Neuropeptide Y-, substance P- and VIP-immunoreactive nerves in cat spleen in relation to autonomic vascular and volume control

Summary Neuropeptide Y (NPY)-immunoreactive (IR) nerve fibres were found around both arteries and veins and in smooth muscle trabeculae of the cat spleen with the highest density on the arterial side. Considerably more tyrosine hydroxylase (TH)- and dopamine--hydroxylase (DBH)-positive than NPY-IR nerves were seen in the trabeculae and splenic capsule. The NPY-IR nerves in the spleen most likely originated in the coeliac ganglion, since (1) splanchnic nerve sectioning did not change the splenic NPY-IR nerves, (2) most neurones in the coeliac ganglion were NPY-IR, as well as DBH- and TH-positive, and (3) NPY-IR was transported axonally from the coeliac ganglion towards the spleen via the splenic nerve. Local NPY infusion in the isolated, blood-perfused cat spleen caused a marked increase in splenic vascular resistance and a small volume reduction. NA caused a comparatively larger reduction in splenic volume than NPY in addition to vasoconstriction. VIP-IR cell bodies in the coeliac ganglion were NPY- and TH-negative. VIP-IR nerves were seen both around the splenic artery and vein as well as around arterioles and within venous trabeculae of the spleen. VIP infusion caused reduction of splenic perfusion pressure (i.e. vasodilation) as well as an increase in splenic volume. Substance P-IR nerves, most likely of splanchnic afferent origin, were present in the coeliac ganglion around the splenic artery and arterioles of the spleen. Infusion of substance P induced marked reduction in perfusion pressure and a reduction in splenic volume. Enkephalin-immunoreactive nerves of splanchnic origin surrounded some TH- and NPY-positive, coeliac ganglion cells.It is concluded that several vasoactive peptides are located in splenic nerves. NPY is present in noradrenergic neurones and causes mainly increased vascular resistance. VIP occurs in non-adrenergic neurones of sympathetic origin and induces vasodilation and relaxation of the capsule. Finally, substance P is present in peripheral branches of spinal afferent nerves and causes vasodilation and capsule contraction. Stimulation of the splenic nerves may thus release several vasoactive substances in addition to noradrenaline, exerting a variety of actions.     

A comparative study of endothelin and sarafotoxin action in vascular and non-vascular smooth muscle

The effects of endothelin and sarafotoxin on smooth muscle tone have been examined in the rat aorta and anococcygeus muscle and their actions compared to those of norepinephrine. The contractions elicited by endothelin and sarafotoxin (10 nM), or norepinephrine (1 μM) were approximately equieffective in terms of tension development and correspond to EC₅₀ values and these concentrations were thus used throughout the study. In calcium-free Krebs the three agonists generated approximately similar levels of tone in the aorta and the anococcygeus corresponding to 20 and 5% of the maximum response, respectively. Nifedipine, 10 μM, significantly inhibited responses to endothelin and norepinephrine in the aorta but only norepinephrine in anococcygeus; the responses to sarafotoxin were however not significantly affected in either tissue. A combination of 10 μM ryanodine and nifedipine caused near complete inhibition of the response to endothelin in the aorta and also significantly reduced the response to both endothelin and norepinephrine in the anococcygeus. The lipoxgenase inhibitor, nordihydroguaiaretic acid, inhibited the response to endothelin in the aorta and endothelin and norepinephrine in the anococcygeus muscle. The cyclooxygenase inhibitor, indomethacin, however, had no effect on the responses to any of the three agonists in either the aorta or anococcygeus. At concentrations greater than 30 nM both endothelin and sarafotoxin induced myogenic activity in normally quiescent anococcygeus muscle. As determined by the loss of myogenic activity the tissues recovered more rapidly from sarafotoxin than endothelin with complete recovery apparent after 2.62 ± 0.85 and 5.22 ± 0.06 h respectively. Omitting Ca²⁺ from the Krebs solution reduced recovery times to 1.62 ± 0.2 and 2.4 ± 0.51 h respectively.

Overall the results suggest that endothelin and sarafotoxin activate different cell signaling systems to differing extents in rat aorta versus anococcygeus suggesting that the membrane receptors mediating the responses to endothelin and sarafotoxin are not necessarily identical.     

