Frequency- and reserpine-dependent chemical coding of sympathetic transmission: differential release of noradrenaline and neuropeptide Y from pig spleen

The importance of impulse pattern and stimulation frequency for the release of noradrenaline (NA) and the coexisting peptide neuropeptide Y (NPY) in relation to vasoconstriction (perfusion-pressure increase) was studied in the blood-perfused pig spleen in vivo. Splenic nerve stimulation with intermittent bursts at high frequency (20 Hz) caused a several-fold larger release of NPY-like immunoreactivity (-LI) in relation to NA than a continuous stimulation at a low frequency (2 Hz), giving the same total number of impulses. alpha-Adrenoceptor blockade by phentolamine enhanced markedly both NA and NPY release, especially at low stimulation frequency, suggesting prejunctional adrenergic inhibition of release. Addition of propranolol unmasked a large remaining perfusion-pressure response to nerve stimulation. Reserpine treatment reduced the NA content of the spleen as well as the stimulation-evoked NA release by greater than 90%. However, the perfusion-pressure increase in response to nerve stimulation was well maintained. A marked increase in the stimulation-evoked release of NPY-LI occurred after reserpine. Adrenoceptor blockade after reserpine treatment reduced only slightly the perfusion-pressure response in parallel with a decline in NPY output. NPY caused an adrenoceptor-resistant perfusion-pressure increase at plasma concentrations that were in the same range as the maximal increase during nerve stimulations. In conclusion, the present data suggest a frequency-dependent, chemical coding of sympathetic transmission with preferential release of the classical transmitter NA at low, continuous frequencies and release of NPY, mainly at high frequencies. Reserpine treatment enhances markedly NPY release, which may explain why the functional response is largely intact in spite of adrenoceptor blockade and marked NA depletion.     

Possible involvement of neuropeptide Y in sympathetic vascular control of canine skeletal muscle

Sympathetic nerve stimulation (2 min, 2 and 10 Hz) increased perfusion pressure in the blood perfused canine gracilis muscle in situ after pretreatment with atropine, desipramine and beta-adrenoceptor antagonists. This vasoconstriction was accompanied by clear-cut increases in the overflow of endogenous noradrenaline (NA) at both frequencies and, at 10 Hz but not at 2 Hz, also of neuropeptide Y-like immunoreactivity (NPY-LI). The irreversible alpha-adrenoceptor antagonist phenoxybenzamine enhanced the nerve stimulation induced overflows of NA and NPY-LI five- to eightfold and threefold, respectively. The fractional overflows of NA and NPY-LI per nerve impulse were similar in response to the high-frequency stimulation, indicating equimolar release in relation to the tissue contents of the respective neurotransmitter. The maximal vasoconstrictor response elicited by 10 Hz was reduced by about 50% following a dose of phenoxybenzamine which abolished the effect of exogenous NA and the remaining response was more long-lasting. Local i.a. infusion of NPY evoked long-lasting vasoconstriction in the presence of phenoxybenzamine, while the stable adenosine 5(1)-triphosphate (ATP) analogue alpha-beta-methylene ATP was without vascular effects. Locally infused NPY reduced the nerve stimulation evoked NA overflow by 31% (P less than 0.01) at 1 microM in arterial plasma, suggesting prejunctional inhibition of NA release. In conclusion, NPY-LI is released from the canine gracilis muscle upon sympathetic nerve stimulation at high frequencies. There is nerve stimulation evoked vasoconstriction, which is resistant to alpha-adrenoceptor blockade. This may in part be mediated by NPY released together with NA from the sympathetic vascular nerves.     

