8 Hz) during cooling. At 30 degrees C, but not at 37 degrees C, endothelin-1 (1, 3 and 10 nM) potentiated the contraction to electrical stimulation in a dose-dependent way (from 43 +/- 7% to 190 +/- 25% for 8 Hz). 3. This potentiation by endothelin-1 was reduced by the antagonist for endothelin ETA receptors BQ-123 (10 microM) but not by the antagonist for endothelin ETB receptors BQ-788 (10 microM). The agonist for endothelin ETB receptors IRL-1620 (0.1 microM) did not modify the contraction to electrical stimulation. 4. The blocker of L-type Ca2+ channels verapamil (10 microM l-1) reduced (about 72% for 8 Hz) and the unspecific blocker of Ca(2+)-channels NiCl2 (1 mM) practically abolished (about 98%), the potentiating effects of endothelin-1 found at 30 degrees C. 5. Inhibition of nitric oxide synthesis with NG-nitro-L-arginine (L-NOARG, 0.1 mM) increased the contraction to electrical stimulation at 30 degrees C more than at 37 degrees C (for 8 Hz, this increment was 297 +/- 118% at 30 degrees C, and 66 +/- 15% at 37 degrees C). Endothelium removal increased the contraction to electrical stimulation at 30 degrees C (about 91% for 8 Hz) but not at 37 degrees C. Both L-NOARG and endothelium removal abolished the potentiating effects of endothelin-1 on the response to electrical stimulation found at 30 degrees C. 6. These results in the rabbit ear artery suggest that during cooling, endothelin-1 potentiates the contraction to sympathetic stimulation, which could be mediated at least in part by increasing Ca2+ entry after activation of endothelin ETA receptors. This potentiating effect of endothelin-1 may require the presence of an inhibitory tone due to endothelial nitric oxide.     

Gly166 in the NK1 receptor regulates tachykinin selectivity and receptor conformation

We have studied the pharmacological properties of genetically engineered human NK1 tachykinin receptors in which residues at the extracellular surface of the fourth transmembranal domain were substituted with the corresponding amino acids from the NK2 receptor. We show that substitution of G166C:Y167F in the human NK1 receptor induces high affinity binding of a group of tachykinin ligands, known as 'septides' (i.e. neurokinin A, neurokinin B, [pGlu6,Pro9]-substance P6-11 and substance P-methylester). In contrast, binding of substance P and non-peptide antagonists is unaffected by these mutations. This effect parallels that found on the rat receptor and is therefore species specific. Second, we demonstrate that mutation of Gly166 to Cys alone is both necessary and sufficient to create this pan-reactive tachykinin receptor, whereas replacement of Tyr167 by Phe has no detectable effect on the pharmacological properties of the receptor. Furthermore, analysis of the effect of N-ethylmaleimide and dithiothreitol on binding of radiolabelled substance P documents differences in the mode in which this ligand interacts with wild-type and mutant receptors and supports the existence of a mutational induced change in the conformational status of the NK1 receptor.     

Generation of chromophore Tyr-containing mutants of the ribosomal protein L7/L12 from Escherichia coli by site-directed mutagenesis and characterization