The effect of some brain/gut peptides on plasma cholesterol levels in the rat

The effect of substance P (SP), bombesin (BBS), somatostatin (SRIF) and neurotensin (NT) on the plasma cholesterol levels was studied in the rat. SP and BBS had no effect, but SRIF produced a significant transient hypocholesterolemia and NT a significant transient hypercholesterolemia 15 minutes after the intravenous injections. Neither SRIF nor NT influenced the rate of the 7α-hydroxylation of cholesterol. The data suggest a relation of SRIF and NT to the maintenance of the plasma cholesterol concentration.     

The effect of some brain/gut peptides on plasma cholesterol levels in the rat

The effect of substance P (SP), bombesin (BBS), somatostatin (SRIF) and neurotensin (NT) on the plasma cholesterol levels was studied in the rat. SP and BBS had no effect, but SRIF produced a significant transient hypocholesterolemia and NT a significant transient hypercholesterolemia 15 minutes after the intravenous injections. Neither SRIF nor NT influenced the rate of the 7-hydroxylation of cholesterol. The data suggest a relation of SRIF and NT to the maintenance of the plasma cholesterol concentration.     

Effect of 6-hydroxydopamine on neuropeptides in the rat female genitourinary tract

The occurrence and distribution of neuropeptide Y has been determined in the rat female genitourinary tract by radioimmunoassay and chromatographic analysis. Within the bladder, higher concentrations of neuropeptide Y were found in the trigone (48.8±5.2 pmol/g) than in the dome (36.0±2.1 pmol/g). In the genital tract, highest concentrations were identified in the vagina (41.4±2.1 pmol/g). Treatment of rats with 6-hydroxydopamine resulted in significant depletion of neuropeptide Y concentrations in both parts of the bladder, together with vagina, uterine horn and fallopian tube. No change was observed in the cervix, uterine body and ovary. Concentrations of vasoactive intestinal polypeptide were unaffected by treatment with 6-hydroxydopamine except in the area of the cervix where concentrations rose from 64.1±5.7 pmol/g to 133.6±15.1 pmol/g (p<0.05). There was a generalised, but statistically insignificant rise in substance P concentrations.     

Presence and Ontogeny of Enkephalin and Substance P in the Chick Ciliary Ganglion

The avian ciliary ganglion has been reported to contain both enkephalin and substance P in preganglionic terminals. However, extensive biochemical characterization of these antigens has not been completed. Using radioimmunoassays specific for Met5- and for Leu5-enkephalin and for substance P we identified immunoreactive substances in ganglionic extracts that comigrate on HPLC columns with standard Met5- and Leu5-enkephalin and with substance P. The ontogeny of Met5-enkephalin and substance P during embryogenesis was determined in ganglionic extracts and we found that the content of Met5-enkephalin in the ganglion reached a peak at embryonic stage 37 whereas the content of substance P in the ganglion reached its maximum in the adult.     

Peptide-containing neurons in explant cultures of guinea-pig myenteric plexus during development in vitro: Gross morphology and growth patterns

Summary The gross morphology and growth patterns of substance P, enkephalin-, somatostatin and vasoactive intestinal peptide-immunoreactive neurons have been studied in explant cultures of the myenteric plexus taken from beneath the newborn guinea-pig taenia coli, grown for up to 4 weeks in vitro. Substance P and enkephalin-immuno-reactive neurons were more abundant than somatostatin and vasoactive intestinal peptide-immunoreactive neurons. The peptide-containing neuronal cell bodies were clearly visible in culture and exhibited characteristic gross morphologies similar to those described in situ, although some overlap of shape between populations containing different peptides was seen. All four types of peptide-containing fibres were found in the outgrowth and central areas of the cultures. In the case of substance P and somatostatin, the density and pattern of labelling in the central, neuronal area of the cultures resembled that previously seen in the myenteric plexus of the newborn guinea-pig caecum in situ, while the density of the enkephalin-immunoreactive fibres was greater, and that of the vasoactive intestinal peptide-immunoreactive fibres less than that seen in situ. These observations suggest that subpopulations of myenteric neurons containing different peptides may be differentially affected by the culture environment. Possible contributory factors are discussed.     