Co-release of neuropeptide Y and catecholamines upon adrenal activation in the cat

The release of neuropeptide Y (NPY)-like immunoreactivity (-LI) in relation to catecholamines from the cat adrenal was studied in anaesthetized animals. Abdominal surgery increased plasma levels of NPY-LI from 65 +/- 6 to 149 +/- 26 pmol l-1. A positive veno-arterial concentration gradient over the adrenal gland was found for both NPY-LI, adrenaline (Adr) and noradrenaline (NA) during basal conditions. Asphyxia for 2 min increased the output of both NPY-LI and catecholamines from the adrenal. Electrical stimulation of the splanchnic nerve caused a marked increase in adrenal output of NPY-LI and catecholamines. The adrenal content of NPY-LI, as well as the release of NPY-LI from the adrenal, was at least 1000-fold lower on a molar basis than that of catecholamines. The concentration of NPY-LI in the adrenal vein upon splanchnic nerve stimulation was in the nM range. Reversed-phase HPLC characterization revealed that NPY-LI in the adrenal, and in the adrenal venous plasma collected during splanchnic nerve stimulation, was closely related to synthetic porcine NPY. Stimulation with bursts of 20 Hz for 1 S with 10 s intervals for 2 min caused a four-fold higher output of NPY-LI and Adr compared to a continuous stimulation with 2 Hz, giving the same number of impulses. The NA output, however, was only slightly increased by burst stimulation. Guanethidine did not reduce the adrenal output of NPY-LI or catecholamines induced by splanchnic nerve stimulation, while the release was abolished by chlorisondamine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Release and vasoconstrictor effects of neuropeptide Y in relation to non-adrenergic sympathetic control of renal blood flow in the pig

The possible involvement of neuropeptide Y (NPY) in sympathetic control of renal blood flow was investigated in the pig in vivo. Exogenous NPY caused renal vasoconstriction with a threshold effect at an arterial plasma concentration of 164 pmol 6(-1). Stimulation of the renal nerves (0.59, 2 and 10 Hz) in control animals evoked rapid and frequency-dependent reduction in renal blood flow and overflow of NPY-like immunoreactivity (NPY-LI) and noradrenaline (NA) from the kidney, suggesting co-release from sympathetic nerves. Following the administration of the alpha- and beta-adrenoceptor antagonists phenoxybenzamine and propranolol, the vasoconstrictor response to exogenous NA was reduced by 98%, whereas that of NPY was unaltered. The response to nerve stimulation with 0.59 Hz was abolished, whereas relatively slowly developing reductions in renal blood flow by 7 and 28% were obtained upon stimulation with 2 and 10 Hz respectively. The nerve stimulation-evoked overflow of NA at 0.59 and 2 Hz, but not at 10 Hz and not that of NPY-LI, was enhanced after adrenoceptor blockade. Twenty-four hours after reserpine treatment (1 mg kg-1 i.v.) the contents of NPY-LI and NA in the renal cortex were reduced by 80 and 98% respectively. Sectioning of the renal nerves largely prevented the reserpine-induced depletion of NPY-LI, but not that of NA. Nerve stimulation of the denervated kidney with 2 and 10 Hz 24 h after reserpine treatment evoked slowly developing and long-lasting reductions in renal blood flow by 6 and 52% respectively. These responses were associated with overflow of NPY-LI, which was similar to and threefold higher than that observed in controls at 2 and 10 Hz respectively, while no detectable overflow of NA occurred. Repeated stimulation with 10 Hz resulted in a progressive fatigue of the vasoconstrictor response and the associated overflow of NPY-LI, giving a high correlation (r = 0.86, P less than 0.001) between the two parameters. It is concluded that NPY is a potent constrictor of the renal vascular bed. Furthermore, although NA is the likely transmitter mediating most of the responses to low to moderate nerve activation under control conditions, the data suggest that NPY may mediate the non-adrenergic reductions in renal blood flow evoked by high-frequency sympathetic nerve stimulation after reserpine treatment.     

Neuropeptide Y and differential sympathetic control of splenic blood flow and capacitance function in the pig and dog

