Neurokinin A-like Immunoreactivity Research Articles

Neurokinin A and neurokinin B in the human retina

Very recently, the authors found levels of neurokinin (NK) A-like immunoreactivities in the human retina which were more than five times higher than those of substance P (SP). The present study aimed to find out how many of these immunoreactivities can be attributed to NKA and NKB and then the exact distribution pattern of both NKA and NKB was evaluated in the human retina and compared with that of SP. For this purpose, NKA-like immunoreactivities were characterized in the human retina by reversed phase HPLC followed by radioimmunoassay using the K12 antibody which recognizes both NKA and NKB. Furthermore, the retinae from both a 22- and 70-year-old donor were processed for double-immunofluorescence NKA/SP and NKB/SP. The results showed that NKA contributes to approximately two thirds and NKB to approximately one third of the immunoreactivities measured with the K12 antibody. NKA was found to be localized in sparse amacrine cells in the proximal inner nuclear layer, in displaced amacrine cells in the ganglion cell layer with processes ramifying in stratum 3 of the inner plexiform layer and also in sparse ganglion cells. By contrast, staining for NKB was only observed in ganglion cells and in the nerve fiber layer. Double-immunofluorescence revealed cellular colocalization of NKA with SP and also of NKB with SP. Thus, the levels of NKA and NKB are more than three and two times higher than those of SP, respectively. Whereas the distribution pattern of NKA is typical for neuropeptides, the localization of NKB exclusively in ganglion cells is atypical and unique.

Tachykinins (Substance P and Neuropeptide γ) from the Brains of the Pallid Sturgeon, Scaphirhynchus albus and the Paddlefish, Polyodon spathula (Acipenseriformes)

A peptide with substance P-like immunoreactivity was isolated from extracts of the brains of the pallid sturgeon, Scaphirhynchus albus and the North American paddlefish, Polyodon spathula. The primary structure of the peptide (Lys-Pro-Lys-Pro-His-Gln-Phe-Phe-Gly-Leu-Met.NH2) is the same in both species and contains 2 amino acid substitutions (Arg1→Lys and Gln5→His) compared with human substance P and 1 substitution (Arg3→Lys) compared with substance P from the trout (Teleostei). Scyliorhinin I, a tachykinin previously isolated from an extract of sturgeon intestine, was not detected in either brain extract. A peptide with neurokinin A-like immunoreactivity (Ser-Ser-Ala-Asn-Arg-Gln-Ile-Thr-Gly-Lys10Arg-Gln-Lys-Ile-Asn-Ser-Phe-Val-Gly-Leu20Met.NH2) was isolated from sturgeon brain and contains 10 amino acid substitutions compared with human neuropeptide γ (a specific product of the posttranslational processing of γ-preprotachykinin A) but only 4 substitutions compared with trout neuropeptide γ. It was not possible to obtain the paddlefish neurokinin A-related peptide in pure form. The structural similarity between the sturgeon and the trout tachykinins supports the hypothesis that the Acipenseriformes (sturgeons and paddlefish) represent the sister group of the Neopterygii (gars, bowfin, and teleosts).

Neurokinin A-LI release after antigen challenge in guinea-pig bronchial tubes: influence of histamine and bradykinin.

1. Our aim was to determine if antigen challenge stimulates sensory nerves and provokes the release of tachykinins. The involvement of histamine and bradykinin was studied by using specific receptor antagonists. Capsaicin-induced responses were also examined. Experiments were performed in vitro on tracheal and bronchial preparations from ovalbumin-sensitized guinea-pigs. 2. Characterization of ovalbumin-induced contraction, with regard to histamine and bradykinin, was carried out on airway ring preparations in the presence of phosphoramidon. The histamine H1 receptor antagonist pyrilamine reduced allergen-induced bronchial contractions by about 30%, whereas the bradykinin B2 receptor antagonist icatibant (Hoe 140) did not significantly affect the response. Combined treatment with pyrilamine (1 microM) and icatibant (0.1 microM) reduced the contractions by about 80%, indicating a synergistic inhibitory action. Tracheal preparations were not significantly affected by treatments, neither were capsaicin-induced contractions. 3. To study the outflow of tachykinins, we used a perfused bronchial-tube preparation, allowing simultaneous measurement of smooth muscle tension and mediator release. Neurokinin A-like immunoreactivity (NKA-LI) and substance P-like immunoreactivity (SP-LI) were determined by radioimmunoassay. 4. The results of the perfusion study showed an increased outflow of NKA-LI into the perfusate in response to ovalbumin (127% of basal) challenge. SP-LI determined in some of the samples showed a much lower amount (40 to 70 times lower) of SP-LI than NKA-LI. Treatment with icatibant and pyrilamine, separately and in combination, significantly reduced the ovalbumin-induced NKA-LI outflow by 38%, 26% and 22%, respectively. 5. Capsaicin-induced outflow (124% of basal) was not significantly affected by treatments (icatibant 121%, pyrilamine 107% and combined treatment 111% of basal). However, when pyrilamine was present the increased outflow was not statistically significant. 6. In conclusion, we found that allergen provocation of guinea-pig bronchi caused an increased outflow of NKA-LI that was reduced by treatment with both pyrilamine and icatibant. These findings demonstrate that the allergen-induced release of histamine and bradykinin stimulate sensory nerves and thereby increase outflow of tachykinins that contribute to the allergic reaction.