1. The effects of tachykinins and capsaicin were studied by means of intracellular membrane potential and isometric tension recordings in the isolated trachea of the guinea-pig. 2. The basal membrane potential averaged -51 mV, and most preparations demonstrated spontaneous slow waves. Tetraethylammonium (TEA), a potassium channel blocker (8 x 10(-3) M), depolarized the membrane potential to -44 mV and induced a rhythmic activity. 3. In control solution, substance P (10(-8)-10(-6) M), [Nle10]-neurokinin A(4-10) (10(-8)-10(-6) M) and capsaicin (10(-7)-10(-6) M) induced concentration-dependent depolarizations which were statistically significant at the highest concentration tested (depolarization by 10(-6) M: 8, 11 and 16 mV for the NK1 agonist, the NK2 agonist and capsaicin, respectively). 4. In the presence of TEA (8 x 10(-3) M), the three substances induced depolarizations which were statistically significant at the highest concentration tested for substance P (10(-6) M) and at 10(-7) and 10(-6) M for both [Nle10]-neurokinin A(4-10) and capsaicin (depolarization by 10(-6) M: 11, 17 and 10 mV for substance P, [Nle10]neurokinin A(4-10) and capsaicin, respectively). 5. In the presence or absence of tetraethylammonium, [MePhe7]-neurokinin B (10(-8)-10(-6) M) did not induce any significant changes in membrane potential. 6. The depolarizing effects of substance P (10(-6) M) and [Nle10]-neurokinin A(4-10) (10(-6) M) were blocked only by the specific antagonists for NK1 and NK2 receptors, SR 140333 (10(-7) M) and SR 48968 (10(-7) M), respectively. The effects of capsaicin (10(-6) M) were partially inhibited by each antagonist and fully blocked by their combination. 7. Substance P (10(-9) to 10(-4) M), [Nle10]-neurokinin A(4-10) (10(-10) to 10(-5) M), [MePhe7]-neurokinin B and capsaicin (10(-7) to 10(-5) M) evoked concentration-dependent contractions. 8. The contractions to substance P were significantly inhibited by SR 140333 (10(-8) to 10(-6) M) but unaffected by SR 48968 (10(-8) to 10(-6) M). Furthermore, the response to [Nle10]-neurokinin A(4-10) was significantly inhibited by SR 48968 and unaffected by SR 140333 at the same concentrations. Although SR 48968 (10(-7) M) alone did not influence the effects of substance P, it potentiated the inhibitory effect of SR 140333 (10(-7) M). A similar synergetic effect of these two compounds was observed in the inhibition of the contractile response to [Nle10]-neurokinin A(4-10). 9. Neither SR 140333 (10(-7) M) nor SR 48968 (10(-7) M) alone influenced the contractions to [MePhe7]-neurokinin B and capsaicin. However, the combination of the two antagonists abolished the contractions to either peptide. 10. These results demonstrate that the stimulation of both NK1 and NK2 tachykinin-receptors induced contraction and depolarization of the guinea-pig tracheal smooth muscle and that both receptors were stimulated during the endogenous release of tachykinins by capsaicin. There was no evidence for a major role of NK3 receptors in the contractile and electrical activity of the guinea-pig isolated trachea.     

Neurokinin induced inositol phosphate production in guinea pig bladder

PURPOSE: To examine second messenger pathways involved in neurokinin induced bladder contractions. MATERIALS AND METHODS: Neurokinin induced changes in inositol phosphate production and in adenylyl cyclase activity are measured in the guinea pig bladder. RESULTS: Substance P, substance P methyl ester, neurokinin A, and neurokinin B each increase [3H]-inositol phosphate production in the guinea pig bladder. Substance P (10(-6) M) increases [3H]-inositol trisphosphate levels within 30 sec. Substance P and neurokinin A have an additive effect on inositol phosphate production, however substance P (10(-5) M) or neurokinin A (10(-5) M) induced inositol phosphate production is less than that induced by carbachol (10(-5) M). Neurokinin B and to a lesser extent neurokinin A inhibit forskolin-activated adenylyl cyclase activity. CONCLUSIONS: These data are compatible with neurokinin-induced inositol phosphate production being coupled to increases in contractile force of the guinea pig urinary bladder, however more than one second messenger pathway may be involved.     

Two types of modified cardiac Na+ channels after cytosolic interventions at the alpha-subunit capable of removing Na+ inactivation