The roles of different mediators in the sympathetic regulation of the pig and dog spleens were investigated using a preparation with intact vascular perfusion in vivo. Sympathetic nerve stimulation caused overflow of neuropeptide Y-like immunoreactivity (NPY-LI) and noradrenaline (NA), arterial vasoconstriction, increase in venous blood flow and haematocrit. The dog spleen responded to single impulse stimulation, whereas more prolonged stimulation was required to elicit vascular responses in the pig spleen. Furthermore, the maximal splenic capacitance response was about 10 times larger in the dog than in the pig. After depletion of neuronal NA content by reserpine combined with preganglionic denervation, about 70% of the splenic arterial vasoconstrictor responses in the dog and pig still remained at 5 Hz stimulation. Fifty per cent of the capacitance response evoked by nerve stimulation still remained in the pig while in the dog spleen the capacitance response was virtually abolished after reserpine. The stimulation-evoked overflow of NPY-LI in pig spleen was increased several fold after reserpine treatment as compared to controls reaching levels in the venous effluent where exogenous NPY evokes vasoconstriction. In the dog spleen, overflow of NPY-LI was only observed after reserpine. Administration of NA caused arterial vasoconstriction with an initial increase in venous blood flow while NPY mainly reduced arterial blood flow. It is concluded that NA is involved in both the splenic arterial vasoconstriction and the capacitance responses while a non-adrenergic splenic vasoconstriction at least in the pig may be mediated by NPY.     

Sympathetic vascular control of the pig nasal mucosa (III): co-release of noradrenaline and neuropeptide Y

The overflows of noradrenaline (NA) and neuropeptide Y like immunoreactivity (NPYLI) and vascular responses upon sympathetic nerve stimulation were analysed in the nasal mucosa of pentobarbital anaesthetized pigs. In controls, a frequency-dependent increase in NA overflow was observed whereas detectable release of NPY-LI occurred only at 6.9 Hz. Parallel decreases in blood flow in the sphenopalatine artery and vein and in nasal mucosa volume (reflecting blood volume in the venous sinusoids) were observed. The laser Doppler flowmeter signal (reflecting superficial blood flow) increased upon low and decreased upon high frequency stimulation. Twenty-four hours after reserpine pretreatment and preganglionic decentralization, the NA overflow was abolished while a frequency-dependent release of NPY-LI occurred. Forty, 60 and 80% of the vasoconstrictor responses then remained upon stimulation with a single impulse, 0.59 and 6.9 Hz, respectively. Both the vasoconstriction and NPY-LI overflow, however, were subjected to fatigue upon repeated stimulation. In reserpinized animals release of NPY-LI and vasoconstrictor responses were larger upon stimulation with irregular bursts at 0.59 Hz compared to effects seen at stimulation with continuous impulses. Pre-treatment with the a-adrenoceptor antagonist phenoxybenzamine or the monoamine reuptake inhibitor, desipramine, enhanced NA overflow by 2–3 and 1.5 times at 0.59 and 6.9 Hz, respectively. Phenoxybenzamine significantly reduced the nerve-evoked vascular responses while the release of NPY-LI at 6.9 Hz was increased. Desipramine increased the functional responses but reduced the NPY-LI overflow. During tachyphylaxis to the vasoconstrictor effects of the stable adenosine 5′-triphosphate (ATP) analogue α-β-methylene ATP (mATP) in controls, the vasoconstrictor responses as well as the NA and NPY-LI overflow to nerve stimulation were unmodified. In reserpinized animals, however, the vascular responses and the overflow of NPY-LI were reduced after mATP tachyphylaxis. These data show that both NA and NPY are released upon sympathetic nerve stimulation in the nasal mucosa in vivo and this release seems to be regulated via prejunctional a-adrenoceptors. The lack of effect of mATP tachyphylaxis under control conditions makes it less likely that ATP serves as a major mediator of the large nonadrenergic vasoconstrictor component.     

Subcellular storage and axonal transport of neuropeptide Y (NPY) in relation to catecholamines in the cat

