Modulation of vascular contractile responses to α1-and α2-adrenergic and neuropeptide Y receptor stimulation in rats with ischaemic heart failure

In order to evaluate adaptational changes in vascular function in congestive heart failure (CHF), we studied the contractile responses of isolated arterial and venous blood vessels from rats suffering from CHF induced by coronary artery ligature, resulting in a myocardial infarction. The contractile responses of the basilar, femoral and renal arteries and of the iliac vein were examined in relation to adrenergic and neuropeptide Y (NPY) receptor function by the action of the α₁ agonist phenylephrine, the α₂ agonist clonidine and NPY. The contractile force was measured (in mN) and in% of K⁺-induced contraction as well as pD₂ to each agonist. When stimulated by a 60 mM K⁺-buffer solution, the femoral and renal arteries from CHF rats responded with a stronger contraction (E_max; 9.4 ± 0.6 and 9.8 ± 0.6mN) than the corresponding Sham vessels (E_max; 6.2 ± 0.7 and 5.6 ± 0.4 mN respectively, P < 0.001). On the contrary, the iliac vein of CHF responded less to K⁺ than the Sham iliac vein (E_mas 2.5 ± 0.2 and 3.7 ± 0.5 mN, P < 0.01). The CHF iliac vein responded with a weaker contraction when stimulated with phenylephrine (E_max 1.9 ± 0.4 mN) and showed a lower sensitivity (pD₂ 5.6 ± 0.1) than the corresponding sham vessel (E_max 5.7 ± 2.3mN and pD₂ 6.3 ± 0.5, P < 0.05). The CHF renal artery was less sensitive to clonidine (pD₂ 6.4 ± 0.6) than the Sham renal artery (pD₂ 7.2 ± 0.1, P < 0.05). The results indicate differences between CHF and Sham vessel segments according to both contractile capacity induced by K⁺-depolarization and to agonist induced contractile capacity and sensitivity. The differences are not of general nature but vary according to the vascular bed examined.     

Effects of subacute treatment with toluene on cerebrocortical α- and β-adrenergic receptors in the rat. Evidence for an increased number and a reduced affinity of β-adrenergic receptors

Subacute treatment with toluene (80–1500 p.p.m.) produces a dose-dependent reduction of affinity and increase in density of the β-adrenergic antagonist [³H]dihydroalprenolol binding sites in the frontoparietal cortex of the male rat, while the binding characteristics of a,-adrenergic ([³H]WB 4101) and α₂^-adrenergic ([³H]p-aminoclonidine) binding sites in the same region is unaffected by this treatment as evaluated in vitro. Therefore, it is suggested that the cortical β-adrenergic receptors are particularly vulnerable to the action of toluene in vivo. It is speculated that as a result cortical β-adrenergic neurotransmission may be altered following exposure to low concentrations of toluene, possibly related to the physico-chemical properties of toluene, leading to changes in membrane fluidity.     

Naturally processed peptides from HLA-DQ7 (α1*0501-β1*0301): influence of both α and β chain polymorphism in the HLA-DQ peptide binding specificity

Self peptides bound to HLA-DQ7 (α1*0501-β1*0301), one of the HLA molecules associated with protection against insulin-dependent diabetes mellitus, were characterized after their acid elution from immunoaffinity-purified HLA-DQ7 (α1*0501-β1*0301) molecules. The majority of these self peptides derived from membrane-associated proteins including HLA class I, class II, class II-associated invariant chain peptide and the transferrin-receptor (TfR). By in vitro binding assays, the specificity of these endogenous peptides for HLA-DQ7 (α1*0501-β1*0301) molecules was confirmed. Among these peptides, the binding specificity of the TfR 215 – 230 self peptide was further examined on a variety of HLA-DQ and DR dimers. Several findings emerged from this analysis: (1) this peptide displayed HLA-DQ allelic specificity, binding only to HLA-DQ7 (α1*0501-β1*0301); (2) when either the DQα or DQβ chain was exchanged, little or no binding was observed, indicating that specificity of HLA-DQ peptide binding was determined by polymorphic residues of both the α and β chains. (3) Unexpectedly, the TfR 215 – 230 self peptide, eluted from DQ, was promiscuous with regard to HLA-DR binding. This distinct DR and DQ binding pattern could reflect the structure of these two molecules as recently evidenced by crystallography.     