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 immunoreactivity, 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.

Tachykinins (Substance P, Neurokinin A and Neuropeptide γ) and Neurotensin from the Intestine of the Burmese Python, Python molurus

Conlon, J. M., T. E. Adrian and S. M. Secor. Tachykinins (substance P, neurokinin A and neuropeptide γ,) and neurotensin from the intestine of the burmese python, Python molurus. Peptides 18(10) 1505–1510, 1997.—Peptides with substance P-like immunoreactivity, neurokinin A-like immunoreactivity and neurotensin-like immunoreactivity were isolated in pure form from an extract of the intestine of the Burmese python ( Python molurus). The primary structure of python substance P (Arg-Pro-Arg-Pro-Gln-Gln-Phe-Tyr-Gly-Leu-Met-NH 2) shows one amino acid substitution (Phe 8 → Tyr) compared with chicken/alligator substance P and an additional substitution (Lys 3 → Arg) as compared with mammalian substance P. The neurokinin A-like immunoreactivity was separated into two components. Python neuropeptide γ (Asp-Ala-Gly-Tyr-Ser-Pro-Leu-Ser-His-Lys-Arg-His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH 2 shows three substitutions (Gly 5 → Ser, Gln 6 → Pro and Ile 7 → Leu) compared with alligator neuropeptide γ and an additional substitution (His 4 → Tyr) compared with mammalian neuropeptide γ. Python neurokinin A (His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met.NH 2) is identical to human/chicken/alligator neurokinin A. Python neurotensin (pGlu-Leu-Val-His-Asn-Lys-Ala-Arg-Pro-Tyr-Ile-Leu) is identical to chicken/alligator neurotensin. The data are indicative of differential evolutionary pressure to conserve the amino acid sequences of reptilian gastrointestinal peptides.

The cellular localization of the neuropeptides substance P, neurokinin A, calcitonin gene-related peotide and neuropeptide Y in guinea-pig vestibular sensory organs: a high-resolution confocal microscopy study

Four neuropeptides, substance P, neurokinin A, calcitonin gene-related peptide and neuropeptide Y, were detected by radioimmunoassay in guinea-pig vestibular end-organs. High-resolution confocal microscopy visualization of immunofluorescence staining was used to determine the cellular localization of these peptides. Substance P- and neurokinin A-like immunoreactivities were found to co-exist in afferent fibers innervating the peripheral regions of both the utricular and ampullar sensory organs. The immunoreactivity was more concentrated in the distal ends of the calyceal-shaped nerve endings that innervate type I sensory cells. While in the guinea-pig, nerve calyces and type I cells are distributed in both the central and peripheral regions of the sensory epithelia, immunoreactive calyces were found only in the peripheral regions. Calcitonin gene-related peptide-like immunoreactivity was localized in small bouton endings situated at the level of the base of the hair cells. These boutons were in a position to make axosomatic contacts with type II sensory cells and axodendritic contacts with afferent nerve endings. Calcitonin gene-related peptide immunoreactivity co-existed with choline acetyltransferase immunoreactivity. The localization and shape of these boutons identified them as the axonal endings of efferent vestibular fibers. Neuropeptide Y-like immunoreactivity was not observed in the actual sensory epithelium but in the underlying connective tissue, where it was located in varicose fibers along blood vessels.The synaptic position of the tachykinins is clearly distinct from that of calcitonin gene-related peptide. This segregation distinguishes the vestibular end-organs from most peripheral tissues where these peptides are co-localized. The tachykinin-immunoreactive afferent fibers are postsynaptic to the hair cells. If, as in somatic sensory endings, these fibers can be triggered to release the neuropeptides by an axon reflex type of activation, then the tachykinins could interfere directly with the function of type I and type II vestibular hair cells. Calcitonin gene-related peptide co-exists with acetylcholine in the efferent axonal endings that are presynaptic to type II hair cells and to afferent fibers. Calcitonin gene-related peptide can thus interfere by direct synaptic action with type II hair cells only. It may also regulate the activity of the tachykinin-containing afferents.

