Neuropeptide Y as a stimulator of Na+-dependent Ca2+ efflux from freshly isolated adult rat cardiomyocytes

Several physiological stimuli cause a rise in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in cardiomyocytes. This increased [Ca²⁺]_i must be restored to physiological resting level to ensure response to further stimuli. In the present study, we examined the effect of neuropeptide Y (NPY), which is secreted from certain adrenergic or non-adrenergic neurons, on Ca²⁺ efflux from freshly isolated, quiescent adult rat cardiomyocytes. The isolated cardiomyocytes were preloaded with ⁴⁵CaCl₂ for 1 h. Then, the fractional release of ⁴⁵Ca²⁺ from the cells was measured. NPY stimulated the efflux of ⁴⁵Ca²⁺ from isolated adult rat cardiomyocytes in a concentration-dependent manner (10^–8 M to 10^–6 M). NPY (10^–6 M)-induced Ca²⁺ efflux was 2.0 ± 0.16% of the total cellular content. The ⁴⁵Ca²⁺ efflux from the cells was also stimulated by Y₁ receptor agonist, [Leu³¹, Pro³⁴]NPY, but not by Y₂ receptor agonist, NPY_13–36. The effect of NPY was inhibited by a peptide NPY inhibitor, NPY_18–36 and a non-peptide NPY inhibitor, benextramine to a similar extent. From these results, it is conceivable that the effect of NPY on Ca²⁺ efflux from cardiomyocytes is mediated through Y₁ receptors. It was also observed that NPY caused a rise in [Ca²⁺]_i to almost 150 nM. NPY-stimulated ⁴⁵Ca²⁺ efflux was not affected by removal of extracellular Ca²⁺, but was dependent on the presence of extracellular Na⁺. Moreover, NPY caused a ²²Na⁺ influx into the cells of about 1.6-fold over the basal value which was inhibited by amiloride and 5-(N,N-dimethyl)-amiloride, known Na⁺/Ca²⁺ exchange inhibitors. In addition, isoproterenol also caused ⁴⁵Ca²⁺ efflux from the cells and which was enhanced by the addition of NPY. These results suggest that NPY stimulates extracellular Na⁺-dependent ⁴⁵Ca²⁺ efflux from freshly isolated adult rat cardiomyocytes, probably through its stimulatory effect on plasma membrane Y₁ receptors with which NPY may couple during Na⁺/Ca²⁺ exchange. Received: 21 May 1997 / Accepted: 26 August 1997     

A role for neuropeptide Y in the gender-specific gastrointestinal, corticosterone and feeding responses to stress

BACKGROUND AND PURPOSE

Exposure to an acute stress inhibits gastric emptying and stimulates colonic transit via central neuropeptide Y (NPY) pathways; however, peripheral involvement is uncertain. The anxiogenic phenotype of NPY^−/− mice is gender-dependent, raising the possibility that stress-induced gastrointestinal (GI) responses are female-dominant through NPY. The aim of this study was to determine GI transit rates, corticosterone levels and food intake after acute restraint (AR) or novel environment (NE) stress in male and female NPY^−/− and WT mice.

EXPERIMENTAL APPROACH

Upper gastrointestinal transit (UGIT) (established 30 min after oral gavage) and corticosterone levels were determined under basal or restrained conditions (30 min) and after treatment i.p. with Y₁ antagonist BIBO3304 or Y₂ antagonist BIIE0246. Faecal pellet output (FPO) was established after AR and treatment i.p. with NPY in the NE, as were colonic bead expulsion rates.

KEY RESULTS

UGIT and FPO were similar in unrestrained male and female mice. NPY^−/− females displayed significantly slower UGIT than NPY^−/− males after AR, but both genders displayed significantly higher FPO and reduced food intake relative to WT counterparts. Peripheral NPY treatment increased bead expulsion time in WT mice. AR male NPY^−/− mice had higher levels of corticosterone than male WT mice; whilst in AR WT mice, after peripheral Y₁ and Y₂ receptor antagonism in males, and Y₂ antagonism in females, corticosterone was significantly elevated.

CONCLUSIONS AND IMPLICATIONS

NPY possesses a role in the gender-dependent susceptibility to stress-induced GI responses. Furthermore, NPY inhibits GI motility through Y₂ receptors and corticosterone release via peripheral Y₁ and Y₂ receptors.     