The subcellular storage of neuropeptide Y-like immunoreactivity (NPY-LI) in peripheral sympathetic neurons and adrenal gland as well as its axonal transport in the sciatic nerve was studied in relation to catecholamines in the cat. In the subcellular fractions from different parts of sympathetic neurons, i.e. cell bodies (coeliac ganglia), axons (sciatic nerve) and terminals (spleen), the NPY-LI was found together with noradrenaline (NA) in heavy fractions assumed to contain large dense-cored vesicles. In addition, minor lighter fractions in the coeliac ganglion contained NPY-LI. The molar ratio between vesicular NA and NPY was high in the terminal regions (150 to 1) and much lower in axons and cell bodies (10 to 1), thus reflecting the different mechanisms of resupply for classical transmitter and peptide. In the adrenal gland the NPY-LI was mainly located in the catecholamine-storing chromaffin-granule fraction and also to a smaller extent in lighter fractions. Using reversed-phase HPLC, one molecular form of NPY-LI corresponding to porcine NPY was found in the coeliac ganglion, while the adrenal medulla also contained minor peaks with NPY-LI in addition to the main form, which co-eluted with porcine NPY. NA was stored both in light and heavy fractions in the spleen, while it was mainly found in heavier fractions in the sciatic nerve. In the coeliac ganglion, most of the noradrenaline was present in a non-particulate form. The anterograde transport rate for NPY-LI in the sciatic nerve was estimated to be about 9 mm h-1. A minor retrograde transport of NPY-LI was also detected. In conclusion, the present data suggest that NPY, a peptide with sympathoactive actions, is co-stored with NA in heavy fractions corresponding to large dense-cored vesicles, while light fractions with small dense-cored vesicles probably contain NA but not NPY-LI. The main resupply of NPY to terminals is, in contrast to NA, most likely by axonal transport, which implicates differences in the storage, turnover and release of these co-existing substances in the sympathoadrenal system.     

Time-dependent effects of ischaemia on neuropeptide Y mechanisms in pig renal vascular control in vivo

We have investigated the effects of ischaemia on neuropeptide Y (NPY) mechanisms involved in sympathetic vascular control of the pig kidney in vivo. Reperfusion after 2 h of renal ischaemia was associated with local overflow of noradrenaline (NA) but not of NPY-like immunoreactivity (-LI). Renal sympathetic nerve stimulation 10 min into reperfusion evoked markedly reduced vasoconstrictor effects and significantly less overflow of NA (reduced by 70% from the pre-ischaemic conditions), whereas NPY-LI overflow was unaltered. Renal vasoconstrictor responses to exogenous peptide YY (PYY), phenylephrine and angiotensin II were strongly attenuated after this ischaemic period, while vasoconstriction to α,β-methylene ATP was maintained to a larger extent. The renal vascular responses and NA overflow had become partially normalized within a 2 h recovery period. In contrast, the renal vasoconstrictor response and the overflow of NPY-LI upon sympathetic nerve stimulation were enhanced after 15 min of renal ischaemia. In parallel, the PYY-evoked renal vasoconstriction was selectively and markedly prolonged after the 15 min of ischaemia. In the presence of the NPY Y₁ receptor antagonist BIBP 3226, the augmented vascular response to nerve stimulation was significantly attenuated. We conclude that reperfusion after 2 h of renal ischaemia is associated with local overflow of NA, whereas the sympathetic nerve-evoked release of NA and the reactivity of the renal vasculature to vasoconstrictor stimuli are reversibly reduced. Furthermore, possibly due to an impaired local degradation, the role of neurogenically released NPY in renal sympathetic vasoconstriction is enhanced after short-term (15 min) ischaemia compared with control conditions.     

Neuropeptide Y and sympathetic control of heart contractility and coronary vascular tone

The effects of neuropeptide Y (NPY) on contractility of the spontaneously beating guinea-pig atrium and transmural nerve stimulation (TNS)-induced efflux of tritium-noradrenaline (3H-NA) were studied in vitro. NPY induced a moderate positive chronotropic and inotropic atrial response, which was resistant to metoprolol. TNS at 2 Hz for 2 s caused an increase in rate and contractile force. These effects were significantly reduced by NPY. NPY also reduced the TNS induced (2 Hz for 20 s), fractional [3H]NA release by 40% without affecting the contractile response. The contractile effects of exogenous NA on the guinea-pig atrium were not affected by NPY. NPY caused a long-lasting increase in coronary perfusion pressure, and also, in high doses, an inhibition of ventricular contractility in the isolated, perfused guinea-pig heart. The perfusion pressure increase to NPY, which most likely reflects coronary vasoconstriction, was resistant to alpha- and beta-adrenoceptor blockade but sensitive to the calcium antagonist nifedipine. A 50% reduction of the vascular NPY response occurred at 10(-9) M nifedipine, which did not influence cardiac contractility per se or the contractile effects of NA. NPY did not modify the increase in ventricular contractility induced by NA. Noradrenaline did not influence coronary perfusion pressure after beta-blockade. Since NPY is present together with NA in cardiac nerves, it may be suggested that NPY is involved in the regulation of NA release as well as the sympathetic control of atrial contractility and coronary blood flow.     

