Elizabeth Burcher

Neurochemical classification of myenteric neurons in the guinea-pig ileum

A strategy has been developed to identify and quantify the different neurochemical populations of myenteric neurons in the guinea-pig ileum using double-labelling fluorescence immunohistochemistry of whole-mount preparations. First, six histochemical markers were used to identify exclusive, non-overlapping populations of nerve cell bodies. They included immunoreactivity for the calcium binding proteins calbindin and calretinin, the neuropeptides vasoactive intestinal polypeptide, substance P and somatostatin, and the amine, 5-hydroxytryptamine. The sizes of these populations of neurons were established directly or indirectly in double-labelling experiments using a marker for all nerve cell bodies. Each of these exclusive populations was further subdivided into classes by other markers, including immunoreactivity for enkephalins and neurofilament protein triplet. The size of each class was then established directly or by calculation. These distinct, neurochemically-identified classes were related to other published work on the histochemistry, electrophysiology and retrograde labelling of enteric neurons and to the simple Dogiel morphological classification. A classification scheme, consistent with previous studies, is proposed. It includes 14 distinct classes of myenteric neurons and accounts for nearly all neurons in the myenteric plexus of the guinea-pig ileum.

Muscarinic receptor subtypes in human bladder detrusor and mucosa, studied by radioligand binding and quantitative competitive RT-PCR: changes in ageing.

1 We investigated muscarinic receptors in the detrusor and mucosa of the human bladder body. Radioligand‐binding studies with [3H]QNB were conducted using specimens collected from patients (36–77 years) with normal bladder function, undergoing surgery. For RT–PCR, biopsies of normal bladder were obtained from patients (30–88 years) undergoing check cystoscopy. 2 Binding of [3H]QNB in detrusor (n=20) was of high affinity (KD 77.1 (55.2–99.0) pM) and capacity (Bmax 181±7 fmol mg protein−1). Similar values were obtained in mucosa (n=6) (KD 100.5 (41.2–159.9) pM; Bmax 145±9 fmol mg protein−1). 3 Competition‐binding experiments in detrusor membranes with muscarinic receptor antagonists including trospium, darifenacin, 4‐DAMP, methoctramine, AQ‐RA 741, AF‐DX 116 and pirenzepine indicated a receptor population of 71% M2, 22% M3 and 7% M1. In the mucosa, 75% of sites were M2 receptors, with 25% M3/M5. 4 Using RT–PCR, expression of M1, M2, M3 and M5 mRNA was demonstrated in both detrusor and mucosa. 5 The presence of a high density of mainly M2 muscarinic receptors in the mucosa appears to be a novel finding and raises the question of their physiological significance and the source of their endogenous ligand. 6 There was a negative correlation of receptor number (Bmax) with age in detrusor muscle from male patients (P=0.02). Quantitative competitive RT–PCR demonstrated a selective age‐related decrease in mRNA for muscarinic M3 but not M2 receptors, in both male (P<0.0001) and female (P=0.019) detrusor. These findings correspond with reports of decreased detrusor contractility with ageing.

The tachykinins: a family of peptides with a brood of ‘receptors’

Abstract Current evidence indicates that there are three major mammalian tachykinins: substance P, substance K, and neuromedin K. Investigations using in-vitro smooth muscle preparations have provided evidence for at least two distinct types of tachykinin receptors. Recently, three distinct types of radiolabeled-tachykinin binding sites have been identified: P-type, K-type, and E-type. Each of these appears to have a preferential affinity for one of the three mammalian tachykinins. Stephen Buck and Elizabeth Burcher describe these sites and attempt to relate their existence to the known biological effects of the tachykinins in peripheral tissues.

Pharmacologic characterization and autoradiographic distribution of binding sites for iodinated tachykinins in the rat central nervous system

P-type, E-type, and K-type tachykinin binding sites have been identified in the mammalian CNS. These sites may be tachykinin receptors for which the mammalian neuropeptides substance P, neuromedin K, and substance K are the preferred natural agonists, respectively. In the present investigation, we have compared the pharmacology and the autoradiographic distribution of CNS binding sites for the iodinated (125I-Bolton-Hunter reagent) tachykinins substance P, eledoisin, neuromedin K, and substance K. Iodinated eledoisin and neuromedin K exhibited an E-type binding pattern in cortical membranes. Iodinated eledoisin, neuromedin K, and substance K each labeled sites that had a similar distribution but one that was considerably different from that of sites labeled by iodinated substance P. CNS regions where there were detectable densities of binding sites for iodinated eledoisin, neuromedin K, and substance K and few or no sites for iodinated substance P included cortical layers IV-VI, mediolateral septum, supraoptic and paraventricular nuclei, interpeduncular nucleus, ventral tegmental area, and substantia nigra pars compacta. Binding sites for SP were generally more widespread in the CNS. CNS regions where there was a substantial density of binding sites for iodinated substance P and few or no sites for iodinated eledoisin, neuromedin K, and substance K included cortical layers I and II, olfactory tubercle, nucleus accumbens, caudate-putamen, globus pallidus, medial and lateral septum, endopiriform nucleus, rostral thalamus, medial and lateral preoptic nuclei, arcuate nucleus, dorsal raphe nucleus, dorsal parabrachial nucleus, parabigeminal nucleus, cerebellum, inferior olive, nucleus ambiguus, retrofacial and reticular nuclei, and spinal cord autonomic and somatic motor nuclei. In the brainstem, iodinated substance P labeled sites in both sensory and motor nuclei whereas iodinated eledoisin, neuromedin K, and substance K labeled primarily sensory nuclei. Our results are consistent with either of two alternatives: (1) that iodinated eledoisin, neuromedin K, and substance K bind to the same receptor site in the rat CNS, or (2) that they bind to multiple types of receptor sites with very similar distribution.