Anxiolytic activity of NPY receptor agonists in the conflict test

This investigation examined receptor subtype specificity and possible modulation by GABAa receptor ligands of NPY-induced behavioral responses to stressful stimuli. First, a series of NPY receptor agonists were examined for their potential effects on punished responding in a conflict test modified for incremental shock. NPY, peptide YY (PYY) and NPY Y₁ receptor agonists [Leu³¹,Pro³⁴]-NPY and [Gly⁶, Glu²⁶,Lys²⁹,Pro³⁴]-NPY produced increases in punished responding in the conflict test. No significant effects on unpunished responding were noted. The pattern of responding was similar to that observed with the benzodiazepine agonist chlordiazepoxide. Neither pancreatic peptide (PP) nor the Y₂ agonists NPY_13–36 or [Glu^2,32,Ala⁶,Dpr²⁷,Lys²⁸]-NPY significantly altered punished or unpunished responding. Of significance, the atypical Y₁ agonist [Cys^7,21,Pro³⁴]-NPY produced negligible effects on punished responding, consistent with the presence of a subclass of Y₁ receptors. Second, the anxiolytic effects of NPY were subjected to treatments that block actions at the GABAa receptor complex. The increase in punished responding produced by NPY was not altered by administration of the benzodiazepine antagonist flumazenil and only partially blocked by the picrotoxinin receptor ligand isopropylbicyclophosphate (10 and 15 μg/kg). These findings further support the hypothesis that the pharmacologic substrates for the anxiolytic-like actions of NPY may be mediated by the Y₁ receptor subtype and suggest that these actions are independent of either the benzodiazepine or picrotoxinin binding sites of the GABA/benzodiazepine receptor complex. Received: 1 February 1996/Final version: 16 January 1997     

The Y1 antagonist BIBP 3226 inhibits potentiation of methoxamine-induced vasoconstriction by neuropeptide Y

We investigated the interaction of neuropeptide Y (NPY) with the α₁-adrenoceptor agonist, methoxamine, in control of mean arterial pressure, renovascular resistance and mesenteric vascular resistance in anaesthetized rats. Infusion of 3.0 but not 0.3μg/kg/min NPY enhanced the elevations of all three haemodynamic parameters caused by bolus injections of methoxamine (10–100μg/kg). These enhancements largely involved a prolongation of the methoxamine effects. While infusion of the Y₁ NPY receptor-selective antagonist, BIBP 3226 (10μg/kg/min), alone did not alter methoxamine-induced vasoconstriction, it inhibited the potentiation by NPY. We conclude that NPY can potentiate methoxamine-induced vasoconstriction in vivo. This is mediated predominantly, if not exclusively, via the Y₁ receptor. Endogenously released NPY does not appear to reach sufficient concentrations to cause tonic systemic vasoconstriction or potentiation thereof in the anaesthetized rat. Received: 30 May 1997 / Accepted: 25 July 1997     

Neuropeptide Y-induced potentiation of noradrenergic vasoconstriction in the human saphenous vein: involvement of endothelium generated thromboxane