Plasma neuropeptide Y-like immunoreactivity and catecholamines during various degrees of sympathetic activation in man

Neuropeptide Y-like immunoreactivity (NPY-LI) and catecholamine concentrations in plasma were analysed during and after 60 min of physical exercise at a work load corresponding to 70% of individual maximal oxygen uptake in nine healthy men of average physical fitness. Systemic plasma NPY-LI increased progressively from 18 +/- 3 to 81 +/- 19 pmol X 1(-1) in parallel with a 10-fold increase in noradrenaline (NA) concentration. The increase in plasma NPY-LI during exercise and the decrease after completion of exercise were much slower than the corresponding changes in NA concentration. This difference is probably related to a slower diffusion of NPY into systemic circulation after release, as well as to a longer half-life of NPY than of NA in plasma. Reversed phase HPLC and sephadex G-50 gel-filtration chromatography revealed that the main component of NPY-LI in plasma during exercise eluted in a similar position as synthetic human NPY. During exercise plasma NPY-LI correlated well with the plasma concentration of NA (r = 0.80), but not with that of adrenaline (ADR), suggesting a neuronal origin of NPY. The self-ratings of perceived exertion (RPE) were well correlated with the plasma concentrations of both NPY-LI and NA. No clear-cut veno-arterial concentration difference was observed for NPY-LI. Isometric handgrip and orthostatic test doubled plasma NA concentrations but did not cause any increase in plasma NPY-LI. No change in plasma tachykinin-like immunoreactivity was detected during exercise. The present data suggest that NPY is released together with NA during strong, but probably not during mild, sympathetic activation under physiological conditions in man.     

Mechanisms underlying changes in the contents of neuropeptide Y in cardiovascular nerves and adrenal gland induced by sympatholytic drugs

Neuropeptide Y (NPY) is a recently isolated vasoactive peptide, which is present, together with catecholamines, in sympathetic nerves and in the adrenal medulla. In the present study, we report that pretreatment with sympatholytic agents influences the tissue levels of NPY-like immunoreactivity (NPY-LI) in the guinea-pig. Thus, 24 h after reserpine not only noradrenaline (NA), but also NPY-LI, was depleted in the heart, spleen and the adrenal gland. The levels of NPY-LI in the vas deferens and stellate ganglia, however, were unaffected by reserpine in spite of marked depletions of NA. The reserpine-induced depletion of NPY-LI was probably caused by enhanced nerve-impulse flow and subsequent release from cardiovascular nerves in excess of resupply, since it could be prevented by the ganglionic-blocking agent chlorisondamine. Long-term (6 days) treatment with chlorisondamine reduced the levels of NPY-LI in the stellate ganglion. Short-term treatment (48 h) with guanethidine partially prevented the reserpine-induced depletion of NPY-LI, probably due to inhibition of NPY release. Long-term guanethidine treatment depleted not only NA, but also NPY-LI from the spleen. Pretreatment with the alpha-receptor antagonist phenoxybenzamine did not influence the NA levels but reduced the content of NPY-LI in the spleen via a mechanism that was dependent on intact ganglionic transmission. Since NPY has several cardiovascular actions, changes in NPY mechanisms may contribute to the pharmacological and therapeutical effects of sympatholytic agents.     

Neuropeptide Y may mediate effects of sympathetic nerve stimulations on colonic motility and blood flow in the cat

