Characterization of tachykinin NK2 receptor on dog proximal colon. Antagonism by MEN 10,627 and SR 48,968

The nature of the tachykinin receptors involved in the contraction of the circular muscle of dog colon has been investigated. The following rank order of potency for agonists was obtained: [Sar9,Met(O2)11]substance P > or = neurokinin A > [beta-Ala8]neurokinin A-(4-10) > [MePhe7]neurokinin B. The efficacy of the tachykinin NK2 receptor agonists was significantly greater than that of the tachykinin NK1 receptor agonists and of carbachol. A concentration-dependent rightward shift of the motor response to neurokinin A (obtained in the presence of (+/-)-CP 96,345) was induced by peptide and non-peptide tachykinin NK2 receptor antagonists with this rank order: MEN 10,627 = SR 48,968 > L 659,877 > MEN 10,376 > MDL 28,564. MEN 10,627 and SR 48,968 affinities were similar to those measured in human tissues. In conclusion, the tachykinin NK2 receptor plays a predominant role in tachykinin-induced contraction of the canine colonic circular muscle and this tissue could be useful to predict the pharmacological actions of MEN 10,627 and SR 48,968 in human colon.     

Renal effects of intracerebroventricularly injected tachykinins in the conscious saline-loaded rat: receptor characterization

1. The effects of intracerebroventricularly (i.c.v.) injected substance P (SP), neurokinin A (NKA) and [MePhe7]neurokinin B (NKB) were investigated on renal excretion of water, sodium and potassium in the conscious saline-loaded rat. The central effects of [MePhe7]NKB were characterized with selective tachykinin antagonists for NK1 (RP 67580), NK2 (SR 48968) and NK3 (R 820) receptors. 2. Whereas SP or NKA (65 or 650 pmol) failed to modify the renal responses, [MePhe7]NKB (65-6500 pmol) produced dose-dependent and long-lasting (30-45 min) decreases in renal excretion of water (maximal reduction at 65 pmol: from 66.14 +/- 7.62 to 21.07 +/- 3.79 microliters min-1), sodium (maximal reduction at 65 pmol: from 10.19 +/- 2.0 to 1.75 +/- 0.48 mumol min-1) and potassium (maximal reduction at 65 pmol: from 4.31 +/- 1.38 to 0.71 +/- 0.27 mumol min-1). While 650 pmol [MePhe7]NKB elevated urinary osmolality, neither 65 pmol nor 6.5 nmol [MePhe7]NKB altered this parameter. 3. Both the antidiuresis and antinatriuresis induced by [MePhe7]NKB (65 pmol) were significantly blocked by the prior i.c.v. injection of R 820 (1.3 nmol, 5 min earlier), although the potassium excretion was only partially reduced. However, R 820 did not affect the antidiuresis and antinatriuresis elicited by endothelin-1 (1 pmol, i.c.v.). On its own, R 820 decreased renal potassium excretion with no effect on urinary osmolality and renal excretion of water and sodium. The i.c.v. co-injection of RP 67580 and SR 48968 (6.5 nmol each, 5 min earlier) failed to modify the renal responses to [MePhe7]NKB in a similar study. 4. The central effects of [MePhe7]NKB (65 pmol) on renal excretion were blocked by the prior i.v. administration of a linear peptide vasopressin V2 receptor antagonist (50 micrograms kg-1, 5 min earlier). 5. These results suggest that the central NK3 receptor, probably located in the hypothalamus, is implicated in the renal control of water and electrolyte homeostasis through the release of vasopressin in the conscious saline-loaded rat.     

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.     

