Hans Vilhardt

Substance P in the Median Eminence and Pituitary of the Rat: Demonstration of Immunoreactive Fibers and Specific Binding Sites

The distribution of substance P was demonstrated by immunohistochemical methods, and receptor binding with 125I-8-iodinated 8-tyrosine-substance P as an analog was used to describe possible specific binding sites in the hypothalamo-hypophysial system of the rat. The immunohistochemical experiments showed fibers exhibiting substance P-immunoreactivity in the external and internal layer of the median eminence. In the external layer some of the fibers were located in apposition to the portal capillaries. In the posterior pituitary substance P-immunoreactive fibers were identified. These terminals varied in size and were located near the capillaries as well as in the neuropil. The anterior lobe contained a number of substance P-immunoreactive fibers distributed around the pituitary cells. The kinetic analysis on crude membrane fractions from anterior and posterior pituitaries revealed the presence of a specific binding site for substance P in the anterior pituitary. These data indicate that substance P may be released from the median eminence and posterior pituitary to the vascular system and reach specific receptors in the anterior pituitary. However, a direct innervation of the anterior pituitary is possible. The lack of binding sites in the posterior pituitary indicates that fibers in this organ release substance P into the blood stream.

Basic Pharmacology of Desmopressin

SummaryDesmopressin is an analogue of the antidiuretic hormone vasopressin. The structural changes introduced in the molecule have eliminated the pressor activity of vasopressin and potentiated the antidiuretic action. The metabolic stability of the peptide is also increased. Thus, the plasma half-life for desmopressin is 55 minutes compared with a few minutes for vasopressin. The potent antidiuretic activity of desmopressin makes the peptide ideal for the treatment of cranial diabetes insipidus. However, it may also be used for nocturnal enuresis and as a diagnostic agent in determining renal concentrating capacity. Desmopressin has recently been shown to be successful in the treatment of certain bleeding disorders.

Oxytocin receptor binding and uterotonic activity of carbetocin and its metabolites following enzymatic degradation

Metabolites of the analogue 1-deamino-1-carba-2-tyrosine(O-methyl)-oxytocin (carbetocin) following incubation with a rat kidney homogenate were isolated and their pharmacodynamic properties investigated. Apart from the parent compound two metabolites were identified namely desGlyNH2-carbetocin (carbetocin metabolite I) and desLeuGlyNH2-carbetocin (carbetocin metabolite II). Both carbetocin, carbetocin metabolite I and carbetocin metabolite II displayed binding affinities to the myometrial oxytocin receptor of a similar magnitude as oxytocin. Carbetocin was found to have agonistic properties on isolated myometrial strips and it was found to exert this effect through generation of inositol phosphates, as is the case for oxytocin. However, maximal contractile effect of carbetocin was approximately 50% lower than that of oxytocin (2.70 +/- 0.12 g compared to 5.22 +/- 0.26 g) and EC50 was approximately ten times higher (48.0 +/- 8.20 nM compared to 5.62 +/- 1.22 nM). Neither carbetocin metabolite I nor carbetocin metabolite II were able to contract isolated myometrial tissue. All three compounds displayed antagonistic properties against oxytocin in vitro, with carbetocin being the strongest inhibitor (pA2 = 8.21) and carbetocin metabolite II (pA2 = 8.01) being stronger than carbetocin metabolite I (pA2 = 7.81). These results indicate that carbetocin is a partial agonist/antagonist to the oxytocin receptor while the two metabolites carbetocin metabolite I and carbetocin metabolite II are pure antagonists. All three analogues bound to the myometrial vasopressin V1 receptor, albeit with much lower affinities than to the oxytocin receptor. Carbetocin metabolite II showed the weakest binding affinity of 33.7 +/- 7.34 nM compared to 7.24 +/- 0.29 nM for carbetocin and 9.89 + 2.80 nM for carbetocin metabolite I. Only carbetocin bound to the renal vasopressin V2 receptor though the binding affinity was very low (61.3 +/- 14.6 nM).

