Sune Rosell

Tachykinin multiplicity in rat central nervous system as studied using antisera raised against substance P and neurokinin A

Antisera were raised in rabbits against the tachykinins neurokinin A (NKA) and substance P (SP). All NKA-antisera tested cross-reacted markedly with NKB, kassinin and eledoisin in radioimmunoassay (RIA), but virtually not with SP and physalaemin. Also when used for immunohistochemistry, one of the NKA-antisera was found to be virtually without cross-reactivity with SP. The most specific SP-antiserum did not cross-react with NKA but to some extent with NKB at the immunohistochemical level. Using these two antisera, the same distribution pattern of immunoreactivity was seen in both the rat substantia nigra and dorsal spinal cord. In neutral extracts of the substantia nigra, all NKA-antisera used for RIA detected a major component which eluted at the position of NKA in reverse phase high performance liquid chromatography, while no or only little immunoreactivity was detected at the position of NKB. A major component of substance P-like immunoreactivity (SPLI) co-eluting with SP and one or two minor SPLI-components were also detected in these extracts. An SP-antiserum, which cross-reacted markedly with physalaemin, detected an additional rather prominent component. In neutral water extracts of dorsal spinal cord the component detected with the NKA-antisera at the position of NKB, as well as one of the SPLI-components not eluting in the position of SP, were much more prominent than in the corresponding extracts of substantia nigra. In acetic acid extracts of both tissues, only one major SPLI-component co-eluting with SP could be detected, while only very small amounts of immunoreactivity eluting at the position of NKA and NKB (dorsal spinal cord only) could be detected using the NKA-antisera. The present results illustrate the importance of the extraction method used in immunochemical studies and demonstrate that the relative proportions of various tachykinins are markedly different in the rat substantia nigra and dorsal spinal cord.

Inhibition of gastric acid secretion in dogs by neurotensin.

Abstract Neurotensin is a tridacapeptide which has been isolated from bovine hypothalamus. The action of synthetic neurotensin was studied on gastric acid secretion in dogs provided with gastric pouches. Intravenously infused neurotensin, 50 ng × kg −1 × min −1 , was found to produce a considerable inhibition of pentagastrin stimulated gastric acid secretion. On the other hand, there was no sign of inhibition of histamine induced gastric acid secretion. The experiments show that neurotensin, isolated from the central nervous system is a potent gastric secretory inhibitor and that it has a selective action in inhibiting gastric acid responses to pentagastrin but not to histamine.

Antisera raised against eledoisin and kassinin detect immunoreactive material in rat tissue extracts: tissue distribution and chromatographic characterization

Radioimmunoassays were developed for the tachykinins eledoisin (ELE) and kassinin (KAS) using antisera raised in rabbits. The antisera exhibited low (less than 0.1%) cross-reactivities to substance P (SP) and physalaemin (PHY), but crossreacted (with one exception, antiserum K7) to varying extents with neurokinin A (NKA) and neurokinin B (NKB). In the rat, the tissue distribution of the immunoreactive material detected by antiserum (E7) raised against ELE and by another antiserum (K1) raised against KAS both resembled that previously described for SP. Using the highly KAS-specific antiserum K7, no or only very low levels of immunoreactivity could be detected in extracts of various rat tissues. Gel permeation chromatography and ion-exchange chromatography of tissue extracts indicated that all antisera (except K7) detected the same population of immunoreactive molecules. One of the components was chromatographically indistinguishable from NKA. The tissue distribution of this component also resembled that of SP. Another immunoreactive component co-chromatographed with NKB at cation exchange chromatography. Acid tissue extracts, but not neutral tissue extracts, were found to contain immunoreactive components which appeared more basic than NKA and NKB. The total levels of immunoreactivity were higher in neutral than in acid tissue extracts. However, the ratio between the amounts of immunoreactivities in the two types of extracts varied considerably between tissues, indicating that tachykinin immunoreactive components may be present in different relative proportions in various tissues.