Characterization of the tachykinin receptors involved in spinal and supraspinal cardiovascular regulation

The pharmacological characterization of the tachykinin receptors involved in spinal and supraspinal cardiovascular regulation is reviewed in this report. In conscious rats, substance P (SP), neurokinin A (NKA), neurokinin B (NKB), neuropeptide K (NPK), and neuropeptide gamma (NP gamma) were injected either intrathecally (i.t.) or intracerebroventricularly (i.c.v.), and their effects were assessed on mean arterial blood pressure (MAP) and heart rate (HR). Moreover, selective antagonists for NK1 ((+/-)-CP-96045 and RP-67580), NK2 (SR-48968), and NK3 (R-486) receptors were tested against the agonists. I.t. tachykinins elicited dose-dependent increases in MAP and HR (NPK > NP gamma > SP > NKA > NKB). The cardiovascular response to i.t. SP, NPK, and NP gamma was significantly attenuated by the prior i.t. administration of (+/-)-CP-96345 and RP-67580 but not by SR-48968 and R-486. By the i.c.v. route, tachykinins also elicited pressor and tachycardiac responses dose dependently (NPK > NP gamma > SP > NKA > NKB). Senktide and [MePhe7]NKB, two NK3-selective agonists, were slightly more potent than NKB on both parameters. Whereas the cardiovascular response to NPK was largely blocked by (+/-)-CP-96345 and RP-67580, that to SP was reduced by 40-50%. This treatment had no effect on the cardiovascular response to NKA and [MePhe7]NKB. Conversely, SR-48968 reduced by 40-50% the NKA-induced cardiovascular changes without affecting the central mediated effects of NPK, SP, and [MePhe7]NKB. However, when coadministered, RP-67580 and SR-48968 abolished the effects to SP and NKA while leaving untouched those induced by [MePhe7]NKB. Finally, the central effects mediated by [MePhe7]NKB, senktide, and NKB were blocked by R-486. These findings suggest that the i.t. action of tachykinins on the rat cardiovascular system is mediated by a NK1 receptor in the spinal cord, while NK1, NK2, and NK3 receptors are likely involved in the supraspinal (hypothalamus) effects of these neuropeptides. It is also concluded that NPK is a pure and powerful NK1 agonist, in contrast to SP and NKA, which are not selective for NK1 and NK2 receptors, respectively.     

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.     

Effect of early cyclosporine levels on kidney allograft rejection

Experiments were performed on strips of mouse stomach and urinary bladder to characterize the receptors involved in the contractile responses of these tissues to neurokinins (substance P (SP), neurokinin A (NKA), neurokinin B (NKB), and neuropeptide gamma (NP gamma). The neurokinin receptors were characterized by using assays with selective agonists as well as peptide and nonpeptide antagonists and by applying the two Schild criteria for receptor classification, namely, the order of potency of agonists and the apparent affinity of competitive antagonists. The mouse stomach contains primarily NK1 and NK2 functional sites and possibly some NK3 receptors, whereas the urinary bladder possesses only the NK2 receptor. The rank order of potency of agonists in the stomach is Ac[Arg6,Sar9,Met(O2)11]SP-(6-11) > NKA > SP > [beta-Ala8]NKA-(4-10) > NKB > [MePhe7]NKB. Among the selective agonists, Ac[Arg6,Sar9,Met(O2)11]SP-(6-11) is more active than SP and [Sar9,Met(O2)11]SP on the NK1 receptor, whereas the order of potency on the NK2 receptor is NKA > NP gamma > or = [beta-Ala8]NKA-(4-10) > [Nle10]NKA-(4-10). The order of potency of agonists in the bladder is NP gamma > NKA > [beta-Ala8]NKA-(4-10). The myotropic responses mediated by NK1 selective agonists are blocked by SR 140333 (pA2 8.57) and those mediated by the NK2 selective agonists are inhibited by SR 48968 (pA2 9.05). RP 67580 (pA2 8.41) is more active than CP 99994 (pA2 6.06) on the mouse NK1 receptor. The NK1 receptor of the mouse shows, therefore, a pharmacological profile similar to that of the NK1 receptor of the rat. Similarly, MEN 10627 (pA2 9.20) is more active than R 396 (pA2 6.21), suggesting that the mouse NK2 receptor is similar to that of the rabbit. The mouse NK2 receptor of the urinary bladder shows similarity with that of the stomach, but is less sensitive to [beta-Ala8]NKA-(4-10).     