Effects of Repeated Sensory Stimulation (Electro‐acupuncture) and Physical Exercise (Running) on Open‐field Behaviour and Concentrations of Neuropeptides in the Hippocampus in WKY and SHR rats

The effects of repeated sensory stimulation (electro-acupuncture) and physical exercise (running) on open-field behaviour and on hippocampal concentrations of neuropeptide Y, neurokinin A, substance P, galanin and vasoactive intestinal peptide (VIP)-like immunoreactivities were studied in WKY (wistar-Kyoto) and SHR (spontaneously hypertensive) rats. Significantly higher concentrations of substance P-like immunoreactivity, neurokinin A-like immunoreactivity and neuropeptide Y-like immunoreactivity were found in the hippocampus immediately after 3 weeks of treatment (electro-acupuncture and running), but not 1 week after the last (tenth) changes in neuropeptide concentrations were similar in the two rat strains. Open-field behaviour was significantly reduced during the treatment period in both strains. There were significant negative correlations between behaviour and neuropeptide concentrations in SHR rats, suggesting interdependency with sympathetic activity. It is proposed that the effects of electro-acupuncture and physical exercise in rats are related to increases in neuropeptide Y, neurokinin A and substance P in the hippocampus.

Isolation, localization, and cardiovascular activity of tachykinins from the stomach of the bowfin Amia calva

The bowfin is an extant representative of an ancient group of ray-finned fish with evolutionary connections to modern teleosts. A peptide with substance P-like immunoreactivity was isolated from an extract of bowfin stomach and its primary structure was established as Ser-Lys-Ser-His-Gln-Phe-Tyr-Gly-Leu-Met-NH2. This amino acid sequence resembles mammalian substance P only in the COOH-terminal region of the peptide. A second tachykinin with neurokinin A-like immunoreactivity isolated from the extract comprises 23 amino acid residues and shows limited structural similarity to mammalian neuropeptide-gamma. A randomly distributed population of cells in the gastric glands of the bowfin were immunostained with an antiserum raised against substance P, but no immunopositive structures were identified in the surface epithelium, lamina propria, or the nerve plexuses of the submucosa. Bolus injections of synthetic bowfin substance P (0.1-10 nmol/kg) into the bulbus arteriosus of unanesthetized bowfin resulted in a significant and dose-dependent rise in vascular resistance and arterial blood pressure (P < 0.01) and a fall in cardiac output (P < 0.05) without change in heart rate. After 5-10 min, arterial pressure and vascular resistance returned to preinjection levels, but cardiac output significantly (P < 0.05) increased over baseline values. The response to the peptide was unaffected by pretreatment of the animals with phentolamine. The study has shown that the stomach of the bowfin synthesizes tachykinins with novel structural features that display cardiovascular activity in this species.

Cigarette smoke-induced bronchoconstriction and release of tachykinins in guinea pig lungs

Two series of experiments were carried out to determine whether the release of tachykinins is involved in the bronchoconstriction induced by inhalation of cigarette smoke in guinea pigs. In the first series, cigarette smoke consistently induced bronchoconstriction (ΔR l = + 203% and ΔCdyn = −46%) in anesthetized guinea pigs, and the response was only partially blocked by bilateral cervical vagotomy. However, the smoke-induced bronchial constriction was completely abolished in animals receiving a systemic capsaicin pretreatment to destroy the tachykinin-containing C-fiber afferents. In the second series, the bronchoconstrictive effect of cigarette smoke was increased by approx. three times in isolated perfused guinea pig lungs when phosphoramidon (3 × 10 −6 M) was added to the perfusate to prevent the degradation of tachykinins after their release. Moreover, the enhanced bronchomotor response to smoke was accompanied by an overflow of neurokinin A-like immunoreactivity (LI) and calcitonin gene-related peptide -LI in the pulmonary effluent. These studies showed that cigarette smoke triggers the release of tachykinins in the lungs, which plays an important role in the smoke-induced bronchoconstrictive effect in guinea pigs.