Adrenergic stimulation of diacylglycerol production in genital tract smooth muscle myocytes

Summary Diacylglycerol (DAG) production has not been reported in previous studies that have characterized inositol phosphate production during alpha-1 adrenergic receptor signal transduction in the DDT₁ MF-2 genital tract myocytes. The current study sought to measure norepinephrine (NE)-stimulated DAG production in these transformed myocytes utilizing thin layer chromatography. DAG production was characterized as an alpha-1 adrenergic mediated event utilizing subtype specific adrenergic agonist and antagonists. DAG production occurred in response to physiologic concentration of NE, was apparent by 30 s and was significantly increased by 2 min. Maximal DAG production was unaffected by pretreatment of the myocytes for 96 h with testosterone, which has previously been shown to induce a doubling of alpha-1 adrenergic receptors in these cells. In contrast, testosterone pretreatment did result in a shift of the dose-response curve resulting in a significantly lower EC₅₀ for NE in the treated cells compared to control myocytes. In conclusion, these studies have confirmed that DAG production occurs as a component of alpha-1 adrenergic signal transduction in the DDT₁ MF-2 myocytes; transduction events that were modulated by testosterone resulting in increased agonist sensitivity.     

Nerves containing substance P,vasoactive intestinal polypeptide,enkephalin or somatostatin in the guinea-pig taenia coli

Summary The guinea-pig taenia coli is rich in peptide-containing nerves. Nerve fibres containing substance P (SP), vasoactive intestinal peptide (VIP), or enkephalin, were numerous in the smooth muscle while somatostatin fibres were very few. Nerve fibres displaying SP or VIP immunoreactivity were numerous in the myenteric plexus. Enkephalin nerve fibres were fairly numerous in the plexus while somatostatin nerve fibres were sparse. Nerve cell bodies containing immunoreactive SP or VIP were regularly seen in the plexus. Delicate varicose elements of the different types of nerve fibres were found to ramify around nerve cell bodies in a manner suggestive of innervation.In the electron microscope the various peptide-storing nerve fibres (i.e., elements containing SP, VIP or enkephalin) were found to contain a varying number of fairly large, electron-opaque vesicles in the varicose swellings. These vesicles represent the storage site of the neuropeptides.The isolated taenia coli responded to electrical nerve stimulation with a contraction. After cholinergic and adrenergic blockade the contractile response was replaced by a relaxation followed by a contraction upon cessation of stimulation. SP contracted the taenia while VIP caused a relaxation. The enkephalins raised the resting tension slightly while somatostatin had no effect. These observations are compatible with a role for SP as an excitatory neurotransmitter and for VIP as an inhibitory one, and with the view that both SP neurones and VIP neurones act as motor neurones. In preparations contracted by SP the electrically induced contractions were reduced in amplitude while the electrically induced relaxations seen after adrenergic and cholinergic blockade were enhanced in amplitude. In preparations relaxed by VIP there was an increased contractile response to electrical stimulation, while in the atropine + guanethidine-treated preparation the electrically induce relaxations were reduced in amplitude. The enkephalins reduced the contractile response to electrical stimulation, while somatostatin induced a very small reduction in the amplitude of such responses. These observations suggest that SP neurones and VIP neurones may play additional roles as interneurones. Somatostatin neurones probably act as interneurones. Enkephalin-containing fibres may serve to modify the release of transmitter from other nerves in the smooth muscle, perhaps through axo-axonal arrangements. Alternatively, the enkephalin nerve fibres in the smooth muscle are afferent elements involved in mediating sensory impulses to the myenteric plexus.     

Evidence for Ascending and Descending Intraspinal as Well as Primary Sensory Somatostatin Projections in the Rat Spinal Cord

Somatostatin distribution was measured quantitatively in the rat spinal cord by radioimmunoassay. Rostro-caudally, somatostatin content was about 50% higher in lumbar-sacral cord than in cervical or thoracic levels. The dorso-ventral distribution is more uneven: somatostatin is highest in the dorsal horn, where the peptide is 15 times as concentrated as it is in the ventral white matter, the region of lowest concentration. However, measurable amounts of the peptide were found in all regions studied. Dorsal root ganglionectomy decreased somatostatin levels in the dorsal cord, supporting the previously proposed role for this peptide as a primary sensory neurotransmitter or modulator; but somatostatin content also was decreased both rostral and caudal to spinal transection, indicating the presence of ascending and descending somatostatin pathways within the spinal cord. Brain levels did not change. Met-enkephalin and substance P were also measured after the above surgical manipulations. Met-enkephalin content was not altered and substance P content was lowered significantly only after ganglionectomy. Although this study confirms the primary sensory neuron as the origin of a part of spinal cord somatostatin, it further indicates the presence of ascending and descending somatostatin pathways within the rat spinal cord.     