The present study investigated sympathetic mechanisms involved in the regulation of colonic motility and blood flow in the cat. Infusion of neuropeptide Y (NPY) close i.a. produced an inhibition of colonic motility and a vasoconstriction of long duration but no post-infusion vasodilatation. In contrast to NPY, porcine pancreatic polypeptide did not evoke any vascular or motility response. On a molar basis, NPY was 25 times more potent than noradrenaline in producing 50% reduction of the colonic blood flow. These vascular and motility effects of NPY were resistant to guanethidine, phentolamine, phenoxybenzamine and propranolol. Thus, the action of NPY on vascular and colonic smooth muscle did not seem to be mediated via adrenergic receptors. Noradrenaline administered close i.a. produced inhibition of colonic motility, and vasoconstriction followed by a rapid vasodilatation. These effects were completely blocked by combined alpha- and beta-adrenoceptor blockade. Electrical stimulation of the splanchnic and lumbar colonic nerves produced an overall inhibition of colonic motility, and vasoconstriction of the proximal and distal colon, respectively, with a rapid post-stimulatory vasodilatation. After combined alpha- and beta-adrenoceptor blockade the inhibitory effect of the nerve stimulations on colonic motility partly remained together with a marked vasoconstriction, which was most pronounced upon lumbar colonic nerve stimulation. All vascular effects of sympathetic nerve stimulation were eradicated by guanethidine, which also abolished the inhibitory motility response to splanchnic nerve stimulation. However, lumbar colonic nerve stimulation elicited a colonic contraction, possibly due to stimulation of afferent C-fibres. The present findings indicate the existence of a sympathetic nonadrenergic neuronal mechanism mediating vasoconstriction and inhibition of colonic motility in the cat. Thus, NPY may be released from noradrenergic neurons to act on colonic smooth muscle and vessels.     

Renal sympathetic nerve activation in relation to reserpine-induced depletion of neuropeptide Y in the kidney of the rat

The effect of reserpine treatment on renal sympathetic nerve activity and tissue levels of neuropeptide Y (NPY)-like immunoreactivity (LI) and noradrenaline (NA) were studied in rats. Injection of reserpine (1 mg kg-1 i.v.) caused a clear-cut (about 50%) increase in rectified activity of the post-ganglionic sympathetic nerves to the kidney within 15 min in chloralose-anaesthetized rats compared to a saline-treated control group. This increase in nerve activity was still maintained 120 min after the reserpine injection. The renal nerve activation was accompanied by a progressive fall in mean arterial blood pressure and an initial tachycardia. In a separate group of conscious rats, the levels of NPY-LI (1.3 +/- 0.06 pmol g-1) and NA (1.6 +/- 0.07 nmol g-1) in the kidney were significantly reduced (by 74 and 83%, respectively) 24 h after reserpine treatment (1 mg kg-1 i.v.). The reserpine-induced depletion of NPY-LI, but not that of NA, was inhibited by pretreatment with the ganglionic blocking agent chlorisondamine or the alpha 2-adrenoceptor agonist clonidine, both of which are known to decrease renal sympathetic nerve activity. The tissue content of NPY-LI in the right atrium (16.3 +/- 0.7 pmol g-1) was not reduced by reserpine. Arterial plasma NPY-LI in the rat was high (222 +/- 5 pmol l-1), and this value did not change after pretreatment with reserpine, chlorisondamine or clonidine, indicating that, in the rat, circulating NPY-LI is not a good indicator of sympatho-adrenal activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of ATP in non-adrenergic sympathetic vascular control of the nasal mucosa in anaesthetized cats and dogs.

1. In anaesthetized cats and dogs, local intra-arterial injection of noradrenaline and alpha, beta-methylene adenosine 5'-triphosphate (mATP) reduced both nasal arterial blood flow and nasal mucosal volume (a measure of capacitance vessel function). The responses to mATP were not modified by pretreatment with the adrenoceptor antagonists phentolamine and propranolol or the purinoceptor antagonist suramin. The vascular effects of noradrenaline were not altered by suramin, but were virtually abolished by adrenoceptor antagonists. 2. After adrenoceptor blockade, frequency-dependent reductions in nasal arterial blood flow with sympathetic nerve stimulation were reduced by 25 and 39% in cats and dogs, respectively; whereas the volume response was reduced by 56% in cats and 54% in dogs. The remaining non-adrenergic sympathetic nerve-evoked vascular responses were not influenced by suramin. 3. During desensitization to mATP induced by local intra-arterial infusion for 5 min, the remaining non-adrenergic nasal blood flow and volume responses to sympathetic nerve stimulation were reduced in the dog but not in the cat. 4. It is suggested that both adrenergic and non-adrenergic mechanisms are involved in the sympathetic control of the nasal mucosa vascular bed of both species. Since desensitization to mATP markedly reduces the remaining non-adrenergic nasal vasoconstriction evoked by sympathetic nerve stimulation in the dog, ATP is a possible sympathetic mediator in the nasal vascular bed in this species.     