1. We investigated the potentiating effect of low concentrations of neuropeptide Y (NPY) on the vasoconstriction induced by transmural nerve stimulation (TNS) and noradrenaline (NA) in human saphenous veins. The effects of (i) endothelium removal; (ii) the addition of the NO pathway precursor L-arginine; (iii) the ET_A/ET_B endothelin receptor antagonist Ro 47-0203; (iv) the cyclo-oxygenase inhibitor, indomethacin; (v) the selective thromboxane A₂ (TxA₂) receptor antagonists Bay u3405 and ifetroban, and (vi) the TxA₂ synthase inhibitor, UK 38485, were studied in order to gain information about the mechanisms of NPY-induced potentiation.
2. Contractile response curves for TNS (0.5–8 Hz) and for exogenously administered NA (0.1–3 μM) were obtained in superfused saphenous vein rings. The contractions induced by both TNS and NA at all tested frequencies and concentrations, respectively, were significantly potentiated by 50 nM NPY in endothelium intact veins. Conversely, in endothelium-denuded vessel rings the contractile-response curves to TNS and NA overlapped both in the absence and presence of NPY, thus suggesting that a release of vasoactive substances from endothelial cells could account for the noradrenergic NPY-induced potentiation.
3. In vessels with intact endothelium, the potentiating action of NPY on TNS and NA was unaffected by the presence of high concentrations of the NO precursor L-arginine (3–10 mM) or the non-selective ET_A/ET_B endothelin receptor antagonist, Ro 47-0203 (10 μM). These data indicate that the NPY-induced effect does not involve either the endothelium-derived vasodilator nitric oxide or the vasoconstrictor endothelin. Conversely, in the presence of the cyclo-oxygenase inhibitor, indomethacin (30 μM), NPY failed to potentiate the vasoconstrictions produced by either nerve stimulation or by exogenous NA, thus providing evidence that arachidonic acid metabolites through the cyclo-oxygenase pathway are mainly responsible for the potentiation evoked by NPY.
4. When the TxA₂ receptor antagonists, Bay u 3405 (1 μM) and ifetroban (1 μM) were added to the superfusing medium, NPY did not alter either the frequency- or the concentration-response curves for either TNS or NA. Accordingly, both TNS- and NA-induced contractions were not potentiated by NPY in the presence of the TxA₂ synthase inhibitor, UK 38485 (10 μM). This clearly demonstrates the pivotal role of TxA₂ in NPY-induced potentiation.
5. In superfused vein rings with endothelium, a subthreshold concentration (0.2 nM) of the TxA₂ mimetic U 46619 potentiated both TNS- and NA-induced vasoconstrictions. This potentiation was higher at low stimulation frequencies and low NA concentrations, and resembled that produced by NPY.
6. Our results indicate that in the human saphenous vein NPY potentiates the contractions produced by sympathetic nerve stimulation acting at the postjunctional level, primarily on endothelial cells. In particular, the NPY-induced release of a cyclo-oxygenase metabolite, namely TxA₂, may have a synergistic effect on the vasoconstriction induced by the noradrenergic mediator. Thus, such a mechanism may play a key role in the maintenance of the sympathetic tone of large human capacitance vessels.

     

Neuropeptide Y and neuropeptide Y18-36 Structural and biological characterization

Neuropeptide Y (NPY), a 36-residue peptide amide, has been shown by numerous studies to be a potent vasoconstrictor. In order to gain an appreciation of the structural requirements for this action, we have previously synthesized a number of fragments of NPY. It had been shown that sequential deletions from the N-terminus resulted in peptides with decreasing hypertensive activity. In the present study we present data supporting the unexpected finding of two fragments, NPY_17-36 and NPT_18-36 with substantial hypotensive action in vivo. This action was dose dependent (data not shown) and was also observed to a lesser extent with NPY_19-36 but not NPY_16-36 or NPY_20-36. It was, however, slower in onset and of longer duration than the hypertensive action of NPY. These differing kinetics of action may suggest that NPY and NPY_18-36 act through different mechanisms. Structural studies using circular dichroism were performed. While NPY was found to assume an ordered helical structure in both aqueous buffer and trifluoroethanol (TFE), 30% TFE in aqueous buffer was required to induce substantial helicity for NPY_18-36. This structural investigation suggests that both NPY and NPY_18-36 assume an ordered conformation upon reaching the lipid rich receptor environment.     

Effects of noradrenaline and neuropeptide Y on rat mesenteric microvessel contraction

We have studied the contractile effects of the sympathetic transmitter noradrenaline and its cotransmitter neuropeptide Y (NPY) given alone and in combination on isolated rat mesenteric resistance vessels (200–300 m diameter). Noradrenaline and NPY each concentration-dependently contracted rat mesenteric microvessels (EC₅₀ 800 nM and 10 nM, respectively), but noradrenaline caused considerably greater maximal effects than NPY (14.3 mN vs. 3.5mN). A low antagonistic potency of yohimbine indicated that the response to noradrenaline did not involve ₂-adrenoceptors, and the subtype-selective antagonists 5-methylurapidil, tamsulosin and chloroethylclonidine indicated mediation via an _1A-adrenoceptor. Shallow Schild regressions for prazosin and 5-methylurapidil indicated that an ₁-adrenoceptor subtype with relatively low prazosin affinity might additionally be involved. Studies with the NPY analogues PYY, [Leu³¹, Pro³⁴]NPY and NPY_18–36 demonstrated that NPY acted via a Y₁ NPY receptor. In addition to its direct vasoconstricting effects NPY also lowered the noradrenaline EC₅₀ but did not appreciably affect maximal noradrenaline responses indicating possible potentiation. The potentiating NPY response occured with similar agonist potency as the direct contractile NPY effects and also via a Y₁ NPY receptor. The Ca²⁺ entry blocker nitrendipine (300 nM) reduced direct contractile responses to noradrenaline and NPY but did not affect the potentiation response to NPY.     