Widespread distribution of substance P-and somatostatin-immunoreactive elements in the spinal cord of the neonatal rat

The distribution of substance P (SP)- and somatostatin (SOM)-immunoreactive elements in the spinal cord of the neonatal rat was examined. With few exceptions, the distribution of SP-immunoreactive elements is similar to that described for the adult. A major difference is the obvious presence of SP-immunoreactive fibers in all funiculi of neonatal cords. In addition, an obvious small bundle of longitudinal SP immunoreactive fibers is seen in the base of the dorsal horn at rostral cervical levels. Unlike that of the adult, the neonatal spinal cord shows a widespread distribution of SOM-immunoreactivity. SOM-immunoreactive fibers are present in all funiculi. SOM-immunoreactive perikarya of various shapes and sizes are widely dispersed throughout the gray matter. The cell density is increased in the superficial laminae of the dorsal horn, in a region ventral-lateral to the central canal and in the ventral horn. SOM-immunoreactive varicosities are present in moderate amounts in the superficial laminae of the dorsal horn but are extremely sparse in other regions of the gray matter. A few SOM-immunoreactive fibers course longitudinally at the base of the dorsal horn at rostral levels of the cord. These fibers are found in the same region occupied by the longitudinal SP-immunoreactive fibers referred to above.     

Corticotropin releasing factor-like immunoreactivity in sensory ganglia and capsaicin sensitive neurons of the rat central nervous system: colocalization with other neuropeptides

Immunohistochemistry and radioimmunoassay (RIA) revealed that corticotropin releasing factor (CRF)-like immunoreactivity was found to be colocalized with substance P (SP)-, somatostatin (SST)- and leu-enkephalin (LENK)-like immunoreactivity in the dorsal root- and trigeminal ganglia, the dorsal horn of the spinal cord (laminae I and II), the substantia gelatinosa, and at the lateral border of the spinal nucleus and in the tractus spinalis of the trigeminal nerve. These peptides were also located in fast blue labeled cells of the trigeminal ganglion following injection of the dye into the spinal trigeminal area. This indicates that there are possible sensory projections of these peptides into the spinal trigeminal area. Capsaicin treatment of neonatal rats resulted in a marked decrease in the density of CRF-, SP-, VIP- and CCK-containing neurons in the above mentioned hindbrain areas, whereas SST- and LENK-immunoreactivity were not changed. RIA revealed that, compared to controls, CRF, SP and VIP concentrations in these areas were decreased in rats pretreated with capsaicin, while SST levels were increased; CCK and LENK levels were unchanged. It is concluded that the primary afferent neurons of the nucleus and tractus spinalis of the trigeminal nerve are richly endowed with a number of peptides some of which are sensitive to capsaicin action. The close anatomical proximity of these peptide containing neurons suggests the possibility of a coexistance of one or more of these substances.     

Amylase in human lungs and the female genital tract

Summary We investigated the localization of amylase in normal human lungs and the female genital tract using immunohistochemical and histochemical methods. Immunohistochemical procedures were applied to formaldehydefixed, paraffin-embedded specimens as well as to cryostat sections of periodate/lysine/paraformaldehyde (PLP)-fixed tissues. The starch-substrate-film method was used for the histochemical investigation of unfixed frozen sections. Amylase immunoreactivity was observed in ciliated epithelial cells of the bronchus and in serous cells of the bronchial glands but not in the alveolar epithelium. Immunoreactive amylase was also found in the cytoplasm of the ciliated epithelium of the fallopian tubes, especially in the apical part of the cytoplasm and in ciliary vesicles. Immunoreactive amylase was also found to be present in the surface epithelial cells and glands of the uterine cervix, as well as in the superficial part of the endometrial glands. The distribution of amylase activity revealed using histochemistry was similar to that observed in cryostat sections of PLP-fixed tissues after immunohistochemical staining. Amylase amtigenicity was better preserved in cryostat sections of PEP-fixed materials than in formaldehyde-fixed, paraffinembedded specimens. The results are discussed in relation to pulmonary and female-genital-tract diseases.This work was supported by a grant from the Tokyo Metropolitan Government     