Repeated oesophageal acidification is a definitive feature of gastro-oesophageal reflux disease, which in turn is caused by relaxation of the lower oesophageal sphincter (LOS). This study in anaesthetised ferrets investigates the reflex pathways involved in effects of oesophageal acidification on motor function of the LOS, with particular focus on the role of tachykinins. LOS pressure was monitored with a perfused micromanometric sleeve assembly. Oesophageal acidification reduced LOS pressure by 48 +/- 5% until washout with saline. This reduction became larger with repeated tests, and was unaffected in amplitude by acute bilateral vagotomy, although the response became slower in onset. Intra-oesophageal capsaicin (0.5% solution) caused a 68 +/- 17% decrease in LOS pressure which remained unchanged with repeated tests. The NK-1 receptor antagonist CP96,345 (1-5 mg/kg intravenous (i.v.) blocked the post-vagotomy LOS responses to both intra-luminal acid and capsaicin. Close intra-arterial (i.a.) injections of capsaicin (1-100 micrograms) gut induced LOS relaxation which was neither vagally nor NK-1 receptor-mediated. Substance P or the selective NK-1 receptor agonist [Sar9, Met(O2)11] substance P (25-500 ng close i.a.) caused a biphasic LOS response, consisting of initial brief contraction followed by prolonged, dose-dependent relaxation. Tetrodotoxin (10 micrograms/kg close i.a.) changed the biphasic response to substance P to excitation only. The neurokinin-1 (NK-1) receptor antagonist CP96,345 (0.3-10 mg/kg i.v.) dose-dependently reduced the inhibitory response to substance P. The excitatory phase of the response to substance P was larger and prolonged after guanethidine (5 mg/kg, i.v.), or propranolol (1 mg/kg, i.v.). L-NAME (100 mg/kg i.v.) reduced the inhibitory phase. The selective NK-2 receptor agonist [beta-Ala8] neurokinin A(4-10) caused LOS excitation only. These data indicate that intra-oesophageal acid causes substance P release from extrinsic afferent nerve endings which activates local inhibitory pathways to the LOS via NK-1 receptors.     

Interaction of Escherichia coli heat-stable enterotoxin B with cultured human intestinal epithelial cells

Binding of Escherichia coli heat-stable enterotoxin B (STb) to the human intestinal epithelial cell lines T84 and HT29 and to polarized T84 cells was studied to define the initial interaction of this peptide toxin with target cells. Equilibrium and competitive binding isotherms showed that 125I-STb bound specifically to T84 and HT29 cells; however, the toxin-epithelial cell interactions could be characterized by low-affinity binding (< or = 10(5) M(-1)) to a high number of binding sites (> or = 10(6) per cell). STb binding to T84 and HT29 cells as a function of 125I-STb concentration did not approach saturation at levels well above the effective biological concentration of STb for fluid secretion. Treatment of the 125I-STb-bound T84 and HT29 cells with an acidic saline solution to remove surface-bound toxin revealed that only approximately 55% +/- 10% of 125I-STb could be removed by this treatment at 4 degrees C, suggesting that approximately half of the bound STb was stably associated with the plasma membrane and/or internalized into the cytoplasm. Similar results were obtained when binding and internalization experiments were conducted at 22 and 37 degrees C. Immunofluorescence studies demonstrated that the strongest signal for STb appeared in the plasma membrane even after acid treatment. Toxin-treated cells also displayed diffuse cytoplasmic staining, indicating that once cell bound, STb did not appear to preferentially associate with membrane vesicles or cellular organelles. Binding and subsequent internalization of 125I-STb were not affected by treatment of the cells with trypsin, endoglycosidase F/peptide N-glycosidase F, Vibrio cholerae neuraminidase, tunicamycin, or 5 mM sodium chlorate, which blocks sulfation of surface proteoglycans. In addition, the internalization process was not altered by preincubation of the cells with the cytoskeleton inhibitors cytochalasin D and colchicine or cellular perturbants (i.e., 0.45 M sucrose and 5 mM sodium azide), indicating that cell surface proteins or carbohydrates did not function as STb receptors. The binding of 125I-STb to polarized T84 cells was also examined, and the total and nonspecific binding isotherms were found to overlap, indicating that the apical surface of polarized T84 cells did not contain a specific receptor for STb. In comparison to undifferentiated cells, twice the amount of bound STb (approximately 80% +/- 10%) was removable from polarized T84 cells after treatment with acidic solution. The percentage of surface-bound STb to polarized T84 cells did not vary significantly with the transepithelial electrical resistance of the cells or when STb was applied basolaterally. Together, our results indicate that STb binds with relatively low affinity to the plasma membrane of cultured intestinal epithelial cells and polarized T84 cells, probably to membrane lipids, and becomes stably associated with the lipid bilayer. The fact that a significant portion of the bound STb becomes free in the cytoplasm, even at a low temperature, suggests that the bound toxin may directly traverse the membrane bilayer.     