Facilitatory effects of testosterone on hypothalamic tachykinin levels and release

We have reported earlier that central administration of tachykinins profoundly altered the release of pituitary LH and the direction of the LH response was modulated by gonadal steroids in male rats. To further understand the relationship between androgens and hypothalamic tachykinins, we have examined the effects of castration and of testosterone (T)-replacement on levels of Neurokinin A-like immunoreactivity (NKA-li) in microdissected regions of the rat hypothalamus and on in vitro release of hypothalamic NKA-li. Results showed that castration decreased NKA-li levels specifically in the median eminence (ME) and arcuate (ARC) regions as compared to those in intact rats. T replacement immediately following castration prevented these site-specific decreases in NKA-li. Similarly, basal efflux of NKA-li was significantly reduced from the entire medial basal hypothalamus (including the ME and ARC) and preoptic area (POA) of castrated rats vs. that from the hypothalami of intact and T-replaced castrated rats. These results show that T can modulate levels of NKA-li selectively in sites previously implicated in the control of LH release and since NKA can inhibit LH and hypothalamic LH-releasing hormone (LHRH) release, these observations are in accord with our view that afferents to LHRH neurons are the targets of gonadal steroid feedback on pituitary LH release.

Neurokinin a like immunoreactivity (NKA-LI) is in primary afferent neurons supplying the renal pelvis in the rat

Neurokinin a like immunoreactivity (NKA-LI) is in primary afferent neurons supplying the renal pelvis in the rat

Novel tachykinins from the brain of the sea lamprey, Petromyzon marinus, and the skate, Raja rhina

Using radioimmunoassays for mammalian tachykinins, peptides with substance P-like immunoreactivity and neurokinin A-like immunoreactivity were identified in an extract of the brain of the longnose skate, Raja rhina (elasmobranch) but only a peptide with neurokinin A-like immunoreactivity was identified in the brain of the sea lamprey, Petromyzon marinus (agnathan). The primary structure of the skate peptide with substance P-like immunoreactivity (Ala-Lys-His-Asp-Lys-Phe-Tyr-Gly-Leu-Met-NH 2) shows one amino acid substitution (Phe 3 → His) compared with scyliorhinin I, previously isolated from dogfish brain and gut. The skate neurokinin A-related peptide (His-Lys-Leu-Gly-Ser-Phe-Val-Gly-Leu-Met-NH 2) shows two substitutions (Thr 3 → Leu and Asp 4 → Gly) compared with mammalian neurokinin A. Although the COOH-terminus of the lamprey tachykinin (Arg-Lys-Pro-His-Pro-Lys-Glu-Phe-Val-Gly-Leu-Met-NH 2) resembles neurokinin A, the presence of the strongly conserved Lys/Arg-Pro-Xaa-Pro motif at the NH 2-terminus of the peptide indicates greater structural similarity with substance P. The additional arginine residue at position 1 in the peptide suggests that the lamprey is utilizing a site of posttranslational processing in the tachykinin precursor that is different from the equivalent site in mammalian and other lower vertebrate preprotachykinin(s).

Fluorescence immunohistochemical localization of neurokinin A-like and neurokinin B-like immunoreactivity in human gut, pancreas, gallbladder and liver

Fluorescence immunohistochemical localization of neurokinin A-like and neurokinin B-like immunoreactivity in human gut, pancreas, gallbladder and liver

Neuropeptide γ‐(l‐9)‐Peptide: A Major Product of the Posttranslational Processing of γ‐Preprotachykinin in Rat Tissues