Roles of neuronal NK1 and NK3 receptors in synaptic transmission during motility reflexes in the guinea-pig ileum.

1 The role of NK1 and NK3 receptors in synaptic transmission between myenteric neurons during motility reflexes in the guinea‐pig ileum was investigated by recording intracellularly the reflex responses of the circular muscle to distension or compression of the mucosal villi. Experiments were performed in a three‐chambered organ bath that enabled drugs to be selectively applied to different sites along the reflex pathways. 2 When applied in the recording chamber, an NK1 receptor antagonist, SR140333 (100 nm), reduced by 40–50% the amplitudes of inhibitory junction potentials (i.j.ps) evoked in the circular muscle by activation of descending reflex pathways. This effect was abolished when synaptic transmission in the stimulus region was blocked with physiological saline containing 0.1 mm Ca2+ plus 10 mm Mg2+, leaving only the component of the descending reflex pathway conducted via long anally directed collaterals of intrinsic sensory neurons. 3 SR140333 (100 nm) had no effect on descending reflex i.j.ps when applied to the stimulus region. Ascending reflexes were also unaffected by SR140333 in the stimulus region or between the stimulus and recording sites. 4 Septide (10 nm), an NK1 receptor agonist, enhanced descending reflexes by 30–60% when in the recording chamber. [Sar9,Met(O2)11]substance P had no effect at 10 nm, but potentiated distension‐evoked reflexes at 100 nm. 5 A selective NK3 receptor antagonist, SR142801 (100 nm), when applied to the stimulus region, reduced the amplitude of descending reflex responses to compression by 40%, but had no effect on responses to distension. SR142801 (100 nm) had no effect when applied to other regions of the descending reflex pathways. 6 SR142801 (100 nm) only inhibited ascending reflexes when applied at the recording site. However, after nicotinic transmission in the stimulus region was blocked, SR142801 (100 nm) at this site reduced responses to compression. 7 Contractions of the circular muscle of isolated rings of ileum evoked by low concentrations of septide, but not [Sar9,Met(O2)11]substance P, were potentiated by tetrodotoxin (300 nm). 8 Contractile responses evoked by an NK3 receptor agonist, senktide, were non‐competitively inhibited by SR142801. After excitatory neuromuscular transmission was blocked, senktide produced inhibitory responses that were also antagonised by SR142801, but to a lesser extent and in an apparently competitive manner. 9 These results indicate that tachykinins acting via NK1 receptors partly mediate transmission to inhibitory motor neurons. NK3 receptors play a role in transmission from intrinsic sensory neurons and from ascending interneurons to excitatory motor neurons during motility reflexes.

Multiple tachykinin binding sites in hamster, rat and guinea-pig urinary bladder

Binding of the 125I-Bolton-Hunter labelled tachykinins substance P, substance K, eledoisin and neuromedin K (BHSP, BHSK, BHE, BHNK) was examined in urinary bladders of hamster, rat and guinea-pig using crude membrane suspensions and by autoradiography. High-affinity binding of BHSK was observed in hamster and rat bladder and high-affinity binding of BHSP was seen in rat and guinea-pig bladder. Characterization of this binding showed that the hamster bladder contains very large numbers of K-type binding sites, where BHSK is displaced by substance K greater than kassinin greater than eledoisin greater than neuromedin K greater than substance P greater than physalaemin, and has very few P-type binding sites, where BHSP is displaced by substance P greater than substance K much greater than neuromedin K. In contrast, the rat bladder contains moderate and approximately equal numbers of both K (KD, 0.74 nM; Bmax 2.9 fmol/mg wet weight tissue) and P (KD, 0.12 +/- 0.01 nM; Bmax 2.6 +/- 0.2 fmol/mg wet weight tissue) sites. The guinea-pig bladder possesses predominantly P sites. Most tachykinin binding sites are localized over smooth muscle and probably represent functional receptors mediating the direct contractile effects of tachykinins in these tissues. Few E-type binding sites, as previously described in rat brain, were found, although some BHNK binding sites were seen in the mucosa of guinea-pig bladder.

