Epinephrine but not vasopressin attenuates the airway response to anaphylactic shock in rats.

PURPOSE: The two life-threatening signs of anaphylactic shock (AS) are severe arterial hypotension and bronchospasm. Guidelines recommend epinephrine as first-line treatment. Arginine vasopressin (AVP) has been proposed as an alternative if epinephrine does not correct arterial hypotension. These two drugs may have beneficial, neutral or deleterious effects on airflow either directly or by modifying factors that regulate vasodilatation and/or edema in the bronchial wall. AIM OF THE STUDY: To compare the effects of epinephrine and AVP on airflow and airway leakage in a rat model of AS. MATERIALS AND METHODS: Thirty-two ovalbumin-sensitized rats were randomized into four groups: control (CON), AS without treatment (OVA), AS treated with epinephrine (EPI), and AS treated with AVP (AVP). Mean arterial pressure (MAP), respiratory resistance and elastance and microvascular leakage in the airways were measured. RESULTS: All OVA rats died within 20 minutes following ovalbumin injection. Ovalbumin induced severe arterial hypotension and airway obstruction (221 ± 36 hPa.s.L-1 vs. vehicle 52 ± 8 hPa.s.L-1; p < 0.0001) associated with microvascular leakage distributed throughout the trachea, bronchi and intra-pulmonary airways. EPI and AVP extended survival time; EPI restored a higher level of MAP than AVP. Airway obstruction was attenuated by epinephrine (146 ± 19 hPa.s.L-1; p < 0.0001), but not by AVP (235 ± 58 hPa.s.L-1; p = 0.42). CONCLUSIONS: Epinephrine was superior to AVP for alleviating the airway response in a rat model of AS. When bronchospasm and severe arterial hypotension are present during AS, epinephrine should be the drug of choice.

Altered agonist sensitivity of a mutant v2 receptor suggests a novel therapeutic strategy for nephrogenic diabetes insipidus.

Loss-of-function mutations of the type 2 vasopressin receptor (V2R) in kidney can lead to nephrogenic diabetes insipidus (NDI). We studied a previously described, but uncharacterized, mutation of the V2R (N321K missense mutation) of a patient with NDI. The properties of the mutant receptor were evaluated. We constructed a highly sensitive Epac-based bioluminescence resonance energy transfer biosensor to perform real-time cAMP measurements after agonist stimulation of transiently transfected HEK293 cells with V2Rs. ß-Arrestin binding of the activated receptors was examined with luciferase-tagged ß-arrestin and mVenus-tagged V2Rs using the bioluminescence resonance energy transfer technique. Cell surface expression levels of hemagglutinin-tagged receptors were determined with flow cytometry using anti-hemagglutinin-Alexa 488 antibodies. Cellular localization examinations were implemented with fluorescent tagged receptors visualized with confocal laser scanning microscopy. The effect of various vasopressin analogs on the type 1 vasopressin receptor (V1R) was tested on mouse arteries by wire myography. The N321K mutant V2R showed normal cell surface expression, but the potency of arginine vasopressin for cAMP generation was low, whereas the clinically used desmopressin was not efficient. The ß-arrestin binding and internalization properties of the mutant receptor were also different than those for the wild type. The function of the mutant receptor can be rescued with administration of the V2R agonist Val(4)-desmopressin, which had no detectable side effects on V1R in the effective cAMP generating concentrations. Based on these findings we propose a therapeutic strategy for patients with NDI carrying the N321K mutation, as our in vivo experiments suggest that Val(4)-desmopressin could rescue the function of the N321K-V2R without significant side effects on the V1R.

Insulin potentiates AVP-induced AQP2 expression in cultured renal collecting duct principal cells.