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.     

The fifth transmembrane segment of the neuromedin B receptor is critical for high affinity neuromedin B binding

The two bombesin receptor subtypes, neuromedin B (NMB-R) and gastrin releasing peptide (GRP-R) receptors, bind their respective ligands with high affinity. To identify molecular components mediating high affinity NMB binding, four mutant receptors were constructed, in which different parts of the NMB-R were replaced with the corresponding regions of the GRP-R. When stably expressed in Balb 3T3 fibroblasts, all four NMB-R/GRP-R chimeras were functional and showed NMB-induced stimulation of inositol phosphate (IP) formation. Results of 125I-[D-Tyr0]NMB displacement assays using unlabeled NMB for competition indicated that high affinity NMB binding was determined by amino acid sequences in transmembrane domain V (TM-V) of the NMB-R. To identify which amino acid(s) in TM-V of NMB-R contributed to high affinity NMB binding, four additional NMB-R mutants were constructed where non-conserved amino acids in TM-V of NMB-R were replaced by the corresponding GRP-R amino acids. Three of the mutations, TyrPheLeu220-222-->PheTyrVal, Ile230-->Val, and His234-->Phe, did not affect high affinity NMB binding. The Ile216-->Ser substitution, however, abolished high affinity NMB binding and severely impaired the ability of the mutant receptor to stimulate NMB-dependent inositol phosphate formation. These results suggest that ILe216 in TM-V of NMB-R may be critical for high affinity NMB binding.     

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.     

Heterogeneity of tachykinin receptors in the rabbit lung

The tachykinins, substance P (SP) and neurokinin A (NKA), are agonists for the NK(1) and NK(2) receptors, respectively. Tachykinins have various respiratory effects, including bronchoconstriction. This study characterizes tachykinin binding sites in the rabbit lung. We hypothesize that (2-[(125)I]iodohistidyl(1))Neurokinin A ([(125)I]NKA) interacts with NK1 and NK2 binding sites in the rabbit lung. The K d determined from saturation isotherms was 0.69 times/divided by 1.14 nM (geometric mean times/divided by SEM) and the B max was 4.15 + or - 0.22 femtomole/mg protein (arithmetic mean + or - SEM). Competitive inhibition studies with NKA, SP and various selective tachykinin agonists showed the rank order of potency; [beta-Ala(8)]-Neurokinin A 4-10 = SP > NKA > [Sar(9),Met(02)11]-Substance P. [beta-Ala(8)]-Neurokinin A 4-10, a selective NK(2) agonist, and SP inhibition of [(125)I]NKA binding were best described using a two-site model. Competitive inhibition studies using the selective nonpeptide NK(2) antagonist (SR 48968) and the selective nonpeptide NK(1) antagonist (CP 96,345) revealed Ki's of 5.5 nM and 8.1 nM, respectively. Our data therefore suggest that [(125)I]NKA binds to both the NK(1) and NK(2) receptors in the lung.     

The unpredicted high affinities of a large number of naturally occurring tachykinins for chimeric NK1/NK3 receptors suggest a role for an inhibitory domain in determining receptor specificity

Three chimeric receptors were constructed by exchanging exon sequences between human NK1 and NK3 receptor genes. The resulting chimeric receptors not only retained high affinities for their natural ligands substance P and neurokinin B but also exhibited surprisingly high affinities for other naturally occurring tachykinins including neurokinin A, neuropeptide K, neuropeptide gamma, eledoisin, kassinin, physalaemin, and phyllomedusin. In contrast, these chimeric receptors displayed a wide range of variability in their affinities for non-naturally occurring ligands including selective agonists and antagonists of NK1, NK2, and NK3 receptors. Since the only common feature among these naturally occurring neurokinin peptides is the conserved C-terminal sequences, our data suggest that these conserved sequences must play the major role in conferring high affinity binding to the chimeric receptors. To explain the apparently "improved" affinities of these naturally occurring ligands for the chimeric receptors as compared with their affinities for the parent NK1 and NK3 receptors, we are proposing that certain inhibitory domains that are present in the NK1 and/or NK3 receptors are compromised in these chimeric receptors. Upon disruption of these inhibitory domains during the formation of chimeras, the naturally occurring ligands can interact more favorably with chimeric receptors through their conserved C-terminal sequences. Based on this hypothesis, the binding affinities of natural tachykinin ligands may be largely determined by their conserved C-terminal sequences, whereas receptor selectivities of these ligands are influenced more by the presence or absence of inhibitory domains rather than specific binding domains on their target receptors.     