The peptide binding motif of the disease associated HLA-DQ (α 1* 0501, β 1* 0201) molecule

To identify the binding motifs of peptides which bind to the celiac disease and insulin-dependent-diabetes-mellitus (IDDM)-associated DQ2 molecule, peptides were eluted from affinity-purified DQ2 molecules. The eluted peptides were separated by reverse-phase HPLC. Prominent peptide peaks and the remaining pool of peptides were sequenced by Edman degradation. Truncated variants of eight different peptides with a length of 9–19 amino acids were identified; among them class II-associated invariant chain peptides (CLIP) and peptides that stem from HLA class I α, HLA-DQα1*0501, Ig and CD20 molecules. Data from the pool sequencing and the biochemical binding analyses of synthetic variants of an eluted high-affinity ligand (HLA class I α 46–60), indicate that the side chains of amino acid residues at relative position P1 (bulky hydrophobic), P4 (negatively charged or aliphatic), P6 (Pro or negatively charged), P7 (negatively charged) and P9 (bulky hydrophobic) are important for binding of peptides to DQ2. Computer modeling of the DQ2 with variants of the high-affinity ligand in the groove suggests that peptides bind to DQ2 through the primary anchors P1, P7 and P9 and making additional advantageous interactions using the P4 and P6 positions.     

Substitution of aspartic acid at β57 with alanine alters MHC class II peptide binding activity but not protein stability: HLA-DQ (α1*0201, β1*0302) and (α1*0201, β1*0303)

In class II major histocompatibility complex (MHC) proteins, residue β57 is usually aspartic acid. Alleles carrying serine, valine, or alanine at this position are strongly correlated with the development of insulin-dependent diabetes mellitus (IDDM). Aspβ57 participates in a conserved salt bridge that bridges the α and β subunits in the peptide-binding site. It has been proposed that the correlation between IDDM and MHC alleles lacking Aspβ57 may be due to an instability of the protein caused by loss of this salt bridge. Using a pair of HLA-DQ proteins (α1*0201, β1*0302) and (α1*0201, β1*0303) differing only in having aspartic acid or alanine at position β57, we show that the polymorphism does not have a significant effect on protein stability for either the empty or peptide-loaded forms. However, the circular dichroism spectra indicate that empty and peptide-loaded Alaβ57 proteins display slightly different secondary structures relative to their Aspβ57 counterparts. A set of three peptides shows different binding affinities for DQ(α1*0201, β1*0302) relative to DQ(α1*0201, β1*0303). We propose that substitution of Aspβ57 residue causes a local rearrangement within the DQ peptide-binding site that alters the peptide-binding specificity. This rearrangement may help to explain the previously observed differences in SDS stability between Asp and non-Aspβ57 DQ proteins.     

Substance P-induced respiratory excitation is blunted by δ-receptor specific opioids in the rat medulla oblongata