In the renal collecting duct (CD), water reabsorption depends on the presence of aquaporin-2 (AQP2) in the apical membrane of principal cells. AQP2 expression and subcellular repartition are under the control of AVP. Some pieces of experimental evidence indicate that additional hormonal factors, including insulin, may also control AQP2 expression and thereby CD water permeability. We have previously shown that AVP induces endogenous AQP2 expression in cultured mouse mpkCCD(cl4) CD principal cells (23). In the present study, we investigated the effect of insulin on AQP2 expression in mpkCCD(cl4) cells. Addition of insulin to the basal medium of cells grown on filters slightly increased AQP2 mRNA and protein expression, whereas insulin potentiated the effect of AVP. The potentiation of AVP-induced AQP2 expression by insulin was abolished by actinomycin D, a transcriptional inhibitor. Analysis of AQP2 protein expression under conditions of AVP washout and/or in the presence of chloroquine, a lysosomal degradation inhibitor, revealed that insulin did not significantly alter AQP2 protein degradation. Inhibition of ERK, p38 kinase, and phosphatidylinositol 3'-kinase (PI 3-kinase) activities prevented the insulin-induced stimulation of AQP2 expression, whereas inhibition of PKC has no effect. Taken together, our results indicate that insulin increased AQP2 protein expression mostly through increased AQP2 mRNA levels in cultured mpkCCD(cl4) cells. This effect most likely relies on increased AQP2 gene transcription in response to MAPK and PI 3-kinase activation.

Identification and pharmacological characterization of native, functional human urotensin-II receptors in rhabdomyosarcoma cell lines.

1 In an effort to identify endogenous, native mammalian urotensin-II (U-II) receptors (UT), a diverse range of human, primate and rodent cell lines (49 in total) were screened for the presence of detectable [125I]hU-II binding sites. 2 UT mRNA (Northern blot, PCR) and protein (immunocytochemistry) were evident in human skeletal muscle tissue and cells. 3 [(125)I]hU-II bound to a homogenous population of high-affinity, saturable (Kd 67.0+/-11.8 pm, Bmax 9687+/-843 sites cell(-1)) receptors in the skeletal muscle (rhabdomyosarcoma) cell line SJRH30. Radiolabel was characteristically slow to dissociate (< or =15% dissociation 90 min). A lower density of high-affinity U-II binding sites was also evident in the rhabdomyosarcoma cell line TE671 (1667+/-165 sites cell(-1), Kd 74+/-8 pm). 4 Consistent with the profile recorded in human recombinant UT-HEK293 cells, [125I]hU-II binding to SJRH30 cells was selectively displaced by both mammalian and fish U-II isopeptides (Kis 0.5+/-0.1-1.2+/-0.3 nm) and related analogues (hU-II[4-11]>[Cys(5,10)]Acm hU-II; Kis 0.4+/-0.1 and 864+/-193 nm, respectively). 5 U-II receptor activation was functionally coupled to phospholipase C-mediated [Ca2+]i mobilization (EC50 6.9+/-2.2 nm) in SJRH30 cells. 6 The present study is the first to identify the presence of 'endogenous' U-II receptors in SJRH30 and TE671 cells. SJRH30 cells, in particular, might prove to be of utility for (a) investigating the pharmacological properties of hU-II and related small molecule antagonists at native human UT and (b) delineating the role of this neuropeptide in the (patho)physiological regulation of mammalian neuromuscular function.

[Localization of proline-rich peptide (galarmin) in brain structures of rats (immunohistochemical study)]. / Lokalizatsiia obogashchennogo prolinom peptida (galarmina) v strukturakh mozga krys (immunogistologicheskoe issledovanie).