Impermeability of the blood-cerebrospinal fluid barrier to 1-deamino-8-D-arginine-vasopressin (DDAVP) in patients with acquired, communicating hydrocephalus

Abstract. The passage of the vasopressin analogue, 1‐deamino‐8‐D‐arginine‐vasopressin (DDAVP) into the cerebrospinal fluid (CSF) following intravenous (i.v.) administration was studied in human subjects. After i.v. injection of DDAVP, resulting in plasma levels exceeding normal plasma concentrations of vasopressin (AVP) by 100‐fold, serial blood and ventricular CSF specimens were sampled during 2 h. Plasma and CSF were analysed by radioimmunoassay using two different AVP antisera: one specific against natural AVP and another cross‐reacting 100% with DDAVP. No changes in the joint concentrations of DDAVP + AVP in CSF were found following i.v. infusion of DDAVP when compared with the concentration before the infusion. Neither was any difference between CSF concentrations of AVP and DDAVP + AVP observed at any sampling time following the i.v. DDAVP administration. Hence, it is concluded that no measurable amounts of DDAVP can be found in the CSF for up to 2 h after i.v. administration of the peptide, and that for all practical purposes a blood‐CSF barrier to DDAVP exists.

Growth Hormone Releasing Hexapeptide (GHRP-6) Activates the Inositol (1,4,5)-Trisphosphate/Diacylglycerol Pathway in Rat Anterior Pituitary Cells

Growth hormone-releasing hexapeptide (GHRP-6) is known to stimulate secretion of growth hormone (GH) in vivo and in vitro in a variety of species. However, the cellular effects of GHRP-6 remain largely unknown. We have tested the influence of GHRP-6 on the inositol phospholipid second messenger system in cultured anterior pituitary cells. Cultured pituitary cells responded upon challenge with GHRP-6 with a dose-dependent release of GH. Moreover, incubation of GHRP-6 with pituitary cell cultures labelled with myo-[3H]inositol resulted in a dose-dependent rise in [3H]inositol phosphates. Brief stimulation of pituitary cells with GHRP-6 increased phosphorylation of MBP4-14, a specific protein kinase C substrate, when incubated with the cytosol- or plasma membrane fraction from the stimulated cells. Furthermore, introduction of MBP4-14 into the cytosol in digitonin permeabilized pituitary cells caused increased phosphorylation of this substrate. GHRP-6 induced a rise in intracellular Ca2+ in individual somatotrophs loaded with the Ca2+ indicator, Fura-2. Preincubation (3 min) with somatostatin (SRIF) diminished the Ca2+ spike elicited by GHRP-6, while no effect of SRIF was observed when added simultaneously with GHRP-6. These results indicate that GHRP-6-stimulated GH-secretion involves the diacylglycerol/inositol(1,4,5)trisphosphate pathway with a resulting rise in cytosolic Ca2+.

Antidiuretic response in conscious dogs following peroral administration of arginine vasopressin and its analogues

Conscious dogs were subjected to a constant water load causing water diuresis. When arginine vasopressin or analogues of vasopressin were administered through a gastric tube a dose-dependent antidiuretic response occurred. All the peptides tested proved to be antidiuretic in doses of 50 micrograms or less with 1-deamino-8-D-arginine vasopressin and 1-deamino-4-asparagine-8-D-arginine vasopressin being the most potent substances. No pressor response was observed even after 1 mg of AVP.

