Jan M. Lundberg

High levels of neuropeptide Y in peripheral noradrenergic neurons in various mammals including man

Using a highly specific radioimmunoassay for neuropeptide Y (NPY), levels in the peripheral nervous system of guinea-pig, cat, pig and man were measured. In all species very high levels (up to 800 pmol X g-1) were found in sympathetic ganglia and in tissues which receive a dense sympathetic innervation, such as vas deferens, heart atrium, blood vessels and spleen. By immunocytochemistry, NPY-immunoreactive (-IR) principal ganglion cells in sympathetic ganglia and the pelvic plexus were also found to contain dopamine-beta-hydroxylase (DBH) and tyrosine hydroxylase (TH), strongly suggesting that the NPY-IR cells are noradrenergic. NPY- and DBH-IR nerves had a roughly parallel occurrence in the heart, spleen, kidney, respiratory and urogenitary tracts, around blood vessels and within visceral smooth muscle. Considerably more NPY-IR than DBH-IR nerve fibres were seen in the gastrointestinal tract from the oesophagus to the anal sphincter. In addition, NPY-IR local ganglion cells were observed in the submucous and myentric plexuses. NPY-like immunoreactivity was also observed in the adrenal medulla of guinea-pig and cat. NPY thus seems to be a major peptide in the sympathetic nervous system, supporting its proposed role in sympathetic neurotransmission.

Co-existence of substance P and calcitonin gene-related peptide-like immunoreactivities in sensory nerves in relation to cardiovascular and bronchoconstrictor effects of capsaicin.

Immunohistochemical studies showed that substance P (SP) and calcitonin gene-related peptide (CGRP) immunoreactivity co-exist in capsaicin-sensitive primary sensory neurons. Varicose SP- and CGRP-immunoreactive nerve fibres with a similar distribution pattern were seen in the lower airways and heart. The functional analysis revealed that CGRP caused cardiac stimulation and had, together with SP and neurokinin A, potent hypotensive effects. Vascular permeability was increased by SP and neurokinin A, and the bronchial smooth muscle was particularly sensitive to neurokinin A. Thus, multiple peptides stored in an possible released from the same nerve endings by capsaicin may exert differential effects in various target tissues.

Immunoreactive calcitonin gene-related peptide and substance P coexist in sensory neurons to the spinal cord and interact in spinal behavioral responses of the rat

Using immunohistochemistry evidence was obtained for the coexistence of calcitonin gene-related peptide (CGRP)- and substance P (SP)-like immunoreactivity in spinal sensory neurons. Analysis of caudally directed biting and scratching (CBS) behavior was carried out after intrathecal administration of CGRP and SP alone or in combination. Thus, SP (up to 20 micrograms) alone caused CBS only for a few minutes after injection, whereas SP (10 micrograms) plus CGRP (20 micrograms) caused a response with a duration up to 40 min. CGRP (20 micrograms) alone had no effects in this model. These findings provide support for a possible interaction of the two peptides at synapses in the dorsal horn of the spinal cord.

Coexistence of peptides and classical neurotransmitters

In the present article the fact is emphasized that neuropeptides often are located in the same neurons as classical transmitters such as acetylcholine, 5-hydroxy-tryptamine, catecholamines, γ-aminobutyric acid (GABA) etc. This raises the possibility that neurons produce, store and release more than the one messenger molecule. The exact functional role of such coesisting peptides is often difficult to evaluate, especially in the central nervous system. In the periphery some studies indicate apparently meaningful interactions of different types with the classical transmitter, but other types of actions including trophic effects have been observed. More recently it has been shown that some neurons contain more than one classical transmitter, e.g. 5-HT plus GABA, further underlining the view that transfer of information across synapses may be more compex than perhaps hitherto assumed.