Pancreatic polypeptide family (APP,BPP, NPY and PYY) in relation to sympathetic vasoconstriction resistant to α-adrenoceptor blockade

Electrical stimulation of the cat cervical sympathetic trunk caused submandibular salivary secretion and vasoconstriction simultaneously with a contraction of the nictitating membrane. Following α- and β-adrenoceptor blockade by phentolamine or phenoxybenzamine combined with propranolol, the salivary response and the nictitating membrane contraction upon sympathetic stimulation were almost abolished. A considerable vasoconstrictor response (up to 40% of control) however still remained in the submandibular gland. This yasoconstriction, which persisted after α-adrenoceptor blockade, was rather slow in onset and had a long duration without any poststimulatory hyperemia. Local intra-arterial infusions of noradrenaline caused submandibular vasoconstriction, salivary secretion and nictitating membrane contraction. The blood flow response to exogenous noradrenaline did, however, not mimic the effects of sympathetic nerve stimulation with regard to vascular escape. Whereas the vascular escape after nerve stimulation was followed by a prolonged vasoconstriction with a gradual decline, the escape after noradrenaline infusions was accompanied by a normalization of blood flow. Local intra-arterial infusions of pancreatic polypeptide (PP)-related peptides caused a slowly developing vasoconstriction with a long duration in the submandibular gland, but no salivary secretion or contraction of the nictitating membrane. The relative molar potencies as vasoconstrictory agents were about PYY: 1, neuropeptide Y (NPY): 5, avian and bovine pancreatic polypeptid 100. The vasoconstrictor effects of PP-related peptides were resistant to α-adrenoceptor blockade and present also in sympathectomized animals, suggesting a direct action on vascular smooth muscle. Combined local infusions of noradrenaline and NPY caused a vascular response in the submandibular salivary gland which was similar to that seen upon sympathetic nerve stimulation. PYY and NPY caused increase in systemic arterial blood pressure upon systemic administration which indicates general vasoconstrictor actions. This effect was accompanied by a transient bradycardia which was due to inhibition of sympathetic tone, since it was absent in animals treated with propranolol. In conclusion, the present findings illustrate the differential sensitivity to α-adrenoceptor antagonists of the submandibular vasoconstriction and salivation as well as smooth muscle contraction of the nictitating membrane induced by sympathetic nerve stimulation. This remaining vasoconstriction may be explained by release of a nonadrenergic, PP-related transmitter such as NPY which may be present together with noradrenaline in the vascular nerves. Release of an additional vasoconstrictory factor may also account for the finding that infusions of noradrenaline do not mimic the vascular effects of sympathetic nerve stimulation in vivo.     

Neuropeptide Y (NPY) and sympathetic control of blood flow in oral mucosa and dental pulp in the cat

Neuropeptide Y (NPY) immunoreactivity (-IR) was found to be present in perivascular nerves in the cat dental pulp and oral mucosa. Many ganglion cells in the superior cervical ganglion also contained NPY-IR. Ligation of the inferior alveolar or lingual nerves produced an accumulation of NPY-IR in axons proximal to the site of ligation, suggesting an anterograde axonal transport of the peptide. After unilateral sympathectomy the NPY-IR disappeared in the dental pulp and oral mucosa on the ipsilateral side. Reversed phase high performance liquid chromatography showed that the main peak of NPY-like immunoreactivity found in the superior cervical ganglion co-chromatographed with synthetic porcine NPY. Changes in blood flow in dental pulp or oral mucosa were measured indirectly by recording local clearance of ¹²⁵I during electrical stimulation of the sympathetic nerve or during close intra-arterial infusion of noradrenaline or NPY. All three procedures resulted in a pronounced decrease in local blood flow of a similar magnitude in both tissues. After a-adrenoceptor blockade with phentolamine, the vasoconstrictor effect of noradrenaline was abolished. However, the effect of sympathetic stimulation after phentolamine was only partially reduced (23–54%) and the response to NPY was almost unaffected by the a-receptor blockade. The remaining effect of sympathetic stimulation after phentolamine was abolished by guanethidine. However, the response to NPY was not changed by the latter drug. In conclusion, the vasoconstrictor response in the dental pulp and oral mucosa caused by activation of sympathetic nerves is more resistant to phentolamine than the response induced by infusion of exogenous noradrenaline. Since NPY is probably co-localized with noradrenaline in the sympathetic perivascular nerves and NPY reduces local blood flow,it is proposed that this peptide is involved in sympathetic vascular control in oral tissues.     