Oxytocin receptors and contractile response of the myometrium after long term infusion of prostaglandin F2α, indomethacin, oxytocin and an oxytocin antagonist in rats

Binding of [3H]oxytocin to isolated myometrial plasma membranes was not affected by the presence of prostaglandin (PG)F2 alpha or E2 in the incubation medium. Long-term treatment with PGF2 alpha or indomethacin had no effect on oxytocin receptor concentrations and dissociation constants of myometrial plasma membranes nor on maximal contractility or KM values of isolated uterine strips exposed to oxytocin. Infusion of oxytocin for 5 days in non-pregnant rats resulted in a decrease in oxytocin receptor concentrations in myometrial plasma membranes whereas the binding affinity to oxytocin was unaffected. Isolated uterine strips from similarly treated rats showed a reduced maximal contractile response to oxytocin and an elevated KM value, possibly indicating an influence of oxytocin on the coupling between receptor occupancy and contractility. Treatment for 5 days with desamino1-[D-Tyr(O-ethyl)2-Thr4-Orn8] oxytocin (an oxytocin antagonist) increased the concentration of myometrial oxytocin receptors. In addition KD values of these receptors were elevated. The present results indicate that prolonged exposure to oxytocin leads to a down-regulation of the myometrial receptor concentration, which is not caused by ligand-receptor interaction in itself. The concerted effect of oxytocin and prostaglandins on myometrial contraction does not appear to involve modulation of the oxytocin receptor by prostaglandins.

Absorption of intragastrically administered DDAVP in conscious dogs

Plasma concentrations of DDAVP were measured after intragastric administration and intravenous infusion in dogs. Oral ingestion of DDAVP led to a dose dependent increase in peak plasma concentrations as well as area under the curve (AUC). Intravenous infusion of DDAVP (0.13 pmol/l min) resulted in a mean steady-state level of 20.3 pmol/l. Elimination half-lives for oral DDAVP were 77.6 and 76.1 min for low and high doses respectively. T1/2 estimated from the ascending part of the i.v. infusion curve was 50 min. A metabolic clearance rate (MCR) of 3.9 ml/kg . min was assessed from the i.v. steady-state level.

Tachykinins Induce Secretion of Prolactin from Perifused Rat Anterior Pituitary Cells by Interactions with Two Different Binding Sites

Substance P and the two other mammalian tachykinins, neurokinin A and B, are accepted to have direct regulating effects at the anterior pituitary level. We have examined the effects of substance P (SP) and neurokinin B (NKB), alone and in combination, on prolactin release from cultured anterior pituitary cells grown on collagen-coated micro beads and placed in a perfusion system. Prolactin (Prl) secretion was observed within 25 s after exposure to either secretagogue and reached a maximum within 60-80 s. Furthermore, the prolactin response induced by SP and NKB was dose-dependent. Prl secretion remained constant for up to 4 h when SP or NKB were perifused and then fell gradually towards basal levels. Simultaneous addition of submaximal concentrations of SP and NKB resulted in an additive response compared with the responses of either secretagogue alone. Continuous (8 h) perifusion with SP did not prevent a normal prolactin response by NKB or TRH. These results indicate that the tachykinins, substance P and neurokinin B, release Prl from perifused female rat anterior pituitary cells by interaction with two different receptors, possibly the NK1 and NK3 tachykinin receptor subtypes.

DDAVP factor VIII concentrate and its properties in vivo and in vitro

DDAVP fraction I-0 was prepared from a plasma pool derived from 80 blood donors who had received DDAVP and tranexamic acid. A control fraction I-0 was prepared from a plasma pool from the same group of blood donors under identical conditions except for the drug treatment. The DDAVP plasma pool as well as the DDAVP fraction I-0 contained 2–3 times as much VIII:C as the controls. VIIIR:Ag increased to a lesser extent. No difference in stability of VIII:C was seen. In vivo studies showed that infusion of the DDAVP fraction I-0 to 3 patients with severe haemophilia A (<0.01 i.u. VIII:C/ml) caused a 2–3 times higher increase in VIII:C than after infusion of the same volume of the control fraction I-0. No difference in disappearance rate of VIII:C was seen. Both factor VIII concentrates normalised the prolonged bleeding time and caused a secondary VIII:C increase in VWD. It is concluded that administration of DDAVP to blood donors prior to collection of blood increases the content of VIII:C in the ensuing factor VIII concentrate and secondly, that such stimulation has no demonstrable qualitative effect on the factor VIII obtained.