Neurotensin Increases Colonic Motility

The effects of neurotensin on colonic motility were investigated in 6 healthy volunteers (rectosigmoid area) and 7 patients (ascending colon and at the splenic flexure). Neurotensin (12 pmol/kg X min) infused intravenously for 30 min increased the duration of the contractions to 76% in the ascending colon and 46% in the rectosigmoid area. In the postinfusion period, the values were 42% and 67%, respectively. The motor activity did not change significantly at the splenic flexure. During the infusion period, the motility index increased from 870 to 4500 in the ascending colon and from 332 to 1700 in the rectosigmoid area. In the rectosigmoid area, however, a statistically significant increase was recorded first after cessation of the infusion. All subjects reported increased sensation of intestinal movement after intravenous infusion of neurotensin, and the patients discharged a median volume of 600 ml of bowel contents 20 min after cessation of the infusion. The data show that neurotensin causes an increase in colonic motility in the ascending colon of patients and also, after a latent period, in the rectosigmoid area of healthy subjects.

Characterization of human plasma neurotensin-like immunoreactivity after fat ingestion

The concentration of neurotensin-like immunoreactivity in plasma (p-NTLI) increases after the ingestion of food, and fat seems to be the most important nutrient. It is essential to characterize the NT species that are responsible for this postprandial rise of p-NTLI. After an overnight fast, two male and two female subjects therefore ingested 300 ml of cream (containing 40% (w/w) milk fat). Unextracted plasma samples were subjected to column chromatography and the eluates were analysed using four NT antisera having different specificities. The concentration of chromatographically identified NT(1-13) in peripheral plasma increased significantly from 3 pM in the fasting state to 26 pM 30 min after the ingestion of fat. The concentration of NT(1-8), which is probably a metabolite of NT(1-13), also increased markedly. No significant increase of smaller COOH-terminal sequences of NT was found. The results show that the plasma concentration of NT(1-13) may increase about tenfold following the ingestion of fat. This is further support for the hypothesis that NT(1-13) may function as a hormone.

(Gln4)-Neurotensin Changes the Motility Pattern of the Duodenum and Proximal Jejunum From a Fasting-Type to a Fed-Type

The effect of (Gln4)-neurotensin on the motor activities of the duodenum and small proximal jejunum was investigated in 8 healthy volunteers who fasted for at least 8 h. Motor activity was monitored by measuring the intraluminal pressure at three levels. The proximal site for pressure recording was in the first part of the duodenum, the middle was at the angle of Treitz, and the distal was 25 cm further down. Under control conditions the median time between the migrating motility complexes was 106.3 min. (Gln4)(-Neurotensin, 3 or 6 pmol/kg . min, infused intravenously for 200 min inhibited the migrating motor complexes for 220.4 min (median time) and they were replaced by irregular pressure waves with a frequency of 4-7 waves/min. The plasma concentration of neurotensinlike immunoreactivity increased from 13 +/- 2.8 pM to 131 +/- 6 pM and 231 +/- 16 pM, respectively. Ingestion of fat (55 ml 20% Intralipid) inhibited the migrating motor complex for 196.1 min (median time) and the migrating motor complexes were replaced by irregular pressure waves. The data show that (Gln4)-neurotensin alters the motility pattern in the duodenum and proximal jejunum in man from a fasting- to a fed-type. They further support the contention that neurotensin may function as an endocrine hormone participating in the postprandial regulation of intestinal motility.

Substance P- antagonists: a new type of pharmacological tool

tain large amounts of substance PI’. Kevoked release of substance P has been demonstrated in several tissues including the isolated spinal cordIs. Furthermore. stimulalion of trigeminal nerves causes a netease of substance P in the dental pulp”. The functional significance of substance P is at ppsent under intensive study in several laboratories and it has been assigned a neumtransmitter or neuromodulatory mle in the CNS and in peripheral organs. substall@Psnologucsasan~ Obviously. the availability of specific substance P-antagonists would facilitate the analysis of the physiological function of substance P and also be of importance for a better understanding of the role of sub stance P in pathological conditions. Moreover, on the basis of what we knou about the distribution and effect of sub stance P, it is obvious that substance P-antagonists may fom the basis for a new type of drug. Reports on substance P antagonistic actions of various substances such as arfonad and baclofen’* have been published but subsequent studies indicate that these drugs do not block the effects of substance P specifically in all tissues. In 1975 we started a project with the aim of developing specifK substance Pantagonists on the basis of substance Panalogues. The first step was then to achieve a more or less total loss of agonist uctivity by means of structural suhstitutions. Studies with substance P (Aq’-ProiLys’ - Pm’ - Gins - Gin” - Phe’ - Pheb - Gly” Leu’“-Met”-Ntll) and smaller sequer+ ces of it revealed the imponancc of chain length and of the amino acid residues Phe’. Leu’” and Met” ft>r the smooth muAc activity of substance P. which is confined to the C-terminal hexapeptide sequence of substance P. Among scver;ll suhstanee P-analogues with low agonist activity (&Phe’)_SPand @Leu@‘. bPhe”&SPwcrc found to slightly inhibit the substance P-induced contractionof the terminal ileum