Evidence for involvement of neuropeptide Y receptors in the regulation of food intake: studies with Y1-selective antagonist BIBP3226

1. Experiments were conducted to evaluate the effects of the novel non-peptide neuropeptide Y Y₁ receptor antagonist, BIBP3226 (N²-(diphenylacetyl)-N-[(4-hydroxy-phenyl)methyl]-D-arginine amide) on spontaneous, fasting-induced and NPY-induced food intake in rats. In addition to consumption of regular chow, the effects of BIBP3226 on consumption of highly palatable sweetened mash were monitored in a 1 h test on first exposure and after familiarization with novel food.
2. BIBP3226 (10.0 nmol, i.c.v.) had no effect on the consumption of regular chow, but reduced significantly the intake of highly palatable diet and the food intake stimulated by fasting (24 h). Neuropeptide Y (NPY, 1.0 nmol, i.c.v.) significantly increased the consumption of regular rat chow. This orexigenic effect of NPY was blocked by BIBP3226 (10.0 nmol, administered i.c.v. 5 min before NPY) at 30  min and 4  h, but not at 1 and 2  h. When animals were pretreated with diazepam (0.5 mg kg⁻¹, i.p., 20 min before NPY), BIBP3226 failed to suppress NPY-induced feeding.
3. An NPY Y₁ and Y₃ receptor agonist, [Leu³¹,Pro³⁴]NPY and a Y₅ receptor agonist human peptide YY_3–36 (hPYY_3–36, both 30 pmol), microinjected into the paraventricular nucleus of the hypothalamus (PVN) increased the consumption of regular rat chow. BIBP3226 (0.4 nmol, into the PVN) completely blocked the stimulatory effect of [Leu³¹,Pro³⁴]NPY but not that of hPYY_3–36. BIBP3226 (0.4 nmol) alone failed to modify the consumption of the regular chow. Higher doses of BIBP3226 (1.0 and 2.0 nmol) injected into the vicinity of the PVN reduced the consumption of the sweetened mash.
4. These results suggest that both the NPY Y₁ and Y₅ receptors in the PVN are involved in the regulation of food intake. The stimulatory effect of exogenous NPY is probably mediated through an NPY receptor subtype that is not identical with the Y₁ receptor (possibly Y₅ receptor). However, the NPY Y₁ receptors may mediate the effect of endogenous NPY in conditions of increased energy demand or on intake of highly palatable diets.

     

Failure of the putative neuropeptide Y antagonists, benextramine and PYX-2, to inhibit Y2 receptors in rat isolated prostatic vas deferens.

1. The pharmacological activity of neuropeptide Y (NPY) and some analogues in inhibiting the twitch contractions induced by electrical stimulation (single pulses at 25 V, 0.15 Hz, 1 ms) in the prostatic portion of the rat isolated vas deferens was investigated. The rank order of agonist potency was: PYY > NPY2-36 > NPY >> NPY13-36 >> NPY18-36 >> [Leu31,Pro34]NPY = hPP, which is consistent with the activation of a Y2 receptor. 2. The putative Y1 and Y2 antagonist, benextramine (BXT), incubated at 100 microM for 10 or 60 min, was ineffective against PYY-induced inhibition of the twitch response, suggesting that the prejunctional Y2 receptor in this tissue is different from the postjunctional one reported in the literature to be sensitive to BXT blockade. 3. The putative NPY antagonist, PYX-2, incubated at 1 microM for 20 min, was completely ineffective in antagonizing PYY-induced inhibition of twitches. 4. The twitch response was totally inhibited by suramin (100 microM) but was little affected by prazosin (1 microM). Furthermore, NPY was without effect on the dose-response curve to ATP in resting conditions. Taken together, these results suggest that in our paradigm, NPY inhibits the release of a purinergic neurotransmitter which mediates contraction of the prostatic portion of the rat vas deferens.     