Substance P-induced inward current in identified auditory efferent neurons in rat brain stem slices

The effects of substance P (SP) on whole cell currents were studied in neurons of the medial olivocochlear efferent system (MOCS) in the ventral nucleus of the trapezoid body (VNTB) of brain stem slices from neonatal rats. Each neuron was identified by retrograde labeling with Fast Blue injected into the cochlea. Bath application of SP (0.1-10 microM) reversibly induced an apparent inward current in 49 of 63 labeled neurons when voltage clamped at near resting voltages. This apparent inward current was consistent with the SP-induced membrane depolarization observed in current-clamp mode. The SP-induced change in current was dose dependent with a half-maximal response dose of 200 nM. It was mimicked by [Cys3,6, Tyr8, Pro9]-SP, a neurokinin (NK1) receptor selective agonist, whereas [Succinyl-Asp6, MePhe8]-SP 6-11 (Senktide), a NK3 receptor agonist, had no detectable effect. The SP effect was not blocked by 10(-6) M tetrodotoxin (TTX) and persisted when the perfusate contained 30 mM tetraethylammonium (TEA) or 100 microM Cd2+ or was in a 0-Ca solution. In a TTX-containing solution, SP caused a voltage-dependent decrease of membrane conductance, and the SP-evoked current reversed at a potential at around -105 mV. The predicted K+ equilibrium potential was -93.8 mV under the experimental conditions. The SP-induced inward current was attenuated by 66% when the perfusate contained 3 mM Cs+. We conclude that the apparent inward current is partly caused by SP decreasing an outward current normally maintained by the inward rectifier K+ channels in these cells. In the presence of Cs solution in the recording pipette and with a perfusate containing 3 mM Cs+, 0.1 mM Cd2+ and 10(-6) M TTX, a residual SP-induced inward current was observed at test voltages ranging from -120 to 40 mV. This subcomponent reversed its polarity at approximately 20 mV. This inward current was reduced substantially (but not abolished) when all NaCl in the external solution was replaced by TEA-Cl. The results indicate that SP also opens an unknown cation channel, which the available data suggests may be relatively nonselective. The results suggest that MOCS neurons are subject to modulation by SP, which depolarizes the cell membrane by decreasing the activity of inward rectifier K+ channels as well as concurrently activating a separate cation conductance. It also was found that in MOCS neurons responsive to both SP and norepinephrine, the norepinephrine effect was abolished by TTX, suggesting that an interneuronal population excited by norepinephrine converges selectively onto SP-sensitive MOCS neurons in the VNTB.     

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.     

Multivalent cations depress ligand affinity of insulin-like growth factor-binding proteins-3 and -5 on human GM-10 fibroblast cell surfaces

The effect of multivalent cations on [125I]-IGF binding to cell-associated IGFBPs was investigated using human fibroblasts. The major cell-associated binding site for [125I]-IGF-I is IGFBP-3 and for [125I]-IGF-II are IGFBP-3 and IGFBP-5. Lanthanum and chromium did not affect either [125I]-IGF-I or [125I]-IGF-II binding to cell-associated IGFBPs. By contrast, zinc (Zn2+), gold (Au3+), and cadmium (Cd2+) depressed binding of both ligands. Ligand binding resulted in nonlinear Scatchard plots. Assuming a pre-existent asymmetric model with high- (K[aHi]) and low- (K[aLo]) affinity sites, Zn2+ lowered both K(aHi) and K(aLo). Au3+ eliminated K(aHi). Assuming that the nonlinear plots were caused by ligand-induced negative cooperativity, Zn2+ and Cd2+ lowered both Ke and Kf (affinity of unoccupied and saturated IGFBPs, respectively). Au3+ eliminated Ke and reduced Kf. Zn2+ was active at serum levels in lowering IGF binding. Zinc, gold, and cadmium bind to similar regions within proteins (a zinc-binding motif) indicating similar mechanisms of action. A zinc-binding motif is present in the IGFBPs, but not in the IGFs. We demonstrate for the first time that the trace nutrient zinc and related multivalent cations decrease IGF binding to fibroblast-associated IGFBPs by lowering the affinity of the IGF-IGFBP interaction.     