In the present study the immunohistochemical localization of proline-rich peptide, so called galarmin, was examined in the brain structures of intact and galarmin-treated rats. Galarmin (a fragment of neurophysin II C-end glycopeptide) was isolated by A.A. Galoyan and coworkers in 1997, from the neurosecretory granules of bovine neurohypophysis, produced by the hypothalamic magnocellular nuclei. In intact rats galarmin-immunoreactive neurons and nerve fibers were widely distributed in the central nervous system. Single intramuscular injection of galarmin to the rats resulted in the increase of both galarmin-immunoreactivity and the number of galarmin-immunoreactive nerve cells, fibers and capillaries. In control experiments where the antisera against the fragment of immunophilin (a receptor of immunosuppressor macrolide FK-506) and the pancreatic neuropeptide Y were used as the primary antibodies, the significant increase of neuropeptide Y-immunoreactive nerve fibers and immunophilin-positive lymphocytes was revealed in galarmin-treated rats. Based on these results and the data on the motoneurons regeneration in the spinal cord hemisectioned rats given galarmin daily for 3 weeks, galarmin has been suggested to act as an immunomodulator, neurotransmitter and neuroregulator.

Neurophysin stimulates prolactin release from primary cultured rat pituitary cells.

The neurohypophysial hormones, oxytocin and vasopressin, are present as non-covalently bound complexes with their designated neurophysin in the secretory granules of the posterior pituitary. The neurophysins are generally considered to be biologically inert carrier proteins for oxytocin and vasopressin. We have examined the actions of bovine neurophysin-I (bNP-I), bovine neurophysin-II (bNP-II), rat neurophysin (rat NP) and oxytocin on prolactin release using primary cultured rat pituitary cells. A dynamic perifusion system was chosen to test their stimulatory actions. The rat NP and bNP-II stimulated prolactin release. It is a new observation that rat NP and bNP-II stimulate prolactin release from primary cultured rat pituitary cells. The maximum sensitivities, the lowest concentration which stimulate prolactin release, of rat NP, bNP-II, bNP-I and oxytocin in primary cultured cells were 1 nmol/1, 1 nmol/l, 1000 nmol/1 and 1 nmol/1 respectively. The maximum sensitivities of rat NP and bNP-II were within the physiologically relevant concentrations.

The pharmacological potential of neurotrophins: a perspective.

Until recently nerve growth factor (NGF) was the only widely characterized neurotrophic factor which had been shown both in vitro and in vivo to be essential for the survival of selected populations of neurons during development and to be important for maintenance of the differentiated phenotype of mature neurons. The recent cloning of new members of the NGF family, namely brain-derived neurotrophic factor neurotrophin-3 (NT-3), NT-4 and NT-5, has greatly expanded our knowledge of the structural properties and neurotrophic activities of these proteins. Elucidation of their developmental and topographical expression and associated receptors in both the central nervous system and peripheral nervous system is proceeding at a brisk pace, leading to proposals for a potential pharmacological use of these proteins. This possibility will ultimately rely upon a more complete understanding of the roles of these trophic factors in human nervous system physiology and pathology.

The C-terminal glycopeptide of propressophysin potentiates excitatory transmission in the rat lateral septum.

Effects of peptides synthesized from the same precursor as vasopressin, i.e. the C-terminal 39-amino acid long glycopeptide and neurophysin II, were investigated for biological activities in electrophysiological experiments in brain slices of the rat lateral septum. These slices contained the glycopeptide as the predominant form and a fragment of it, amino acid sequence 22-39, as a minor form (8% of the glycopeptide 1-39), as shown by high performance liquid chromatography of extracts and by radioimmunoassay. None of the peptides, neurophysin II, the glycopeptide 1-39 and the fragment 22-39, tested in a concentration of 10(-12) M, had measurable effects on the resting membrane potential of the neurons. The glycopeptide and the fragment 22-39, however, increased, in some cells, for tens of minutes the excitatory postsynaptic potentials evoked in these neurons by stimulation of the fimbria fibers. The increase in input resistance, seen in many septal neurons treated with either of the peptides was not correlated with the excitatory postsynaptic potential increase. Neurophysin II affected neither the excitatory postsynaptic potentials nor the input resistance of the neurons. It is concluded that the glycopeptide 1-39 and the fragment 22-39 possess biological activities amongst which the facilitation of excitatory amino acid transmission on lateral septum neurons. Therefore, these peptides derived from the vasopressin precursor may act in concert with vasopressin to establish facilitation of excitatory transmission in the brain.