Neuropeptide Y (NPY) inhibits spontaneous contraction of the mouse atrium by possible activation of the NPY1 receptor

相似文献   

Different receptors for angiotensin II at pre- and postjunctional level of the canine mesenteric and pulmonary arteries

1. This investigation was undertaken to compare pre- and postjunctional receptors involved in the responses of the canine mesenteric and pulmonary arteries to angiotensin II.
2. In the mesenteric artery, angiotensin II caused an enhancement of tritium overflow evoked by electrical stimulation (EC_30%=5 nM), the maximal effect representing an increase by about 45%. Postjunctionally, angiotensin II caused concentration-dependent contractions (pD₂=8.57). Saralasin antagonized both pre- and postjunctional effects of angiotensin II, but it was more potent at post- than at prejunctional level (pA₂ of 9.51 and 8.15, respectively), while losartan antagonized exclusively the postjunctional effects of angiotensin II (pA₂=8.15). PD123319 had no antagonist effect either pre- or postjunctionally.
3. In the pulmonary artery, angiotensin II also caused an enhancement of the electrically-evoked tritium overflow (EC_30%=1.54 nM), its maximal effect increasing tritium overflow by about 80%. Postjunctionally, angiotensin II caused contractile responses (pD₂=8.52). As in the mesenteric artery, saralasin antagonized angiotensin II effects at both pre- and postjunctional level and it was more potent postjunctionally (pA₂ of 9.58 and 8.10, respectively). Losartan antagonized only the postjunctional effects of angiotensin II (pA₂=7.96) and PD123319 was ineffective.
4. It is concluded that in both vessels: (1) pre- and postjunctional receptors belong to a different subtype, since they are differently antagonized by the same antagonists; (2) postjunctional receptors belong to AT₁ subtype, since they are blocked by losartan but not by AT₂ antagonists; (3) prejunctional receptors apparently belong to neither AT₁ or AT₂ subtype since they are blocked by neither AT₁ nor AT₂ antagonists.

     

Multiple neuropeptide Y receptors are involved in cardiovascular regulation. Peripheral and central mechanisms

• 1.1. Neuropeptide Y (NPY) occurs in both the central and peripheral nervous system. In the periphery, NPY coexists with noradrenaline (NA) in perivascular sympathetic fibers.
• 2.2. NPY has a vasopressor effect, reflecting direct vasoconstriction of blood vessels and potentiation of the NA-evoked response. NPY also suppresses the release of NA from sympathetic fibers.
• 3.3. The post- and pre-junctional NPY receptors are referred to as Y₁ and Y₂, respectively. They recognize not only NPY but also the homologous gut hormone peptide YY (PYY).
• 4.4. The Y₁ and Y₂ receptors have been characterized in numerous test systems using analogs of NPY/PYY. Already the deletion of the first N-terminal amino acid (NPY 2–36) results in a marked loss of potency at the Y₁ receptor. The Y₂ receptor is much less dependent upon an intact N-terminus, and a wide range of C-terminal NPY fragments retain quite high potency.
• 5.5. Recently, yet another NPY receptor, Y₃, that is distinct from Y₁ and Y₂ in that it recognizes PYY poorly, has been demonstrated in the brainstem and in the periphery.
• 6.6. Further attempts to characterize the various receptor types have relied on truncated and substituted analogs of NPY/PYY. Although such studies suggest the existence of at least three types of NPY receptors, the lack of antagonists has represented a problem.
• 7.7. Since NPY may regulate cardiovascular functions via peripheral and central receptors its physiological and possibly pathophysiological significance has attracted much attention.
• 8.8. The responsiveness to NPY seems to be altered in animal models of hypertension and elevated plasma levels of NPY have been found in patients under various conditions of stress and in primary hypertension. A number of studies have suggested that NPY may be a pathogenetic factor behind primary hypertension.
• 9.9. Antagonists for the various NPY receptors would be useful for an analysis of which effects of these peptides are physiologically relevant. It is tempting to predict that both agonists and antagonists of the NPY receptors could be useful as drugs, for instance, in the treatment of primary hypertension.