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.     

In vitro and in vivo pharmacology of S 16474, a novel dual tachykinin NK1 and NK2 receptor antagonist

Since tachykinins released from lung sensory nerve endings are thought to play a role in inflammatory diseases of airways via NK1 and NK2 receptors, dual tachykinin NK1 and NK2 receptor antagonists may have a great therapeutic potential. In vitro, the cyclopeptide S 16474 (cyclo-[Abo-Asp(D-Trp(Suc0Na)-Phe-N-(Me)Bzl)]) bound to both human tachykinin NK1 and NK2 receptors expressed in two lines of transfected Chinese hamster ovary cells (IC50 values 85 nM and 129 nM, respectively), while showing a poor affinity for the rat tachykinin NK1 receptor. S 16474 inhibited the contractions induced by substance P in isolated rabbit vena cava (pA2 7.0) and by neurokinin A in rabbit pulmonary artery (pA2 5.6). In vivo in anaesthetized guinea-pigs, S 16474 was found to dose dependently inhibit the bronchoconstrictions induced by intravenously administered substance P, neurokinin A and capsaicin. Plasma extravasation evoked in bronchi by endogenously released tachykinins under vagus nerve stimulation was abolished by S 16474 (10 mu mol/kg i.v.). These results demonstrate clearly that S 16474 is a tachykinin receptor antagonist exhibiting, in vitro and in vivo, a dual inhibitory effect on NK1 and NK2 receptors.     

Out-of-hospital quantitative monitoring of end-tidal carbon dioxide pressure during CPR

BACKGROUND: Human growth hormone (hGH) binds to both the hGH and human prolactin (hPRL) receptors. Binding to the hPRL receptor, however, is approximately 50-fold tighter and requires a single Zn2+ cation, unlike binding of hGH to the hGH receptor. Previous mutational studies have identified putative ligands from hGH and the hPRL receptor responsible for coordinating the interfacial Zn2+. RESULTS: One of these ligands was introduced at a structurally analogous site in the extracellular domain of the hGH receptor by mutating Asn218 to His, and the resulting mutant protein showed a 20-fold increase in hGH binding in the presence of ZnCl2. Alanine-scanning mutagenesis showed that the binding site on hGH for the Asn218-->His hGH receptor in the presence of Zn2+ resembled that for the hPRL receptor. CONCLUSIONS: It is possible to introduce the metal-binding site from the hPRL receptor into the homologous hGH receptor. More generally, these studies indicate that affinity between two proteins may be enhanced by design of an interfacial metal-binding site.     

The tachykinin NK1 receptor antagonist, RP67580, inhibits the bradykinin-induced rise in intracellular Ca2+ concentration in bovine pulmonary artery endothelial cells