Vasopressin, vasopressin fragments and a C-terminal peptide of the vasopressin precursor share cardiovascular effects when microinjected into the nucleus tractus solitarii.

Arginine-vasopressin (VP), the VP fragments VP-(1-8), [pGlu4,Cyt6]VP-(4-9) and -(4-8), and a fragment of the C-terminal glycopeptide of the VP precursor [CPP-(22-39)] decreased blood pressure and heart rate in anesthetized rats when microinjected bilaterally into the nucleus tractus solitarii (NTS). The magnitude of the effect was similar at doses of 20 pg, except for VP-(1-8) which required about 500 pg. The strongest decrease in blood pressure and heart rate was observed with 100 pg VP. Higher doses of other peptides either were ineffective [CPP-(22-39)] or increased blood pressure [( pGlu4,Cyt6]VP-(4-9) and -(4-8)). VP metabolites thus may participate in the control of cardiovascular functions exerted by VP. The effect of CPP-(22-39) indicates that the C-terminal glycopeptide of the VP precursor contains biological activity, which may support the cardiovascular action of VP in the NTS.

Bovine 'neurophysin II' stimulates growth hormone release in the estradiol-primed male rat.

Basal plasma concentrations of growth hormone (GH) were monitored in both normal and estradiol-primed male rats by the collection of sequential blood samples from freely moving rats, via chronic intraatrial cannulae. Blood was sampled every 2 min for a period of 80 min and plasma GH levels determined by radioimmunoassay. The normal male rats displayed a pulsatile release of GH, while the estradiol-primed male rats exhibited a relatively steady level of plasma GH concentration. The rats exposed to high levels of estradiol (1.59 +/- 0.42 nmol/l plasma) also had a higher mean value of basal GH concentration. An injection of 100 micrograms/kg of bovine neurophysin II did not alter GH release in the normal male rats. However, it did significantly elevate GH levels in the estradiol-primed animals to a mean peak level approximately six times the mean basal level. The administration of 100 micrograms/kg of bovine neurophysin I to estradiol-primed male rats did not produce any change in plasma GH levels and thus eliminates the possibility of the nonspecific stimulation of neurophysin II on GH release.

Effect of neurophysin on enzymatic maturation of oxytocin from its precursor.

We examined the extent to which rates of enzymatic conversion of the oxytocin biosynthetic precursor to mature peptide are modulated by intramolecular and intermolecular assembly of precursor and polypeptide intermediates. The biosynthesized precursor contains hormone and neurophysin sequences linked by a Gly-Lys-Arg sequence and undergoes enzymatic processing reactions which include endoproteolytic cleavage at the Lys-Arg dibasic sequence, carboxypeptidase B-like exoproteolytic cleavage, and enzymatic amidation. We evaluated the effect of neurophysin on such processing reactions using semisynthetic precursors of oxytocin/bovine neurophysin I and synthetic oxytocinyl precursor intermediates as substrates. Neurophysin I at high concentration (0.7 mM) reduced the rates of carboxy-peptidase B-like conversion of oxytocinyl-Gly-Lys-Arg to oxytocinyl-Gly and the enzymatic amidation of oxytocinyl-Gly to mature (C-terminal amidated) oxytocin. The dependence of rate suppression on the concentrations of peptide substrate and neurophysin I suggested that suppression is due to intermolecular formation of hormone-neurophysin complexes which are aggregated at least to dimers. An analogous intramolecular neurophysin effect was found for endoproteolytic processing of semisynthetic precursors. Endoproteinase Lys-C cleaved the Lys11-Arg12 peptide bond in a native-like semisynthetic precursor at a significantly slower rate than it did an assembly-deficient precursor analogue. The difference in semisynthetic precursor endoproteolysis rates is most substantial at the high concentrations at which the native-like precursor would form dimers but the assembly-deficient analogue would not. The native-like semisynthetic precursor was more stable than the assembly-deficient precursor analogue to tryptic digestion. The concentration-dependent effects of neurophysin, both intramolecularly as a precursor domain and intermolecularly as an interacting protein, are likely to occur in the secretory granules in which the biosynthetic precursors are packaged. The molecular organization of both hormone/neurophysin precursors and the noncovalently complexed hormone-neurophysin intermediates can be expected to play a role in modulating enzymatic processing reactions that lead to mature neurohypophysial hormones.