The effects of substance P (SP) and the naturally occurring met-enkephalin and the synthetic μ-specific opioid agonist, DAGO (Tyr-d -Ala-Gly-N-Methy-Phe-Gly-ol) and the δ-specific opioid agonist DADL (Tyr-d -Ala-Gly-Phe-d -Leu) on basal ventilation were investigated in halothane-anaesthetized rats. Local injections of SP (0.75–1.5 nmol) in the ventrolateral medulla oblongata (VLM), e.g. nucleus paragigantocellularis, and nucleus reticularis lateralis increased ventilation because of an elevation of tidal volume. Met-enkephalin induced a short-lasting ventilatory depression mainly because of a depression of tidal volume. Activation of δ- and μ-opioid receptors in the VLM by local application of DADL and DAGO, respectively, induced ventilatory depression, which was later in onset and more long-lasting. Local administration of met-enkephalin into the VLM also produced a long-lasting inhibition of the SP-induced ventilatory excitation. A similar blockade of the SP-induced excitatory ventilatory response could be elicited by DADL but not by DAGO. This antagonistic effect was attenuated by local application of the δ-opioid receptor antagonist ICI 154.129. We conclude that the naturally occurring met-enkephalin as well as synthetic μ- and δ-specific enkephalin analogues (DAGO and DADL, respectively) in VLM depress basal ventilation by an effect on inspiratory drive. There is a functional antagonism between activation of δ-opioid receptors and SP receptors into the VLM in respect to respiratory regulation.     

Decreased responsiveness of vascular postjunctional α1-, α2-adrenoceptors and neuropeptide Y1 receptors in rats with heart failure

Heart failure is associated with increased sympathetic nerve activity. We hypothesized that chronic sympathetic stimulation in heart failure resulted in decreased vascular sympathetic responsiveness. A pithed rat model was employed to evaluate peripheral vascular α-adrenoceptor and neuropeptide Y (NPY) receptor responsiveness. Heart failure was induced in Sprague–Dawley rats by coronary artery ligation. Sham operated rats (Sham) served as controls. Two months after this surgical procedure, both heart failure (n= 30) and Sham (n= 30) rats underwent standard pithing procedure. Pressor responses to preganglionic sympathetic nerve stimulation (PNS) and activation of postjunctional α₁- and α₂-adrenoceptors as well as Y1 receptors were studied. In response to PNS, cardiac index was similar between heart failure and sham rats (P= n.s.). Mean arterial pressure (MAP) increased in a frequency-dependent fashion after PNS in heart failure rats as well as in control rats. All the agonists used, i.e. the α₁-adrenoceptor agonist phenylephrine, the α₂-adrenoceptor agonists clonidine and BHT933 as well as NPY, induced dose-dependent increases in MAP in heart failure and in sham rats. However, in rats with heart failure, the response to all the agonists studied was significantly decreased and the dose response curves were shifted to the right (P < 0.01). We conclude that in vivo vascular response to postjunctional α₁- and α₂-adrenoceptors as well as Y1 receptors are decreased in rats with heart failure.     

Functional α1-adrenoceptors in the rat heart during β-receptor blockade

In absence of β-receptor blocking agents, α-adrenergic inotropic effects could be demonstrated in the rat myocardium for the synthetic α-agonists phenylephrine and methoxamine, but not for the naturally occurring catecholamines, adrenaline and noradrenaline. Other synthetic α-agonists were without effects. In the presence of the β-receptor blocking agent, propranolol or timolol, marked α-effects were demonstrated for adrenaline and noradrenaline in both the right and left atria and the right ventricle. The results indicate that α-receptors may be functionally important in the β-blocked myocardium.     

Effect of H+ on spontaneous neuronal activity in the surface layer of the rat medulla oblongata in vitro

Summary The effect of changing extracellular pH (pH_e) on the spontaneous activity of neurons in brain slices taken from the ventral layer of the rat medulla oblongata was compared to the response of neurons in dorsal slices. In the ventral medulla, more than 50% of the neurons were excited by H⁺. These neurons were found just lateral to the pyramidal tract between the root of the hypoglossal nerve and the trapezoid body. In the dorsal medulla, low pH_e caused an inhibition of activity in most neurons, although a few were excited. The fact that H⁺ elicited excitation predominantly in the ventral neurons showed the special nature of the ventral medullary substrate to respond to pH_e changes.Depression of synaptic transmission within the neuronal network in the slice by reducing the [Ca²⁺]_e and increasing the [Mg²⁺]_e altered the nature of responses of neurons to H⁺: In the ventral medulla, the majority of neurons were inhibited by H⁺, whereas in the dorsal medulla more than 50% of neurons were excited. Therefore, specificity of the ventral medullary neurons seemed to be dependent upon intact synaptic connections. A possible role of acetylcholine-acetylcholinesterase system in the response of ventral medullary neurons to H⁺ is discussed.     