The bradykinin-induced rise in intracellular Ca2+ concentration ([Ca2+]i) and the bradykinin receptor involved in this response were characterized in bovine pulmonary artery endothelial cells. It was found that bradykinin induces an intracellular biphasic Ca2+ response, consisting of a transient peak followed by an elevated plateau phase. Both bradykinin and the bradykinin B1 receptor agonist, des-Arg9-bradykinin, induced a concentration-dependent increase in [Ca2+]i, but the bradykinin-induced rise was much greater. Moreover, the bradykinin-induced [Ca2+]i rise could be inhibited by the bradykinin B2 receptor antagonists, D-Arg0[Hyp3, Thi(5,8), D-Phe7]bradykinin and Hoe 140 (D-Arg[Hyp3, Thi5, D-Tic7, Oic8]bradykinin), but not by the bradykinin B1 receptor antagonist, des-Arg9-[Leu8]bradykinin. From these results it can be concluded that a bradykinin B2 receptor is involved in this response. Furthermore, we found that the tachykinin NK1 receptor antagonist, RP67580 ([imino 1 (methoxy-2-phenyl)-2 ethyl]-2 diphenyl 7,7 perhydroisoindolone-4 (3aR, 7aR)), and its negative enantiomer, RP68651 (2-[1-imino 2-(2 methoxy phenyl) ethyl] 7,7 diphenyl 4-perhydroisoindolone (3aS-7aS)), could inhibit the bradykinin-induced [Ca2+]i response, although no functional tachykinin NK1 receptors were found. Binding studies evidenced no binding of RP67580 or RP68651 to the bradykinin receptor. We conclude that RP67580 inhibits the bradykinin-induced rise in [Ca2+]i via a bradykinin B2 receptor-independent mechanism.     

Expression of rat NK-2 (neurokinin A) receptor in E. coli

With the goal of obtaining sufficient functional protein for structural analysis, rat neurokinin-2 receptor was produced in Escherichia coli by linking it to the periplasmic maltose-binding protein. As a first step, we present a biochemical and pharmacological investigation of the recombinant receptor. Western-blots showed that the fusion protein was associated with the membranes. The agonist [4,5-3H-Leu9]neurokinin A and the NK-2 antagonist [3H]SR48,968 bound to the receptor in a highly specific manner. Saturation binding of the [3H]agonist demonstrated a single class of receptors (KD = 10.5 nM, Bmax = 2.5 pmol/mg protein). The [3H]antagonist bound with higher affinity to a larger receptor population (KD = 0.2 nM, Bmax = 7.2 pmol/mg protein). Competition of [3H]agonist binding with other agonists demonstrated a potency order of: neurokinin A > [Nle10]NKA(4-10) = [beta-Ala8]NKA(4-10) > substance P > senktide Against the [3H]antagonist, agonists were only partially inhibitory. Selective NK-2 antagonists inhibited binding of both [3H]ligands with an identical order of potency: SR48,968 > R396 > MEN10,376, which is consistent with NK-2 receptor pharmacology in rat tissue.     

Tachykinins: receptor to effector

Tachykinins belong to an evolutionarily conserved family of peptide neurotransmitters. The mammalian tachykinins include substance P, neurokinin A and neurokinin B, which exert their effects by binding to specific receptors. These tachykinin receptors are divided into three types, designated NK1, NK2 and NK3, respectively. Tachykinin receptors have been cloned and contain seven segments spanning the cell membrane, indicating their inclusion in the G-protein-linked receptor family. The continued development of selective agonists and antagonists for each receptor has helped elucidate roles for these mediators, ranging from effects in the central nervous system to the perpetuation of the inflammatory response in the periphery. Various selective ligands have shown both inter- and intraspecies differences in binding potencies, indicating distinct binding sites in the tachykinin receptor. The interaction of tachykinin with its receptor activates Gq, which in turn activates phospholipase C to break down phosphatidyl inositol bisphosphate into inositol trisphosphate (IP3) and diacylglycerol (DAG). IP3 acts on specific receptors in the sarcoplasmic reticulum to release intracellular stores of Ca2+, while DAG acts via protein kinase C to open L-type calcium channels in the plasma membrane. The rise in intracellular [Ca2+] induces the tissue response. With an array of actions as diverse as that seen with tachykinins, there is scope for numerous therapeutic possibilities. With the development of potent, selective non-peptide antagonists, there could be potential benefits in the treatment of a variety of clinical conditions, including chronic pain, Parkinson's disease, Alzheimer's disease, depression, rheumatoid arthritis, irritable bowel syndrome and asthma.