Modulation of social memory in male rats by neurohypophyseal peptides.

Adult male rats spend a great amount of time investigating novel juveniles. In contrast, rats re-exposed to the same juvenile 30 min after the initial exposure display little investigatory behavior. If the re-exposure occurs 2 h later, the juvenile is thoroughly investigated. These results have been interpreted to mean that rats form a transient memory for a particular juvenile. In the present study, memory was enhanced when the initial exposure to the juvenile was followed by another exposure to the same juvenile (retroactive facilitation) and impaired when exposure to the original juvenile was followed by exposure to another juvenile (retroactive interference). Arginine vasopressin had retroactive facilitating effects on social memory and these effects were blocked by the vasopressor antagonist dPTyr(Me)AVP. Moreover, the antagonist had retroactive interfering effects, since it impaired the recognition of a familiar juvenile. Oxytocin shared the same inhibitory pattern of action. These results suggest that neurohypophyseal peptides may have a prepotent role in modulating the mnemonic processing of chemosensory information associated with social interactions.

Bovine neurophysin II has prolactin-releasing activity in the estradiol-primed male rat.

In the course of the search for the prolactin-releasing factor (PRF), we noticed that the posterior pituitary contained strong PRF activity and subsequently traced this activity to that of bovine neurophysin-II (NP-II). NP-II prepared in our lab was judged to be a homogeneous preparation according to Sephadex G-75 gel filtration, DEAE ion exchange chromatography and polyacrylamide gel electrophoresis. While neurophysin-I (NP-I) injections of 100 micrograms/kg and 1,000 micrograms/kg elevated plasma prolactin in estradiol-primed male rats to 30% and 50% over the control value, respectively, NP-II doses of 100, 1,000 micrograms/kg increased plasma prolactin concentration in estradiol-primed male rats to 130% and 170% over the control value, respectively. The lowest dose of NP-II needed to increase plasma prolactin concentration was 10 micrograms/kg. Since neurophysin does not stimulate prolactin release from the ectopic pituitary under the kidney capsule nor from lactotrophs in a primary monolayer culture system, neurophysin is believed to act indirectly on the pituitary, presumably through a neurotransmitter and/or hypothalamic releasing (or inhibiting) factor. We propose that NP-II may be one element in the complex chain of the prolactin-releasing mechanism.

Neurohypophyseal peptides maintain tolerance to the incoordinating effects of ethanol.

Arginine vasopressin (AVP), lysine vasopressin (LVP) and [des-9-glycinamide]LVP (DGLVP), administered systemically, delayed the disappearance of functional tolerance to the motor-incoordinating effect of ethanol in mice. This result is consistent with previous findings that AVP and related neuropeptides maintain tolerance to the sedative-hypnotic and hypothermic effects of ethanol, and suggests that the peptides modulate the rate of disappearance of tolerance per se, rather than simply influencing the tests used to evaluate tolerance. However, both the duration of tolerance to the incoordinating effect of ethanol, and the duration of peptide maintenance of this tolerance, were less than those observed for tolerance to the hypnotic and hypothermic effects of ethanol. Tolerance to various effects of ethanol clearly can develop and dissipate at different rates, and our results suggest that the characteristics of the maintenance of ethanol tolerance by neurohypophyseal peptides are influenced, to some extent, by the neural systems which mediate the expression of the functional tolerance which is being investigated.

The Mr 80,000 common forms of neurophysin and vasopressin from bovine neurohypophysis have corticotropin- and beta-endorphin-like sequences and liberate by proteolysis biologically active corticotropin.