     

The pharmacokinetics of oxybutynin is unaffected by gender and contraceptive steroids

Objective and methods: The effect of gender and concomitant use of contraceptive steroids on the absorption and metabolism of oxybutynin was investigated in 49 healthy volunteers, 24 females and 25 males. Serum concentrations of oxybutynin and its active metabolite, N-desethyloxybutynin, were measured for up to 48 h after ingestion of a single dose of 10 mg oxybutynin. Results: Intake of oral contraceptive steroids had no significant effect on the pharmacokinetic parameters of oxybutynin or its metabolite. Both in males and females, the mean area under the curve (AUC_0–t) of N-desethyloxybutynin was about 13 times higher and the peak concentration (C_max) 15 to 19 times higher than the AUC_0–t and C_max of the parent oxybutynin, with no significant differences between males and females. Conclusions: The pharmacokinetics of orally administered oxybutynin shows a considerable interindividual variability, but is unaffected by gender and use of contraceptive steroids. Received: 21 May 1997 / Accepted in revised form: 4 August 1997     

A pharmacological differentiation between postjunctional (AT<Subscript>1A</Subscript>) and prejunctional (AT<Subscript>1B</Subscript>) angiotensin II receptors in the rabbit aorta

The effects of angiotensin II and angiotensin III were compared at prejunctional and postjunctional AT₁ receptors of the rabbit thoracic aorta. Furthermore, the influence of PD123319, losartan and eprosartan on these effects was also compared. To study prejunctional effects, the tissues were preincubated with (³H)-noradrenaline, superfused and electrically stimulated (1 Hz, 2 ms, 50 mA, 5 min). To study postjunctional effects, non-cumulative concentration–response curves were determined. Both angiotensin II and angiotensin III were more potent prejunctionally than postjunctionally. In the case of angiotensin II, the EC₅₀ was 12 times lower at the prejunctional than at the postjunctional level, while that of angiotensin III was 30 times lower prejunctionally. Furthermore, whereas angiotensin II was about 33 times more potent than angiotensin III postjunctionally, it was only 12 times more potent than angiotensin III prejunctionally. Eprosartan did not differentiate between prejunctional and postjunctional effects of both angiotensins. In contrast, PD123319 and losartan did differentiate; however, whereas PD123319 concentration-dependently antagonised the facilitation of tritium release caused by angiotensin II and angiotensin III and had no influence on the contraction of the aortic rings elicited by the peptides, losartan did the opposite: it concentration-dependently antagonised the contractions caused by the peptides on the aortic rings and exerted no influence on the facilitatory effect of angiotensin II and angiotensin III. These results show that prejunctional and postjunctional receptors for angiotensin II and angiotensin III are different and underline the hypothesis that postjunctional AT₁ receptors belong to the AT_1A subtype, while prejunctional AT₁ receptors belong to the AT_1B subtype.     

Receptor subtypes Y1 and Y5 are involved in the renal effects of neuropeptide Y

1. Systemic infusion of neuropeptide Y (NPY) reduces renal blood flow and can concomitantly increase diuresis, natriuresis and calciuresis in anaesthetized rats. The present study was designed to investigate whether the apparently contradictory NPY effects on renal blood flow and urine formation and composition are mediated by distinct NPY receptor subtypes.
2. NPY and its analogues, peptide YY (PYY), [Leu³¹, Pro³⁴]NPY and NPY₁₃₃₆, were infused in incremental doses of 0.3, 1 and 3 μg kg⁻¹ min⁻¹ for 45 min each and the results compared to those obtained in vehicle-infused rats. Renal blood flow was monitored in 15 min intervals, while urine excretion and composition were determined in 15 min collection periods. Plasma renin activity and aldosterone concentrations were measured at the end of the final infusion period.
3. Relative to vehicle NPY, PYY and [Leu³¹, Pro³⁴]NPY dose-dependently reduced renal blood flow and increased diuresis, natriuresis and calciuresis with roughly similar potency; NPY₁₃₃₆ slightly but significantly increased renal blood flow but had no effect on diuresis, natriuresis and calciuresis. None of the peptides significantly affected endogenous creatinine clearance or kaliuresis.
4. Plasma renin activity was significantly reduced by PYY. Quantitatively similar reductions were observed with NPY and [Leu³¹, Pro³⁴]NPY but failed to reach statistical significance with the given number of experiments. NPY₁₃₃₆ did not reduce plasma renin activity. None of the peptides significantly affected plasma aldosterone concentrations.
5. In another series of experiments infusion of PYY₃₃₆ (2 μg kg⁻¹ min⁻¹ for 120 min) did not reduce renal blood flow but significantly enhancd diuresis and natriuresis to a similar extent as the NPY 2 μg kg⁻¹ min⁻¹.
6. In a final series of experiments the Y₁-selective antagonist, BIBP 3226 (1 or 10 μg kg⁻¹ min⁻¹) dose-dependently antagonized reductions of renal blood flow elicited by bolus injections of NPY (0.130 μg kg⁻¹). BIBP 3226 (10 μg kg⁻¹ min⁻¹) also inhibited the effects of a 120 min infusion of NPY (2 μg kg⁻¹ min⁻¹) on renal blood flow but had only minor inhibitory effects on enhancements of diuresis and did not significantly affect enhancements of natriuresis.
7. We conclude that NPY reduces renal blood via a classical Y₁ subtype of NPY receptor. In contrast enhancements of diuresis, natriuresis and calciuresis occur via a distinct subtype which resembles the receptor that mediates NPY-induced enhancement of food intake.