Neurokinin A in rat renal afferent neurons and in nerve fibres within smooth muscle and epithelium of rat and guinea-pig renal pelvis

Neurokinin A-like immunoreactivity of dorsal root ganglion neurons innervating the kidney were studied with retrograde tracing of FluoroGold dye applied to the cut renal nerves. The proportions and sizes of renal afferent neurons with neurokinin A-like immunoreactivity were quantified in T9-L2 dorsal root ganglia from five rats. Of 240 renal afferent neuronal somata examined, 26 +/- 3% (S.E.M.) showed neurokinin A-like immunoreactivity. Compared with the overall size distribution of renal afferent neurons, those staining for neurokinin A were mostly small-sized neurons with a few medium-sized neurons. All somata with neurokinin A-like immunoreactivity were neurofilament-poor as judged by labelling with an anti-neurofilament antibody, RT97, and it is therefore likely that they had unmyelinated fibres. To examine the sites to which the renal afferent fibres with neurokinin A might project, sections of rat and guinea-pig kidney and upper ureter were examined. Fibres with neurokinin A-like immunoreactivity were found beneath and within the transitional epithelium lining the inner surface of the pelvis, and within the smooth muscle layer beneath the transitional epithelium. Epithelial innervation was found only in regions with underlying smooth muscle and loose connective tissue, and not in sites where the epithelium was closely applied to the renal parenchyma. The network of fibres was most dense towards the pelvo-uretic junction. Fibres with neurokinin A-like immunoreactivity were not seen beneath or within the cuboidal/columnar epithelium covering the papilla within the renal pelvis. Furthermore, only very few fibres with neurokinin A were observed penetrating the transitional epithelium of the upper ureter in both rat and guinea-pig. The distribution of fibres labelled with antibodies to substance P and calcitonin gene-related peptide in the renal pelvis was similar to that for fibres with neurokinin A-like immuno-reactivity, although a few fibres penetrated further into the fornices than fibres with neurokinin-A-like immunoreactivity. Thus, many afferent fibres in the renal pelvis may contain neurokinin A as well as substance P and calcitonin gene-related peptide. These fibres may be the source of the neurokinin A, substance P and calcitonin gene-related peptide which can be released by topical capsaicin treatment. In addition they may be the mechano- and chemo-receptive fibres in the renal pelvis that are known to play important roles in renal haemodynamics. The intra-epithelial position of some of these fibres in the epithelial layer suggests a possible chemosensory or osmosensory role.     

Deficient muscle carnitine transport in primary carnitine deficiency

The effects of conditions that either increase or decrease heart rate on the pharmacological properties of adenosine receptors in cultured rat myocytes were examined. Levels of A1 adenosine receptors, following prolonged treatment with electrical stimulation (ES) or the antiarrhythmic drug amiodarone, were determined using radioligand binding with the specific A1 receptor antagonist [3H]1,3-dipropyl-8-cyclopentylxanthine (CPX). The effects of lowering temperature were also explored. Exposure to amiodarone for 4 days reduced the density of A1 receptors by 19% (from 24.7 +/- 0.4 to 20.09 +/- 0.3 fmol/dish) and inhibited the rate of contraction by 60% (from 188 +/- 16 to 76 +/- 30 beats/min), without changing the receptor affinity, protein content, creatine kinase (CK) activity or cell number. Electrical stimulation at 25 degrees C elevated the density of A1 adenosine receptors by 185% (from 4.1 +/- 0.4 to 11.69 +/- 2.1 fmol/dish). Four days of reduced temperature (from 37 degrees C to either 30 or 25 degrees C) lowered the density of A1 adenosine receptors by 69 or 86%, respectively (from 24.1 +/- 1.2 to 7.4 +/- 0.4 or 3.4 +/- 0.3 fmol/dish), with no significant change in the receptor affinity, activity of CK, or lactate dehydrogenase (LDH), protein content or cell number. The observed up- and down-regulation of A1 adenosine receptors in primary myocyte cultures in response to conditions that exogenously alter the rate of contraction, is indicative of the role of adenosine receptors in adaptation of heart cells to stress.     