Total numbers of neurons in myenteric ganglia of the guinea-pig small intestine

Two techniques that are thought to stain all of the neurons in the myenteric ganglia of the intestine are NADH diaphorase histochemistry and immunhistochemistry using a “nerve cell body” antiserum. However, this assumption has never been directly verified. In the present study myenteric ganglia of the guinea-pig ileum were prepared as whole-mounts and stained with either of these techniques. All nerve cells that could be identified in the whole-mounts were counted. The whole-mounts were then embedded flat in resin and serially sectioned at 1 μm. Nerve cells were identified and counted from the serial sections, and the data compared to those obtained from the whole-mounts. NADH diaphorase histochemistry did not reveal all the neurons at incubation times that gave selective staining. In contrast, “nerve cell body” antiserum stained the entire neuronal population. To determine the total number of nerve cell bodies/ganglion and the proportion of nerve cell bodies with calbindin immunoreactivity, whole-mounts that had been processed for calbindin immunohistochemistry were serially sectioned and reconstructed. The total number of neurons per myenteric ganglion was 105±10 (SE). Calbindin-immunoreactive neurons comprised about 20% of the myenteric neurons, which is considerably less than previous estimates, because previously the total population has been underestimated. The spatial density of myenteric neurons in the undistended ileum of the guinea-pig is 17300 nerve cells/cm2.

Binding characteristics of [125I]Bolton-Hunter [Sar9, Met(O2)11]substance P, a new selective radioligand for the NK1 receptor

The selective tachykinin agonist [Sar9,Met(O2)11]substance P (Sar-SP) was radioiodinated with [125I]Bolton-Hunter reagent and the product [125I]Bolton-Hunter-[Sar9,Met(O)2)11]SP (BHSar-SP) purified using reverse phase HPLC. Autoradiographic studies showed dense specific binding of BHSar-SP over the rat submandibular gland and over several regions in rat brain, with very low nonspecific binding, identical with the pattern of binding sites seen in a parallel study with [125I]Bolton-Hunter SP (BHSP). In homogenate binding experiments, BHSar-SP bound with high affinity to a single site in membranes from rat brain (KD 261 pM) and rat submandibular gland (KD 105 pM). Comparative values for BHSP were 495 and 456 pM, i.e. of two and four fold lower affinity than BHSar-SP. Association of BHSar-SP to membranes from brain (k+1 3.7 x 10(9) M-1 min-1) was faster than to membranes from salivary gland (k+1 5.6 x 10(8) M-1 min-1). In competition studies, BHSar-SP was displaced from salivary gland membranes by substance P (SP) approximately physalaemin greater than or equal to Sar-SP approximately SP-(3-11) greater than SP-(5-11) much greater than neurokinin A (NKA) approximately eledoisin = kassinin = SP-methyl ester greater than or equal to neurokinin B (NKB) much greater than [Nle10]NKA-(4-10) greater than [MePhe7]NKB-(4-10). In brain membranes, the rank potency order was SP greater than Sar-SP greater than or equal to physalaemin greater than SP-(3-11) greater than SP-(5-11) greater than NKA greater than or equal to eledoisin much greater than NKB greater than kassinin greater than SP-methyl ester: however [MePhe7]NKB-(4-10) and [Nle10]NKA-(4-10) were ineffective competitors at concentrations up to 1 microM. Both binding patterns are consistent with BHSar-SP binding to an NK1 site. With the exception of SP, Sar-SP, SP-(3-11) and physalaemin, all competitors were 5 to 54 times less potent at BHSar-SP binding sites in brain than in salivary gland. These data reveal some differences in characteristics of NK1 binding sites in brain and submandibular gland. Although of higher affinity, BHSar-SP does not appear greatly more selective than BHSP in its ability to define NK1 binding sites.

Release of ATP from rat urinary bladder mucosa: role of acid, vanilloids and stretch

Background and purpose:  ATP, released from urothelial cells, modulates afferent nerve firing from the urinary bladder. Here, we have characterized ATP release from the rat bladder mucosa in response to acid, capsaicin, electrical field stimulation (EFS) and stretch, using agonists and antagonists at transient receptor potential vanilloid receptor 1 (TRPV1) and acid‐sensing ion channels (ASICs).

The rat submaxillary gland contains predominantly P-type tachykinin binding sites.

The specific binding of the 125I-Bolton-Hunter labeled tachykinins substance K (BHSK), eledoisin (BHE), and substance P (BHSP) was examined in crude membrane suspensions and by autoradiography in rat submaxillary gland. All three ligands at 0.1 nM concentrations exhibited binding that was inhibited by tachykinins in a potency rank order of substance P greater than physalaemin greater than substance K greater than eledoisin greater than kassinin greater than neuromedin K with slope factors essentially equal to unity. All tachykinins were 5 to 10 times more potent in inhibiting BHSK and BHE binding compared to BHSP binding. Autoradiographic visualization of BHSK and BHSP binding sites in the gland revealed extensive labeling of mucous and serous acini. The intensity of labeling was much less for BHSK than for BHSP. The results indicate that the rat submaxillary gland contains predominantly P-type tachykinin binding sites.