Occurrence of neuropeptide Y (NPY)-like immunoreactivity in catecholamine neurons in the human medulla oblongata

We report here the coexistence of a neuropeptide and catecholamines in neurons of the human brain. Using indirect immunofluorescence histochemistry, combined with elution and restaining experiments, neurons in the medulla oblongata of man were demonstrated to contain both a neuropeptide Y-like peptide and the catecholamine synthesizing enzyme tyrosine hydroxylase.     

Expression of phenylethanolamine N-methyltransferase (PNMT) and neuropeptide Y (NPY) in embryonic rat medulla oblongata grown in Oculo

Expression and development of specific markers of the adrenergic phenotype were studied in central neurons grown in transplant system. Medulla oblongata from embryonic day 12.5 (E12.5) or E18 rat was grafted into the anterior chamber of the eye of adult rat hosts. After two months, grafts were examined for the presence of immunoreactivity (IR) and catalytic activity to the epinephrine-synthesizing enzyme, phenylethanolamine N-methyltransferase (PNMT, E.C. 2.1.1.28), a specific adrenergic marker. In addition, grafts were examined for immunoreactivity to neuropeptide Y (NPY) and tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis. In E12.5 grafts, PNMT was expressed de novo, enzyme activity developed to levels similar to those in adult rat brainstem and PNMT-IR neurons were observed. TH-IR and NPY-IR neurons were also observed. In contrast, PNMT-IR was not observed in E18 grafts even though these already contained PNMT-IR neurons at the time of grafting. This was not due to poor growth of E18 grafts, in general, since TH-IR neurons were present and the protein content of the grafts was similar to that of E12.5 grafts. These studies suggest that adrenergic neurons survive well in oculo if they are transplanted prior to the age when neuroblasts have initially expressed the adrenergic phenotype, migrated to their final positions and elaborated processes. In addition, these studies establish a transplant system in which factors required for the development of central adrenergic neurons can be more easily studied than in situ.     

β-adrenergic stimulation of cellular K+ uptake in rat distal colon

We recently demonstrated that the ratio between colonic K⁺ absorptive and K⁺ secretive pathways was higher in infant than in adult rats. To test the hypothesis that hormones selectively affect these pathways during ontogeny we examined the effect of adrenergic agonists on cellular K⁺ uptake in distal colon from infant (10-day-old) and adult (50-day-old) rats. Here we describe that adrenaline (10^?5 M ) increased total and ouabain-insensitive ⁸⁶Rb uptake in both age groups, but it did not affect ouabain-sensitive ⁸⁶Rb uptake. This stimulation was more pronounced in adult than in infant rats. The effect of adrenaline was mediated via β-adrenergic receptors. Incubation in vitro with β-agonist, isoproterenol, stimulated SCH-28080-sensitive, i.e. H⁺,K⁺-ATPase-dependent, ⁸⁶Rb uptake in adult but not in infant rats. The threshold dose of β-agonist was at 10^?7 M , and the maximal activation was observed at 10^?5 M . In vivo inhibition of β-adrenergic system with propranolol caused a significant decrease in H⁺,K⁺-ATPase-dependent ⁸⁶Rb uptake in infant but not in adult colon. In conclusion, this study suggests that the higher colonic K⁺ absorption in infant rats may be as a result of a selective β-adrenergic up-regulation leading to stimulation of the apical H⁺,K⁺-ATPase.     