We have tested the hypothesis that the high M(r) forms common to both neurophysin and vasopressin detected in bovine neurohypophysis extracts (Nicolas, P., Camier, M., Lauber, M., Masse, M.-J. O., Möhring, J. & Cohen, P. (1980) Proc. Natl. Acad. Sci. USA 77, 2587-2591) might also contain the sequences of other known neuropeptides. The following evidence indicates that corticotropin- and beta-endorphin-like sequences are associated with similar high M(r) forms and are included in these M(r) 80,000 molecules. During the fractionation steps of high M(r) material, both corticotropin and beta-endorphin immunoreactive species were found to coelute with the neurophysin and vasopressin ones, either under M(r) 140,000 (in 0.1 M formic acid) or M(r) 70,000-80,000 (in 6 M guanidine) elution volumes. Corticotropin immunoreactivity was found to cofocus at pIs 6.05 and 5.8 with the M(r) 80,000 neurophysin-containing species. This material was submitted to affinity chromatography on purified anti-neurophysin antibodies covalently attached to Sepharose 4B. Both the corticotropin and beta-endorphin immunoreactivities, together with the neurophysin and vasopressin immunoreactivities, were retained on the immunoadsorbent and codesorbed by either a drastic pH change or by selective displacement with an excess of neurophysin. Comparison of the tryptic-digest maps of either the M(r) 68,000 fragment immunoprecipitated by anti-corticotropin antibodies or the M(r) 68,000 fragment released after precipitation of the M(r) 80,000 species by anti-neurophysin antibodies indicated large sequence homologies. Exposure of either the M(r) 80,000 or 68,000 components to mild proteolytic activities resulted in the formation of lower-size fragments. The resulting corticotropin-like immunoreactive material, recovered under the elution volume of standard (125)I-labeled corticotropin-(1-24), was tested for its ability to activate glucocorticoid biogenesis by the amphibian interrenal tissue (adrenal) in perifusion. It was found to exhibit a noticeable activity qualitatively undistinguishable from the one of the reference human corticotropin-(1-39). The name neurohypophyseal "coenophorin" (from the Greek word for common) is proposed for this class of M(r) 80,000 polypeptides that might represent the common precursor store-house for a set of neuropeptides produced in the hypothalamo-neurohypophyseal tract.

Time-dependency of neurohypophyseal peptide attenuation of puromycin amnesia in mice.

[Ile3, Arg8]vasopressin (arginine-vasotocin), as well as the C-terminal tripeptides of the neurohypophyseal hormones arginine and lysine vasopressin, Pro-Arg-Gly-NH2 and Pro-Lys-Gly-NH2, were protective against puromycin-induced amnesia in mice when administered 24h before training. The N-protected tripeptide derivative, Z-Pro-Lys-Gly-NH2, was effective when given 5 days before training. The effectiveness of all peptides to attenuate puromycin-induced amnesia decreased as the interval between training and peptide treatment increased, indicating that the peptides influence memory processes, rather than general arousal. Z-Pro-Lys-Gly-NH2 was active at 24h after training, when the other peptides were no longer effective. Although it seems clear that neurohypophyseal hormones per se can attenuate puromycin-induced amnesia, these results are in line with the possibility that some portion of hormone action may be mediated via formation of longer-lived hormone fragments in the CNS.

Failure to demonstrate a natriuretic effect of neurophysin in rats.

Partly purified rat neurophysin was tested for its possible natriuretic activity in acutely hypophysectomized saline-loaded rats (hypophysectomized 2 h prior to saline infusion). Such animals were unable to excrete the saline load. When partly purified rat neurophysin was injected i. p. in a dose of 40 microgram per animal (which corresponds to its content in one rat posterior pituitary) before saline loading, the sodium excretion was not different from that found in acutely hypophysectomized animals, but it was significantly lower than in normal saline-loaded rats. It was concluded that any apparent natriuretic effect of rat neurophysin could not be established by the present experiments under the experimental conditions used.