Characterization of Ehrlichia risticii binding, internalization, and proliferation in host cells by flow cytometry

The binding, internalization, and proliferation of Ehrlichia risticii in P388D1 cells and equine polymorphonuclear (PMN) leukocytes were studied by immunofluorescent staining and flow cytometric analysis. The binding of ehrlichiae to P388D1 cells at 4 degrees C was dose dependent, and the antigens of bound organisms were susceptible to pronase treatment. Additionally, the binding of ehrlichiae to P388D1 cells was diminished when either P388D1 cells or ehrlichiae were treated with 1% paraformaldehyde for 30 min or 0.25% trypsin for 15 min. These results indicate that the ehrlichial ligand and host cell receptor are likely surface proteins. Following incubation at 37 degrees C, bound E. risticii and/or its antigens were removed with pronase and indirect immunofluorescent staining in the presence of saponin was used to examine intracellular ehrlichiae. Our results indicate that E. risticii was internalized into P388D1 cells within 3 h and proliferated by 48 h of incubation. The microfilament-disrupting agent cytochalasin D and the transglutaminase inhibitor monodansylcadaverine were used to differentiate between phagocytosis (sensitive to cytochalasin) and receptor-mediated endocytosis (sensitive to monodansylcadaverine) of E. risticii by P388D1 cells. In concentrations that produced distinctive morphological changes and inhibited phagocytosis of polystyrene latex beads, cytochalasin D did not suppress the infectivity of E. risticii. Binding, internalization, or proliferation of E. risticii was not affected by cytochalasin D. However, monodansylcadaverine inhibited infection of E. risticii in a dose-dependent manner. The agent did not affect the attachment of ehrlichiae to host cells, but it did suppress internalization and proliferation. These results suggest that E. risticii is internalized by receptor-mediated endocytosis and that productive infection by E. risticii does not depend on phagocytosis by the P388D1 cells. Although E. risticii did not bind to the surface of equine PMN leukocytes at 4 degrees C, organisms were taken up by this cell at 37 degrees C. E. risticii, however, failed to survive in equine PMN leukocytes.     

Agonist-induced internalization of the P2Y2 receptor

The Gq/phospholipase C-linked human P2Y2 receptor was tagged at its amino terminus with the hemagglutinin A (HA) epitope sequence (P2Y2-HA) and stably expressed in 1321N1 human astrocytoma cells. Neither the pharmacological selectivity nor the signaling properties of the receptor were altered by the presence of the epitope. An enzyme-linked immunosorbent assay was developed to quantify cell surface levels of P2Y2-HA receptors using an anti-HA antibody. Incubation of cells with P2Y2 receptor agonists resulted in a concentration of agonist- and time-dependent decrease in cell surface immunoreactivity. Methodology for indirect immunofluorescence confocal microscopy was developed and applied to demonstrate that the agonist-promoted decreases in cell surface immunoreactivity paralleled increases in intracellular immunoreactivity. Agonist-induced internalization of P2Y2 receptors was demonstrated directly by prelabeling P2Y2-HA receptors with antibody before agonist challenge and then quantifying the movement of receptors from a cell surface to intracellular localization in the presence of agonist. Removal of agonist from the medium resulted in recovery of cell surface immunoreactivity to control levels within approximately 1 hr. Incubation of P2Y2-HA receptor-expressing cells with P2Y2 receptor agonists also resulted in receptor-specific desensitization of nucleotide-promoted inositol phosphate accumulation. This loss of responsiveness occurred more rapidly and to a greater extent than did the agonist-promoted loss of surface receptors. Inhibition of receptor internalization by reduction of temperature to 16 degrees had no effect on the capacity of nucleotides to induce P2Y2 receptor-specific desensitization. These results illustrate that the P2Y2 receptor undergoes agonist-promoted movement to an intracellular compartment. This receptor internalization is not required for agonist-induced desensitization.     