Differential changes in messenger RNA expressions and binding sites of neuropeptide Y Y1, Y2 and Y5 receptors in the hippocampus of an epileptic mutant rat: Noda epileptic rat

The anti-convulsive effects of neuropeptide Y have been suggested in several animal models of epilepsy. We have found the sustained increase of neuropeptide Y contents and the seizure-induced elevation of hippocampal messenger RNA in a novel spontaneous epileptic mutant rat: Noda epileptic rat. In the present study, we investigated the change of neuropeptide Y Y1 and Y2 receptor messenger RNA expressions and binding sites in the hippocampus following a spontaneous generalized tonic-clonic seizure of Noda epileptic rat. Furthermore, the binding sites of a more recently isolated receptor subtype, neuropeptide Y Y5 receptors, were also evaluated by receptor autoradiography. A marked elevation of neuropeptide Y immunoreactivity in the mossy fiber, and Y2-receptor up-regulation in the dentate gyrus were observed in the hippocampus of Noda epileptic rat, which coincided with the previous results of the other epileptic models. In contrast, Y1-receptor down-regulation was not found after a spontaneous seizure of Noda epileptic rat while this occurs in kindling and after kainic acid-induced seizures. [125I][Leu31, Pro34]peptide YY/BIBP 3226-insensitive (Y5 receptor) binding sites in CA1 stratum radiatum were significantly decreased following a spontaneous seizure of Noda epileptic rat. The present results suggest that a spontaneous seizure of Noda epileptic rat induces significant changes in neuropeptide Y-mediated transmission in the hippocampus via Y2 and Y5 receptors, but not Y1 receptors. Therefore, specific subset of neuropeptide Y receptor subtypes might be involved in the epileptogenesis of Noda epileptic rat.     

β2-adrenergic control of plasma volume in hemorrhage

Hemorrhage is associated with absorption of extravascular fluid from skeletal muscle to blood in order to compensate for the loss of intravascular volume. Our previous studies have shown that this fluid gain is mainly linked to β-adrenergic microvascular adjustments leading to decrease in capillary hydrostatic pressure and to precapillary ‘sphincter’ mediated increase in the capillary surface area available for fluid exchange. In the present study the importance of β-adrenergic control of plasma volume in bleeding was confirmed by measurement of changes in plasma volume after graded hemorrhage in animals with intact and blocked vascular β₂-adrenoceptors (i. v. administration of the ‘selective’β₂-blocking agent ICI 118, 551). With intact β₂-adrenoceptors plasma volume was gradually restored after bleeding so that about 50% of the shed plasma volume (about 35% of the shed blood volume) had been compensated for at two hours after exsanguination of 20% as well as 40% of the blood volume. The corresponding figures in animals with blocked β₂-adrenoceptors were only 14% of the shed plasma volume and 8% of the shed blood volume at both degrees of hemorrhage.     

Intestinal motility responses to neuropeptide γ in vitro and in vivo in the rat: comparison with neurokinin 1 and neurokinin 2 receptor agonists

We have studied the effect of a novel tachykinin, neuropeptide γ(NPγ) on small intestinal motility in the rat. Experiments were done in vitro on longitudinal muscle strips of duodenum, and in vivo on the migrating myoelectric complex (MMC) of the small intestine. In vitro, contractile effects of NPγ were compared with those of a selective neurokinin 1 (NK1) receptor agonist, substance P methyl ester (SPME), and a selective neurokinin 2 (NK2) receptor agonist, Nle¹⁰-NKA(4–10)(NleNKA). NPγ, SPME and NleNKA caused concentration-dependent contractions (P < 0.001). NPγ was eight-fold more potent than NleNKA, and 118-fold more potent than SPME. Contractile responses to NPγ were reduced by hexamethonium (P < 0.01) and atropine (P < 0.05). The non-selective NK receptor antagonist spantide I only slightly reduced the contractile response to NPγ, as did the selective NK1 antagonist GR 82334, and the selective NK2 antagonist L-659877 and MEN 10376. In vivo, effects of NPγ on the MMC were compared with those of the natural tachykinins substance P (SP) and neurokinin A (NKA). NPy disrupted the MMC and induced irregular spiking in a dose-dependent manner from 25 to 100 pmol kg^-1 min^-1 i.v. (P < 0.05). The effect of NPγ was more prominent than that of NKA at equal doses, while SP had no effect. Our findings show that NPγ exerts potent stimulatory effects on small intestinal motility, most likely mediated directly via distinct NK receptors on smooth muscle cells, but also indirectly via a cholinergic link.     