Substance P-immunoreactive sensory nerves in the lower respiratory tract of various mammals including man

SummaryThe occurrence and origin of substance P (SP)-immunoreactive (IR) nerves in the lower respiratory tract was studied by means of immunohistochemistry in the guinea-pig, rat, cat and man. In addition, biopsies from human material were also analysed by radioimmunoassay. SP-IR nerves were seen in four principal locations: 1) under or within the lining epithelium, 2) around blood vessels, 3) within the bronchial smooth muscle layer, and 4) around local tracheobronchial ganglion cells. Ligation experiments combined with capsaicin pretreatments indicated that all SP-IR nerves in the respiratory tract are sensory. The trachea seems to be mainly supplied by the vagal nerves, while intrapulmonary bronchi and blood vessels receive SP-IR nerves of both vagal and non-vagal (spinal) origin. SP-IR nerves were also found in the human bronchi with principally similar location as in the guinea-pig. The levels of SP-IR in the trachea and peripheral bronchi of man were about 3–4 pmol/g, which is in the same range as the content of corresponding tissues from the guinea-pig.In conclusion, the present experimental findings of SP-IR nerves in the lower respiratory tract in both experimental animals and man support the functional evidence for the importance of SP in the vagal and non-vagal (spinal) control of bronchial smooth muscle tone and vascular permeability.

Occurrence of vasoactive intestinal polypeptide (VIP)-like immunoreactivity in certain cholinergic neurons of the cat: Evidence from combined immunohistochemistry and acetylcholinesterase staining

Abstract Postganglionic sympathetic and parasympathetic neurons of the cat were studied with the indirect immunofluorescenee technique using antiserum to vasoactive intestinal polypeptide (VIP) and with acetylcholinesterase staining. In the stellate and in L7 and S1 sympathetic ganglia some of the principal ganglion cells (10–15% of the total) contained both VIP-like immunorcactivity and intense acetylcholinesterase activity, suggesting the presence of a VIP-like peptide in a population of sympathetic cholinergic neurons. This was corroborated by (1) overlapping accumulations of VIP immunoreactivity and acetylcholinesterase staining around a ligation of the sciatic nerve, (2) very similar distribution patterns of VIP immunoreactive and acetylcholinesterase-containing fibres around sweat glands of the food pad and around some blood vessels in several tissues, and (3) the disappearance of both types of staining of fibres around sweat glands and skeletal muscle vessels after lumbo-sacral sympathectomy. In other sympathetic ganglia, as well as in the parasympathetic pelvic ganglion plexus and in some ganglia of the bladder wall, VIP immunoreactivity was also found in neurons rich in acetylcholinesterase. On the other hand, it should be emphasized that the occurrence of a VIP-like peptide in presumably cholinergic neurons does not seem to be a general phenomenon. No marked overlap was observed between VIP immunoreactive and acetylcholinesterase-rich cell bodies in most ganglia in the urinary bladder wall and nerve terminals of the smooth muscle layer. Furthermore, no obvious correlation in the number of VIP immunoreactive and acetylcholinesterase-containing fibres were seen in certain other nerves such as the spinal ventral roots, the splanchnic nerve and the perivascular superior mesenteric nerves. Thus, three groups of peripheral autonomic neurons have been defined in the present study: neurons rich in acetylcholinesterase that also contain a VIP-like peptide; VIP immunoreactive neurons; and neurons rich in acetylcholinesterase. The functional significance of a VIP-like peptide in probable cholinergic neurons is at present unclear. Preliminary data indicate that, whereas acetylcholine directly stimulates sweat secretion, VIP may cooperate in the production of sweat by increasing local blood flow. Thus, VIP, released together with acetylcholine, may be responsible for the atropine-resistant vasodilatation in sweat glands and other exocrine glands.

A galanin-like peptide in the central nervous system and intestine of the rat

Galanin (GAL), a 29 amino acid peptide, was recently isolated from the small intestine of pigs. In the present study an antiserum towards porcine GAL has been developed and used for radioimmunoassay and immunohistochemical studies. We now report that GAL-like immunoreactivity occurs in wide-spread systems in the rat CNS and intestine.