gamma-Preprotachykinin mRNA is the most abundant tachykinin mRNA in rat tissues, but the pathway of post-translational processing of its translation product is unknown. An antiserum was raised against the synthetic peptide Asp-Ala-Gly-His-Gly-Gln-Ile-Ser-His [neuropeptide gamma-(1-9)-peptide, equivalent to gamma-preprotachykinin-(72-80)-peptide], that showed < 1% reactivity with intact neuropeptide gamma and other tachykinins. Neuropeptide gamma-(1-9)-peptide was detected by radioimmunoassay in relatively high concentrations in extracts of regions of rat brain and gastrointestinal tract. These concentrations correlated with (r = 0.99), but were significantly (p < 0.05) less than, the concentrations of neurokinin A-like immunoreactivity. The neuropeptide gamma-(1-9)-like immunoreactivity in an extract of rat brain was eluted from a reverse-phase HPLC column in a single fraction with the same retention time as synthetic neuropeptide gamma-(1-9)-peptide. The synthetic peptide did not contract or relax isolated rat trachea, superior mesenteric artery, stomach fundus, or ileum, and the peptide did not affect the ability of neuropeptide gamma to contract the rat fundus. It is concluded that, in rat tissues, Lys70-Arg71 in gamma-preprotachykinin is a major site of posttranslational processing, but the resulting product, neuropeptide gamma-(1-9)-peptide, is neither an agonist nor an antagonist at the neurokinin-2 (NK-2) receptor.

Identification of multiple tachykinins in bovine adrenal medulla using an improved chromatographic procedure

Comparison of data based on the reverse-phase HPLC with two ion-pairing reagents, trifluoroacetic acid (TFA) and heptafluorobutyric acid (HFBA), together with the use of two antibodies, has allowed us to identify the various tachykinins in the bovine adrenal medulla. The results show that substance P-like, neurokinin B-like, and neurokinin A-like (including its extended forms, neuropeptide K and neuropeptide γ) immunoreactivity are present in the bovine adrenal medulla. The concentration of SP-like immunoreactivity in the adrenal medulla was found to be substantially higher than that of NKA-like and NKB-like immunoreactivity. The strategy described here, using radioimmunoassay combined with HPLC employing TFA and HFBA as the ion-pairing reagents, should be useful for the identification of tachykinins and other peptides in the central and peripheral nervous system.

Structural characterization of tachykinins (neuropeptide γ, neurokinin A, and substance P) from a reptile, Alligator mississipiensis

An extract of the whole brain of the alligator ( Alligator mississipiensis) contained very high concentrations of substance P-like immunoreactivity (405 pmol/g wet tissue) and neurokinin A-like immunoreactivity (514 pmol/g), as measured with antisera raised against the mammalian peptides. The primary structure of alligator substance P was established as: Arg-Pro-Arg-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH... This sequence is the same as that of chicken substance P and shows one substitution (Arg for Lys 3) as compared with mammalian substance P. The neurokinin A-like immunoreactivity was separated into two components. Neuropeptide y was the most abundant peptide and its primary structure was established as Asp-Ala-Gly-Tyr-Gly-Gln-Ile-Ser-His-Lys-Arg-His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH 2. This sequences shows one substitution (Tyr for His 4) compared with mammalian neuropeptide y. The second component was identical to mammalian neurokinin A. A peptide with the chromatographic properties of mammalian neuropeptide K was not identified in the extract.

Neurokinin A-like immunoreactivity in articular afferents of the cat

Dorsal root ganglion cells with axons innervating the cat's knee joint via the medial articular nerve were retrogradely labelled with Fast blue. Neurokinin A-like immunoreactivity was found in 4.5 ± 1.1% (mean ± of 5 nerves and 695 cells) of the articular afferents. Colchicine treatment of the ganglia increased the percentage of immunopositive cells to 8.5 ± 0.7% (mean ± S.D. of 6 nerves and 554 cells) after 3–22 h. The diameter distribution of the immunopositive somata ranged from 20 to 50 μm with a maximum at 26–30 μm. Comparing the proportions of neurokinin A-immunopositive cells with those of substance P, it can be calculated on the basis of mRNA encoding that neurokinin A is synthetized in about half of the substance P-containing primary articular afferents.

Substance-P-related and neurokinin-A-related peptides from the brain of the cod and trout.