Haemodynamic conditions for renal PGE2 and renin release during α- and β-adrenergic stimulation in dogs

Constriction of the renal artery and infusion of an α-adrenergic agonist induce autoregulated vasodilation and increase prostaglandin E₂ (PGE₂) and renin release. The enhancement of renin release during autoregulated vasodilation might be mediated by prostaglandins. To examine this hypothesis, experiments were performed in three groups of anaesthetized dogs. In six dogs constriction of the renal artery to a perfusion pressure below the range of autoregulation raised renin release from 2 ± 1 to 27 ± 6 μg AI.min^-1 and PGE₂ release from 1 ± 1 to 10 ± 2 pmol. min^-1. After administration of indomethacin (10 mg. kg^-1 b. wt), PGE₂ release was effectively blocked and constriction of the renal artery raised renin release only from 0.1 ± 0.1 to 6 ± 1 μg AI.min^-1. During subsequent continuous infusion of a β-adrenergic agonist, isoproterenol (0.2 μg. kg^-1.min^-1), constriction of the renal artery raised renin release from 0.1 ± 0.1 to 52 ± 11 μg AI.min^-1, although there was no rise in PGE₂ release. In six dogs, intrarenal infusion of phenylephrine, an α adrenergic agonist, increased PGE₂ and renin release before, but not after, indomethacin administration. In six other dogs, phenylephrine infused during isoproterenol infusion increased renin release equally before and after indomethacin administration. Thus the enhancing effect of constricting the renal artery or infusing an α-adrenergic agonist is not dependent upon prostaglandins. We propose that autoregulated dilation enhances renin release whether the stimulatory agent is a prostaglandin or a β-adrenergic agonist.     

Distribution of alpha2-adrenergic binding sites in the parabrachial complex of the rat

The present study describes the distribution of alpha₂-adrenoceptors in the parabrachial and KöllikerFuse nucleus of the rat by employing the tritium-labeled alpha₂-receptor antagonist rauwolscine ([³H]-RAUW) as a ligand. The [³H]-RAUW binding was densitometrically quantified in five nuclei of the parabrachial (PB) complex in serial coronal sections. We found that cytoarchitectonically and anatomically distinct nuclei of the PB complex exhibit different numbers of [³H]-RAUW-binding sites. The largest number of binding sites was observed over the external lateral PB and caudally over the waist area of the PB. Lower numbers of binding sites were found in the remaining lateral PB nuclei, followed by the medial PB and the Kölliker-Fuse nucleus. In addition we disclosed that the internal lateral PB contains a very low number of binding sites while the external medial PB is marked by dense [³H]-RAUW binding. Also, the affinities of the binding sites differed between the PB areas. High affinities were observed in the external lateral PB, the remaining lateral PB nuclei and in the waist area of the PB, while the medial PB and the KöllikerFuse nucleus exhibited only low affinities for the ligand. Furthermore, saturation curves demonstrated non-linear profiles, indicating the presence of more than one population of binding sites in the PB nuclei for the radioligand. Our data demonstrate that the PB exhibits a distinct distribution of alpha₂-adrenergic binding sites. These correlate well with the cytoarchitectonically defined nuclei of the PB complex and with the pattern of ascending axons from the medial nucleus of the solitary tract and the area postrema terminating in the PB. Since a large number of these projection neurons utilize adrenaline or noradrenaline as their transmitters, we conclude that solitary-parabrachial neurotransmission to the forebrain is, at least in part, mediated via alpha₂-adrenoceptors.