Vascular protein leakage in various tissues induced by substance P, capsaicin, bradykinin, serotonin, histamine and by antigen challenge

Summary1.Plasma extravasation was induced in rats or guinea-pigs by intravenous injections of (1) substance P (SP), (2) the C-terminal SP-hexapeptide SP(6-11), (3) serotonin (5-HT), (4) histamine, (5) bradykinin, (6) capsaicin and (7) by antigen challenge.2.Plasma extravasation induced by SP, SP(6-11), by 5-HT and by capsaicin was, with few exceptions, observed in the same tissues. The effect of SP was not blocked by H1 and H2 histamine receptor antagonists. The effect of i.v. capsaicin was absent in capsaicin desensitized animals. Plasma extravasation upon i.v. SP, SP(6-11), 5-HT and capsaicin was seen in the skin and in all organs containing mucous membranes except the intestinal mucosa.3.Plasma extravasation by histamine, bradykinin, and antigen challenge of sensitized guinea-pigs was, in addition, also observed in the stomach and intestine. Plasma extravasation and bronchoconstriction by antigen challenge with 20μg/kg ovalbumin was completely blocked by combined H1 and H2 histamine receptor blockade. Both responses were reduced to about the half capsaicin desensitized guinea-pigs, although the reduction of the permeability response was statistically not significant in all organs.4.In conclusion, several substances including anaphylaxis induce protein leakage in many tissues with differing selective distribution patterns. Anaphylactic histamine release leads to protein leakage partly via activation of sensory neurons. SP is a likely mediator of neurogenic protein leakage in many organs.

Evans blue fluorescence: quantitative and morphological evaluation of vascular permeability in animal tissues

The dye Evans blue was used to monitor vascular protein leakage. Fluorometric measurement of Evans blue in formamide extracts of rat tracheal tissue was performed after induction of protein leakage by electrical vagus nerve stimulation and compared with the widely used colorimetric detection. The fluorescence method was approximately 100 X more sensitive than the colorimetric method. Furthermore, Evans blue fluorescence (excitation at 620 nm, emission at 680 nm) was used for microscopic investigation of cryostat sections of tracheal tissue. Extravasated Evans blue after electrical nerve stimulation was mainly found in the subepithelial layer of the trachea obviously bound to tissue constituents. It is suggested that Evans blue fluorescence can be applied for quantification of protein leakage with high sensitivity which opens the possibility of measuring this reaction in very small regions with high accuracy, as well as for tissue localization of protein leakage at the microscopic level.

Frequency- and reserpine-dependent chemical coding of sympathetic transmission: Differential release of noradrenaline and neuropeptide Y from pig spleen

The importance of impulse pattern and stimulation frequency for the release of noradrenaline (NA) and the coexisting peptide neuropeptide Y (NPY) in relation to vasoconstriction (perfusion-pressure increase) was studied in the blood-perfused pig spleen in vivo. Splenic nerve stimulation with intermittent bursts at high frequency (20 Hz) caused a several-fold larger release of NPY-like immunoreactivity (-LI) in relation to NA than a continuous stimulation at a low frequency (2 Hz), giving the same total number of impulses. alpha-Adrenoceptor blockade by phentolamine enhanced markedly both NA and NPY release, especially at low stimulation frequency, suggesting prejunctional adrenergic inhibition of release. Addition of propranolol unmasked a large remaining perfusion-pressure response to nerve stimulation. Reserpine treatment reduced the NA content of the spleen as well as the stimulation-evoked NA release by greater than 90%. However, the perfusion-pressure increase in response to nerve stimulation was well maintained. A marked increase in the stimulation-evoked release of NPY-LI occurred after reserpine. Adrenoceptor blockade after reserpine treatment reduced only slightly the perfusion-pressure response in parallel with a decline in NPY output. NPY caused an adrenoceptor-resistant perfusion-pressure increase at plasma concentrations that were in the same range as the maximal increase during nerve stimulations. In conclusion, the present data suggest a frequency-dependent, chemical coding of sympathetic transmission with preferential release of the classical transmitter NA at low, continuous frequencies and release of NPY, mainly at high frequencies. Reserpine treatment enhances markedly NPY release, which may explain why the functional response is largely intact in spite of adrenoceptor blockade and marked NA depletion.