Blood Flow, Oxygen Consumption, and Free Fatty Acid Release in Subcutaneous Adipose Tissue during Hemorrhagic Shock in Control and Phenoxybenzamine-Treated Dogs

Dogs were anesthetized with α-D (+)-glucochloralose. After bleeding to a mean arterial pressure of 55 mm Hg, blood flow in subcutaneous adipose tissue decreased from 6 ± 0.9 (mean ± SE) to 0.6 ± 0.21 ml/min/100 g (P < 0.001), and remained at that low level during bleeding to 35 mm Hg for an additional 90-minute period. In five out of nine experiments the blood flow ceased completely. Sixty minutes after reinfusion, the blood flow was significantly lower than control, and in two there was no blood flow after reinfusion. In animals previously treated with phenoxybenzamine (5 mg/kg), the decrease in blood flow was significant only at 35 mm Hg arterial pressure (2.6 ± 0.67 ml/min/100 g). After reinfusion blood flow increased to a mean above 10 ml/min/100 g, significantly higher than resting blood flow (P < 0.05). There was no significant change in arterial FFA concentration or FFA release in subcutaneous adipose tissue during bleeding and after reinfusion. In phenoxybenzamine-treated animals there was a tendency to have higher arterial FFA concentration and FFA release. The oxygen uptake fell from 0.47 ± 0.07 to 0.13 ± 0.05 ml/min/100 g (P<0.01) during 55 mm Hg arterial pressure and remained significantly lower also during 35 mm Hg arterial pressure. Previous treatment with phenoxybenzamine prevented a fall in O2 uptake in subcutaneous adipose tissue. The decrease in blood flow in subcutaneous adipose tissue during bleeding was more pronounced than found in other organs with same hemorrhagic shock procedure. After reinfusion, flow in subcutaneous adipose tissue could not be restored, indicating irreversible vascular damage, α-receptor activity seems to play a significant part in the development of vascular and metabolic changes in subcutaneous adipose tissue during bleeding.

Adrenergic receptors in adipose tissue and their relation to adrenergic innervation

THE vascular responses in adipose tissue seem to depend on whether noradrenaline (NA) is released from the sympathetic nerve terminal system or whether it is reaching the receptors by means of the vasculature. Thus electric stimulation of adrenergic nerves to adipose tissue invariably causes α-receptor-mediated vasoconstriction, whereas infusion of NA intravascularly may induce β-receptor-mediated vasodilatation1–4. Likewise intravenous tyramine in monkeys causes vasoconstriction in adipose tissue whereas intravenous NA induces vasodilatation5. Since tyramine acts by releasing NA from the sympathetic nerve terminal system, these observations also indicate that NA produces qualitatively different effects depending on how it is delivered to the vascular adrenergic receptors. Our results indicate that the vascular adrenergic α-receptors only are located close to the adrenergic nerve terminal system, whereas the vascular β-receptors may have a different distribution, being farther away from the adrenergic nerve terminals. Consequently, the α-receptors may be affected primarily by NA released from the nerve terminals, whereas the β-receptors are primarily stimulated by circulating catecholamines.

Distribution and fate of dihydroxyphenylalanine-2-14C (DOPA) in mice.

Abstract The distribution in whole mice of intravenously injected dihydroxyphenyl-alanine-2-14C (dopa) has been studied with an autoradiographic technique. The radioactivity initially accumulated in the pancreas and other organs characterized by a rapid protein synthesis, and also in the adrenal medulla. Later on, the radioactivity vanished from most tissues but increased in the adrenal medulla. After 4 days the adrenal medulla was the only site where radioactivity was observed. Separation by paper chromatography revealed that in the pancreas most of the activity was related to dopa. A certain amount was also found in the dopamine fraction. In the adrenal medulla, labelled dopamine and noradrenaline could be identified 30 min after administration Four days after the injection of 14C-dopa, the radioactivity in the adrenal medulla was found to represent mostly adrenaline but also to some extent noradrenaline.