     

CHARACTERIZATION OF THE ADRENORECEPTOR ACTIVITIES OF ISOPRENALINE IN THE FIELD STIMULATED RAT VAS DEFERENS: SELECTIVE SUPERSENSITIVITY TO β2-MEDIATED RESPONSES FOLLOWING RESERPINE TREATMENT

1 Low concentrations of isoprenaline (EC₅₀= 45.6 nM) inhibited contractions in the isolated field stimulated rat vas deferens. This inhibitory effect was markedly attenuated by the postjunctional β₂-adreno-receptor antagonist timolol, but not affected by the prejunctional α₂ or postjunctional α₁-adrenoreceptor antagonists rauwolscine and prazosin, respectively. 2 In vas deferens of rats previously treated with reserpine, the postjunctional β₂-adrenoreceptor-mediated inhibitory response to isoprenaline was markedly potentiated. 3 High concentrations of isoprenaline (EC₅₀= 1.5 uM) also inhibited contractility in tissues in which postjunctional β₂-adrenoreceptors were maximally blocked by high concentrations of timolol. This contractile inhibition produced by isoprenaline was abolished by rauwolscine but not significantly altered by prazosin or pretreatment of the rats with reserpine indicating stimulation of prejunctional α₂-adreno-receptors. 4 Rauwolscine pretreatment unmasked an ability of isoprenaline (EC₅₀= 17.1 juM) to produce enhancement of field stimulation-induced contractions. This response was abolished by prazosin but was unaffected by timolol or reserpinization indicating an action upon postjunctional α₁-adrenoreceptors. 5 The data indicate isoprenaline activates adrenoreceptor mechanisms in the field stimulated rat vas deferens by a direct action not dependent upon endogenous catecholamines and with an order of activity of β₂>α₂>α₁. Pretreatment with reserpine produces rapid and selective development of supersensitivity to the postjunctional β₂-mediated inhibitory response of isoprenaline in this preparation.     

Design of the Y1‐receptor‐selective cyclic peptide based on the C‐terminal sequence of neuropeptide Y

Abstract: We designed four cyclic peptides which are mimics of the C‐terminal region of human neuropeptide Y (NPY) on the basis of the structural model of NPY. One of these cyclic peptides, c[D‐Cys²⁹‐L‐Cys³⁴]NPY Ac‐29‐36 (YM‐42454), exhibited significantly higher affinity for the Y₁‐receptor than the corresponding C‐terminal linear fragment, NPY Ac‐28‐36. Interestingly, YM‐42454 showed binding affinity for the Y₁‐receptor in spite of the lack of the N‐terminal sequence of NPY, whereas it did not show any binding affinity for the Y₂‐receptor. This conformationally restricted Y₁‐selective peptide would provide some insights into the bioactive conformation of the C‐terminal region of NPY.     

