Fluorescent Labelled Analogues of Neuropeptide Y for the Characterization of Cells Expressing NPY Receptor Subtypes

Ingenhoven, Nikolaus; Beck‐Sickinger, Annette G.

doi:10.3109/10799899709036617

Cited by 25 publications

(22 citation statements)

References 18 publications

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“…SMS-KAN cells (neuroblastoma, hY 2 ) were grown in 50% DMEM/50% nutrient mix Ham's F12 with 15% fetal bovine serum, 4 mm l-glutamine and 0.2 mm nonessential amino acids [39]. BHK cells transfected with rY 5 -receptors were cultured in DMEM containing 10% fetal bovine serum, and 0.5% geneticin [30].…”

Section: Peptide Synthesismentioning

confidence: 99%

Novel analogues of neuropeptide Y with a preference for the Y₁‐receptor

Söll

Dinger

Lundell

et al. 2001

European Journal of Biochemistry

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In contrast, cyclo S±S [Cys20,Cys24]pNPY was found to be a highly selective ligand at the Y 2 -receptor, binding only threefold less efficiently than NPY. Analogues containing variations of positions 31 and 32 showed highly reduced affinity to the Y 1 -receptor, while binding to the Y 5 -receptor was affected less. Inhibition of cAMP-accumulation of selected peptides with replacements within position 20±23 of NPY showed preserved agonistic properties. The NPY analogues tested give insights into ligand±receptor interaction of NPY at the Y 1 -, Y 2 -and Y 5 -receptor and contribute to our understanding of subtype selectivity. Furthermore, the Y 1 -receptor-preferring peptides are novel tools that will provide insight into the physiological role of the Y 1 -receptor.Keywords: food intake; neuropeptides; NPY; selective ligands; structure±affinity relationship.Obesity, food intake and energy homeostasis are key areas of growing importance because obesity is beginning to replace malnutrition and infectious diseases as the most significant contributor to ill health in the developed world [1]. Neuropeptide Y (NPY) is one of the most important effector molecules of leptin: it is a key molecule in the regulation of food intake, it acts via several different receptor subtypes and elicits several physiological effects. Profound effects on stimulation of food intake, secretion of luteinizing and growth hormone, and insulin release suggest an important role for NPY in the pathophysiology of obesity and diabetes [2±5]. A wide range of other effects of NPY have been reported, such as potent vasoconstriction [6], facilitation of learning and memory [7], modulation of locomotor behaviours [8], induction of hypothermia [9,10], inhibition of sexual behaviour [11], shifts in circadian rhythms [12], modulation of cardiorespiratory parameters[13], anxiolytic potency [14] and inhibition of alcohol consumption and resistance [15].NPY is a 36-amino acid peptide amide belonging to the family of pancreatic polypeptides that includes also pancreatic polypeptide (PP) and peptide YY; it was first isolated from pig brain in 1982 [16].NPY is widely distributed within the central nervous system and in the periphery. [25,26]. A putative Y 3 -receptor has been described only pharmacologically and no specific agonists or antagonists are known [27]. All receptors belong to the G-protein coupled receptor superfamily [28]. The correlation of a certain receptor subtype to a distinct physiological effect is not yet fully understood. Recent studies even suggest that different receptor subtypes redundantly mediate identical actions in a given system, as has been shown for the Y 1 -and Y 5 -receptor in the regulation of food intake [3]. Findings that deficiencies in either the Y 1 -or the Y 5 -receptors do not significantly impair the normal feeding response to fasting or NPY stimulation, strongly indicate that both Y receptors are involved in appetite regulation by NPY [29,30]. Selective receptor agonists or antagonists are useful tools with which to stu...

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Section: Peptide Synthesismentioning

confidence: 99%

Novel analogues of neuropeptide Y with a preference for the Y₁‐receptor

Söll

Dinger

Lundell

et al. 2001

European Journal of Biochemistry

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“…Unfortunately, these NK 3 receptor antibodies do not bind to guinea-pig tissue (Mann et al 1997a). An alternative method to locate receptors, which has similar resolution to fluorescence immunohistochemistry, is to use fluorescently labelled agonists Garland et al 1994;Grady et al 1995;McGrath et al 1996;Ingenhoven and Beck-Sickinger 1997). Agonist binding to tachykinin receptors induces receptor endocytosis, resulting in internalisation of the agonist (with fluorescent label) into neurons bearing the tachykinin receptor.…”

Section: Introductionmentioning

confidence: 99%

Neurons bearing NK 3 tachykinin receptors in the guinea-pig ileum revealed by specific binding of fluorescently labelled agonists