Selective blockade of substance P or neurokinin A receptors reduces the expression of c-fos in trigeminal subnucleus caudalis after corneal stimulation in the rat

Stimulation of the cornea activates neurons in two distinct regions of the spinal trigeminal nucleus: at the transition between trigeminal subnucleus interpolaris and subnucleus caudalis and at the transition between trigeminal subnucleus caudalis and the upper cervical spinal cord as estimated by expression of the immediate early gene, c-fos. To determine if receptors for substance P or neurokinin A, neurokinin 1 and neurokinin 2 receptors, respectively, contribute to the production of Fos-positive neurons in these brainstem regions, receptor-selective antagonists were given intracerebroventricularly 15 min prior to stimulation of the cornea in anesthetized rats. The number of Fos-positive neurons produced in superficial laminae at the trigeminal subnucleus caudalis/cervical cord transition by application of the selective small fiber excitant, mustard oil, to the corneal surface was reduced by the neurokinin 1 receptor antagonist, CP99,994 (5-100 nmol, i.c.v.) and the neurokinin 2 receptor antagonist, MEN10,376 (0.01-1.0 nmol, i.c.v.). Combined pretreatment with CP99,994 and the competitive N-methyl-D-aspartate receptor antagonist, CPP, caused a greater reduction in c-fos expression at the subnucleus caudalis/cervical cord transition than after either drug alone suggesting interaction between receptors for glutamate and substance P. Tachykinin receptor antagonists did not reduce the number of Fos-positive neurons produced at the subnucleus interpolaris/subnucleus caudalis transition. The elevation in plasma concentration of adrenocorticotropin, but not the increases in arterial pressure or heart rate, evoked by corneal stimulation was prevented by pretreatment with CP99,994 or MEN10,376 at doses lower than those needed to reduce c-fos expression. The results indicate that receptors for substance P and neurokinin A contribute to the transmission of sensory input from corneal nociceptors to brainstem neurons in trigeminal subnucleus caudalis and to increased activity of the hypothalamo-pituitary axis that accompanies acute stimulation of the cornea.     

Spatial working memory and reference memory of Brown Norway and WAG rats in a holeboard discrimination task

PURPOSE: To investigate the effects of bradykinin (BK) on the outflow facility (C) of human and bovine perfused anterior segments, the [Ca2+]i of cultured bovine trabecular meshwork (BTM) cells, and the area and major axis of BTM cells. METHODS: Cellular studies were performed using first- through third-passage cultures of BTM cells. For [Ca2+]i and shape change assessment, BTM cells were loaded with fura-2 acetoxymethyl ester, and individual fluorescence images were analyzed after the different experimental manipulations. C studies were performed in vitro using human and bovine anterior segments perfused at constant pressure. RESULTS: Bradykinin at 10(-6) M elicited a [Ca2+]i increase of 8 to 10 times the basal levels in 90% of the studied cells. From the responder cells, 60% elicited a 15%+/-1% reduction of the initial cell area, and 37% showed a 13%+/-2% reduction of their major axis. Bradykinin failed to induce any effect in the presence of the BK-B2 receptor antagonist HOE-140. Zero [Ca2+]o the depletion of intracellular stores with thapsigargin, or the presence of the calmodulin antagonist W13, decreased the BK response significantly (P < 0.001; P < 0.001; and P < 0.05). A second application of BK elicited a significantly lower (P < 0.001) response than the previous one. Perfusion with 10(-6) M BK decreased CD, calculated as the area under the curve, by 13%+/-4% (P < 0.05) in human anterior segments and 12%+/-4% (P < 0.05) in bovine anterior segments. The presence of 10(-6) M HOE-140, a BK-B2 receptor antagonist, completely blocked the decrease in C after perfusion with BK. CONCLUSIONS: The C of human and bovine trabecular meshwork (perfused anterior segments) is decreased by BK, acting through BK-B2 receptors. Primary cultured BTM cells respond to BK stimulation by increasing their [Ca2+]i by mobilization of extracellular and intracellular Ca2+. Moreover, these cells are reduced in area and their major axis shortened after the [Ca2+]i peak elicited by BK through BK-B2 receptors. The [Ca2+]i mobilization and shape changes are calmodulin dependent. Taking into account the [Ca2+]i mobilization, the BTM shape changes, the decrease of C, and the temporal sequence of these parameters, a contraction of trabecular meshwork cells related to the functional role of trabecular meshwork is discussed.