Inhibition of nicotinic acetylcholine receptor channels in bovine adrenal chromaffin cells by Y3-type neuropeptide Y receptors via the adenylate cyclase/protein kinase A system

The effect of neuropeptide Y [NPY(1–36)] and related peptides on the voltage-dependent currents and the nicotinic acetylcholine receptor (nAChR) currents (I_ACh) of bovine adrenal chromafptn cells was investigated using the whole-cell patch clamp technique. Catecholamine release from single chromaffin cells was measured by means of fast cyclic voltammetry. The potency order of these peptides in inhibiting I_ACh evoked by nicotine was NPY(1–36), NPY (16–36) > peptide YY(PYY) > [Leu³¹, Pro³⁴] NPY. NPY(16–36) produced a similar degree of inhibition, irrespective of whether nicotine or an equipotent concentration of acetylcholine was used to evoke I_ACh. NPY(16–36) failed to alter voltage-dependent inward or outward currents. Intracellular cAMP, and extracellular dibutyryl-cAMP, produced a slowly developing increase in I_ACh. Intracellular cAMP, extracellular 8-Br-cAMP or dibutyryl-cAMP, and an inhibitor of cyclic nucleotide phosphodiesterases 3-isobutyl-l-methylxanthine (IBMX), decreased the inhibitory effect of NPY(16–36) on l_ACh. Although the intracellular application of the cAMP-dependent protein kinase A inhibitor [PKI(14–24)amide] alone did not alter I_ACh, it potentiated the effect of NPY(16–36) in interaction experiments. While the NPY(16–36)-induced inhibition of I_ACh was reversed on washout of the peptide, the slightly shorter C-terminal fragment NPY(18–36) caused a long-lasting depression of both I_Ach and catecholamine secretion evoked by nicotine. This depression was smaller in the presence of extracellular 8-Br-cAMP than in its absence. NPY(18–36) did not alter the secretory activity induced by a high concentration of potassium. It appears that, by activating Y₃-receptors, NPY inhibits nAChR-current and the resulting secretion of catecholamines from bovine chromaffin cells. This process may involve a G protein-mediated decrease in intracellular cAMP with a subsequent decrease in the degree of phosphorylation of the nAChR-channel.     

NEUROPEPTIDE Y INDUCES AND MODULATES VASOCONSTRICTION IN INTRACRANIAL AND PERIPHERAL VESSELS OF ANIMALS AND MAN

• 1 Neuropeptide Y (NPY) has recently been reported to coexist with noradrenaline (NA) in central as well as peripheral noradrenergic nerves. NPY-containing nerve fibres are particularly numerous around blood vessels.
• 2 Studies were performed on isolated pial arteries as well as on arteries and veins from several peripheral vascular beds from rabbit, cat and man. NPY induced a varying degree of direct contraction of the vessels with an E_Am up to 15 mN. Pial arteries were more sensitive than peripheral arteries to NPY (mean EC₅₀ = 7.6 × 10^?9 M). The presence of NPY did not cause any consistent or significant potentiation of the contractile response to NA in any of the vessels tested.
• 3 Transmural electrical stimulation of the perivascular nerves (including blockade with tetrodotoxin) was performed mainly with auricular artery from the rabbit. Blocking experiments confirmed that the neurogenic contraction was mediated by noradrenergic-type fibres. NPY caused a concentration-related potentiation of the neurally evoked contractile response. The peptide also potentiated the tetrodotoxin-resistant probably non-neurogenic contractions obtained during enhanced electrical field stimulation.
• 4 It is concluded that NPY interacts with NA during sympathetic nerve activation primarily through a presynaptic effect.

     

Antagonism of pre- and postjunctional responses to neuropeptide Y and sympathetic stimulation by D-myo-inositol-1,2,6-trisphosphate in the anaesthetised dog.

Pre- and postjunctional responses to nerve released or exogenous neuropeptide Y (NPY) were measured in the anaesthetised dog before and after administration of D-myo-inositol-1,2,6-trisphosphate (PP56) a putative NPY antagonist. The inhibition of the increase in pulse interval evoked by vagal stimulation was used as a measure of prejunctional action of NPY and the magnitude of increase in blood pressure was used as a measure of postjunctional action of NPY (direct action or constrictor potentiating). Elevated plasma levels of PP56 were maintained throughout the course of the experiment. PP56 significantly reversed the inhibitory effect of NPY (nerve released or exogenous) on cardiac vagal action, and significantly inhibited the pressor response to exogenous NPY. PP56 did not affect the pressor response to intravenous phenylephrine, a selective alpha-adrenoceptor agonist. PP56 therefore significantly antagonises both pre- and postjunctional effects of NPY (nerve released and exogenous) and, with respect to its postjunctional antagonism, this action is selective for NPY.