Jenkinson¹,

Morgan²,

Furness³

et al. 1999

Histochemistry and Cell Biology

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The localisation of NK(3) tachykinin receptors in guinea-pig ileum was studied using the fluorescently labelled agonists, Cy3. 5-neurokinin A and Cy3.5-kassinin. Binding to nerve cell bodies in the myenteric and submucosal plexuses was visualised using confocal microscopy. Binding to NK(1) receptors was blocked by the NK(1) receptor antagonist, CP-99994. NK(3) receptors, demonstrated by binding in the presence of CP-99994, occurred in 72% of myenteric and 38% of submucosal neurons. Colocalisation with other markers was examined to deduce the classes of neurons with NK(3) receptors. In myenteric ganglia, NK(3) receptors were present on the following: 73% of calbindin-immunoreactive (IR) intrinsic primary afferent neurons, 63% of calretinin-IR excitatory motor neurons and ascending interneurons, 63% of nitric oxide synthase-IR inhibitory motor neurons and descending interneurons, 79% of strongly neuropeptide Y (NPY)-IR secretomotor neurons, 67% of weakly NPY-IR descending interneurons and motor neurons, and 46% of NK(1) receptor-IR neurons. In submucosal ganglia, NK(3) receptors were on 65% of calretinin-IR secretomotor/vasodilator neurons, 81% of NPY-IR cholinergic secretomotor neurons, 2% of vasoactive intestinal peptide-IR non-cholinergic secretomotor neurons and were completely absent from substance P-IR intrinsic primary afferent neurons. The results support physiological studies suggesting that NK(3) receptors mediate tachykinin transmission between myenteric sensory neurons and to interneurons and/or motor neurons in descending inhibitory and ascending excitatory pathways.

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“…SMS-KAN cells (hY 2 receptor) were grown in Dulbecco's modified Eagle's medium/nutrient mix F12 1:1 with 15% fetal calf serum, 4 mM glutamine, and 1% nonessential amino acids (23). Cells were grown to confluency at 37°C and 5% CO 2 .…”

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confidence: 99%

The First Selective Agonist for the Neuropeptide YY5Receptor Increases Food Intake in Rats

Cabrele¹,

Langer²,

Bader³

et al. 2000

Journal of Biological Chemistry

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172

165

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Neuropeptide Y (NPY), 1 a 36-residue peptide amide, is a member of the pancreatic polypeptide (PP) hormone family that also includes PP and peptide YY (PYY) (1). NPY is expressed in the central and peripheral nervous systems and is one of the most abundant neuropeptides in the brain. Several important physiological activities, such as induction of food intake, inhibition of anxiety, increase in memory retention, presynaptic inhibition of neurotransmitter release, vasoconstriction, and regulation of ethanol consumption, have been attributed to NPY (2, 3). Especially, the role of NPY in feeding is of major interest because NPY receptor antagonists are potential anti-obesity drug candidates. Many studies have established the strong central influence of NPY in feeding behavior; for example, injection of NPY into the hypothalamus increases food intake (4, 5), and high NPY levels are correlated with leptin deficiency (6), the hormone that is secreted by adipocytes and regulates body weight and energy balance (7,8). Furthermore, NPY knockout can reduce obesity in leptin-deficient mice (named ob/ob mice) (6).Five distinct Y receptor subtypes that bind NPY, PYY, and PP with different affinities have been identified, cloned, and characterized (9). They all belong to the superfamily of the G-protein- Because highly specific tools to investigate the Y 5 receptor activity are still missing, we have focused our work on the design of NPY receptor agonists with both high affinity and selectivity for the Y 5 subtype. It is well established that the C-terminal part of NPY represents the interaction site with the Y receptors and that amino acid exchange is poorly tolerated in the region 33-36 (49); therefore, we induced a conformational change within the peptide region that mediates receptor binding by introducing the ␤-turn-inducing dipeptide Ala-Aib (aminoisobutyric acid) (19) into positions 31-32 of NPY and some peptides that contain segments of NPY and PP (NPY/PP chimeras). The [Ala 31 ,Aib 32 ]-modified peptides showed high selectivity for the Y 5 receptor. Furthermore, in vitro and in vivo studies clearly proved their NPY receptor agonism as well as stimulation of food intake.

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Fluorescent Labelled Analogues of Neuropeptide Y for the Characterization of Cells Expressing NPY Receptor Subtypes

Cited by 25 publications

References 18 publications

Novel analogues of neuropeptide Y with a preference for the Y₁‐receptor

Novel analogues of neuropeptide Y with a preference for the Y₁‐receptor

Neurons bearing NK 3 tachykinin receptors in the guinea-pig ileum revealed by specific binding of fluorescently labelled agonists

The First Selective Agonist for the Neuropeptide YY5Receptor Increases Food Intake in Rats

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Fluorescent Labelled Analogues of Neuropeptide Y for the Characterization of Cells Expressing NPY Receptor Subtypes

Cited by 25 publications

References 18 publications

Novel analogues of neuropeptide Y with a preference for the Y1‐receptor

Novel analogues of neuropeptide Y with a preference for the Y1‐receptor

Neurons bearing NK 3 tachykinin receptors in the guinea-pig ileum revealed by specific binding of fluorescently labelled agonists

The First Selective Agonist for the Neuropeptide YY5Receptor Increases Food Intake in Rats

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Novel analogues of neuropeptide Y with a preference for the Y₁‐receptor

Novel analogues of neuropeptide Y with a preference for the Y₁‐receptor