An extract of the brain of the rainbow trout, Oncorhynchus mykiss contained high concentrations of both neurokinin A-like immunoreactivity (corresponding to 90 pmol mammalian neurokinin A/g wet tissue) and substance-P-like immunoreactivity (corresponding to 50 pmol mammalian substance P/g wet tissue) measured by radioimmunoassay using antisera directed against the C-terminal regions of the mammalian peptides. In contrast, an extract of the Atlantic cod. Gadus morhua contained only neurokinin-A-like immunoreactivity (151 pmol/g). This apparent paradox was resolved by determination of the primary structures of the fish tachykinins. Trout substance P (Lys-Pro-Arg-Pro-His-Gln-Phe-Phe-Gly-Leu-MetNH2) has the same amino acid sequence in its C-terminal region as that in the corresponding region of mammalian substance P. Cod substance P (Lys-Pro-Arg-Pro-Gln-Gln-Phe-Ile-Gly-Leu-MetNH2), however, contains a substitution at position 8 (Phe----Ile) that abolishes reactivity with the antiserum to substance P but permits reactivity with the antiserum to neurokinin A. The amino acid sequence of cod and trout neurokinin A is the same (His-Lys-Ile-Asn-Ser-Phe-Val-Gly-Leu-MetNH2) and shows two substitutions (Thr3----Ile and Asp4----Asn) compared with mammalian neurokinin A. The data indicate that nervous tissue of teleost fish contain tachykinins that are analogous to the peptides found in mammalian tissues.

Adenosine A1 receptors mediate inhibition of tachykinin release from perifused enteric nerve endings.

A perifused preparation of guinea pig myenteric nerve varicosities (synaptosomes) was used to determine the characteristics of evoked tachykinin release and the inhibition of such release by adenosine analogues. Release of substance P-like immunoreactivity (SP-LI) and neurokinin A-like immunoreactivity (NKA-LI) was evoked by elevated extracellular [K+] in a reversible and repeatable manner. This release was completely abolished in the absence of extracellular Ca2+. Perifusion in the presence of 5'-N-ethylcarboxamidoadenosine (NECA), a nonselective A1/A2 adenosine receptor agonist, decreased K(+)-evoked release of SP-LI and NKA-LI compared with that in the absence of the nucleoside. Similar decrements in peptide release were obtained with N6-cyclopentyl adenosine (CPA), a selective A1 agonist, and 2-[p-(2-carboxyethyl)]phenethylamino-5'-N-ethyl-carboxamidoadenosi ne (CGS 21680), a selective A2 agonist. Response to all nucleosides was graded. Potency order of adenosine analogues was CPA greater than NECA much greater than CGS 21680. Inhibition due to the nucleosides was diminished in the presence of the highly selective A1-receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) while perifusion in the presence of DPCPX alone did not alter evoked release of either peptide. These findings provide direct measurements of inhibitory effects of adenine nucleosides on the release, from enteric nerve endings, of endogenous neuromediators SP and NKA. The findings also directly demonstrate the presence of functional adenosine receptors of the A1 subtype on enteric nerve endings coupled negatively to release of tachykinins. The presence of A2 receptors on enteric nerve endings is neither supported nor excluded.

In Vitro Effects of Bladder Mucosa and an Enkephalinase Inhibitor on Tachykinin Induced Contractility of the Dog Bladder

Tachykinin-induced contractility of smooth muscle strips from dog bladders was studied in vitro, and the presence of substance P-like immunoreactivity and neurokinin A and neurokinin B-like immunoreactivity was examined in bladder sections. Nerve fibers with substance P-like immunoreactivity were present in the mucosa, submucosa and smooth muscle. Fibers were also found in nerves, intramural ganglia, and around blood vessels. Neurokinin A-like immunoreactivity had similar distribution, and no neurokinin B-like immunoreactivity was observed. Removal of the mucosa significantly enhanced the sensitivity and the maximum responses to the tachykinins. After removing the mucosa, the sensitivity to these tachykinins increased 0.4 to 0.5log units (p <0.02). The responses to carbachol were not altered by mucosa removal. The leftward shifts of the concentration-response curves for neurokinin A were of similar magnitude after removal of the mucosa, and after pretreatment with phosphoramidon (10 μΜ), an enkephalinase inhibitor, in the presence of mucosa. However, phosphoramidon did not alter the sensitivity of the bladder strips to neurokinin B, and slightly changed the sensitivity to substance P (0.2log units). Additional shifts of the substance P and neurokinin A curves to the left were observed in the presence of phosphoramidon when the mucosa was removed (0.6 and 0.5log units, p <0.005). The order of potency for the tachykinins (neurokinin A > substance P) was not altered by mucosa removal, addition of phosphoramidon, or both. Neurokinin A was degraded by enkephalinase located in the bladder mucosa and addition of phosphoramidon or mucosa removal resulted in an inhibition or loss of enkephalinase activity. It is concluded that the responses to neurokinin A, which acts on NK-2 type of receptors, prevail on the dog bladder.