Substance P and substance K receptor binding sites in the human gastrointestinal tract: Localization by autoradiography

Quantitative receptor autoradiography was used to localize and quantify the distribution of binding sites for ¹²⁵I-radiolabeled substance P (SP), substance K (SK) and neuromedin K (NK) in the human GI tract using histologically normal tissue obtained from uninvolved margins of resections for carcinoma. The distribution of SP and SK binding sites is different for each gastrointestinal (GI) segment examined. Specific SP binding sites are expressed by arterioles and venules, myenteric plexus, external circular muscle, external longitudinal muscle, muscularis mucosa, epithelial cells of the mucosa, and the germinal centers of lymph nodules. SK binding sites are distributed in a pattern distinct from SP binding sites and are localized to the external circular muscle, external longitudinal muscle, and the muscularis mucosa. Binding sites for NK were not detected in any part of the human GI tract. These results demonstrate that: 1) surgical specimens from the human GI tract can be effectively processed for quantitative receptor autoradiography; 2) of the three mammalian tachykinins tested, SP and SK, but not NK binding sites are expressed in detectable levels in the human GI tract; 3) whereas SK receptor binding sites are expressed almost exclusively by smooth muscle, SP binding sites are expressed by smooth muscle cells, arterioles, venules, epithelial cells of the mucosa and cells associated with lymph nodules; and 4) both SP and SK binding sites expressed by smooth muscle are more stable than SP binding sites expressed by blood vessels, lymph nodules, and mucosal cells.     

Intrathecal substance P and somatostatin in rats: behaviors indicative of sensation

Biochemical, histochemical and neurophysiological data suggest that substance P and somatostatin are neurotransmitters for primary afferent neurons. This study used intrathecal administration of these peptides and others (neurotensin and vasoactive intestinal polypeptide) in chronically catheterized, environmentally adapted, freely moving rats to evaluate their effects on unconditioned behavior. Substance P and somatostatin each elicited behaviors which were dose related. The behaviors included caudally directed biting and licking along with hindlimb scratching, writhing and retching. The behavioral responses were rapid in onset (1 min) and, in the case of substance P, short in duration (3 min). Vehicle, neurotensin and vasoactive intestinal polypeptide were without effect. These results demonstrate the ability of substance P and somatostatin to induce behavior in rats upon intrathecal administration and extend previous studies in mice.     

Distribution and origin of bombesin,substance P and somatostatin in cat spinal cord

Bombesin (BN), substance P-(SP) and somatostatin (SRIF) were measured in individual laminae of the cervical, thoracic and lumbar (L) spinal cord of control cats, and in the L6 segment of cats receiving a spinal hemisection (L2) or deafferentation via dorsal rhizotomy at L6, 7, S1. The interlaminar distribution of BN, SP, and SRIF was remarkably similar. Highest concentrations were found in the superficial dorsal horn, and progressively less was found proceeding ventrally. Some intersegmental variations in peptide concentration within a single lamina were found. Dorsal rhizotomy caused a significant decline in BN, SP and SRIF in lamina I-III, therefore all three peptides appear to be contained in dorsal root ganglion cells. Evidence is presented for the existence of ascending BN and SP projections originating in lamina I-III and VII, for a descending SRIF pathway terminating in lamina VIII, and for an ascending BN path in lamina VIII. Dorsal root afferents to lamina VIII influence levels of BN, SP and SRIF.     

Central effects of neuropeptides on ventilation in the rat

We have examined the effects of centrally applied neuropeptides on ventilation (respiratory rate, tidal volume, and minute ventilation) in urethane-anesthetized rats. TRH caused an increase in respiratory rate, a decrease in tidal volume, but an increase in net minute ventilation. One TRH metabolite, acid TRH, caused similar changes, but no effect was observed from the other TRH metabolite, cHis-Pro. Both bombesin and calcitonin caused increases in minute ventilation due to increases in respiratory rate and tidal volume. Additionally, bombesin induced periodic sighing respirations at rates up to 15/minute which was observed with no other neuropeptide. Substance P, somatostatin, and neurotensin had no effect upon ventilation variables.