Sympathetic vascular control of the pig nasal mucosa (2): Reserpine-resistant, non-adrenergic nervous responses in relation to neuropeptide Y and ATP

The possible occurrence of non-adrenergic mechanisms in the sympathetic vascular control of the nasal mucosa was studied in vivo using reserpine-treated pigs (1 mg kg-1, i.v., 24 h earlier) in combination with pharmacological blockade of alpha-adrenoceptors by local phenoxybenzamine (1 mg kg-1, i.a.) infusion. The nasal mucosal depletion (99%) of the content of noradrenaline (NA) in reserpinized animals was not influenced by preganglionic denervation while the depletion (44%) of neuropeptide Y (NPY) was prevented. Upon stimulation with single shocks, 25% of the arterial blood flow reduction and 47% of the nasal mucosal volume reduction (reflecting contraction of venous sinusoids) were still present after reserpine as compared with controls. In reserpinized animals, the vascular responses were slow developing and long-lasting, and about 60% remained at 0.59 Hz and more than 80% at 6.9 Hz. The vascular effects after reserpine were, however, subjected to fatigue, which may explain why phenoxybenzamine treatment still reduced the functional effects in the absence of NA. Local intra-arterial injections of NA, NPY and the metabolically stable adenosine-5'-triphosphate analogue alpha, beta-methylene ATP (mATP) caused reduction in both arterial blood flow and nasal mucosal volume. The C-terminal fragment of NPY (NPY 13-36) also induced nasal vasoconstriction although with a fivefold lower potency than NPY 1-36. Adenosine-5'-triphosphate caused a biphasic vascular effect with vasodilatatory actions at low doses and a short-lasting vasoconstriction followed by vasodilatation at very high doses (100-fold higher than the threshold response to mATP). In contrast to the response to NA, the long-lasting vascular effects of NPY and mATP were resistant to phenoxybenzamine treatment. In conclusion, although NA is likely to mediate most of the sympathetic vascular responses to low-frequency stimulation in the pig nasal mucosa, a large resistance and capacitance vessel component upon high-frequency stimulation seems to be non-adrenergic and mimicked by NPY rather than ATP.     

Effects of vinblastine on neuropeptide Y levels in the sympathoadrenal system,bone marrow and thrombocytes of the rat

The dose-related and time-related effects of vinblastine on tissue, platelet and plasma content of neuropeptide Y (NPY) were investigated in the rat and compared to the effects on catecholamine (CA) content. CA was quantified by HPLC with electrochemical detection and NPY-like immunoreactivity (LI) was analyzed by radioimmunoassay (RIA). Vinblastine (3.0 mg/kg, i.v.) decreased levels of both NPY-LI and CA after 48 h in the kidney, vas deferens and adrenal gland, whereas in the coeliac ganglion and bone marrow vinblastine induced an increase of NPY-LI which occurred already at a dose of 0.3 mg/kg. Also the content of NPY-LI in platelet-poor plasma and platelets as well as the decapitation-induced increase of plasma levels of noradrenaline (NA) and adrenaline (A) were attenuated by vinblastine (3.0 mg/kg). The elevation of NPY-LI content in the kidney, coeliac ganglion and bone marrow as well as the reduced levels of NPY-LI in platelets and platelet-poor plasma was observed already after 24 hours, whereas the reduction of NPY-LI and CA in the kidney and adrenal gland was present after 2 days. Vinblastine caused a biphasic effect on the content of NPY-LI in the sympathetic nerves of the kidney with an initial increase (by 120% at 24 h) followed by a decrease (by 79% at 4 days). The effect on NA-levels, however, was only a decrease. The axonal transport of NPY-LI as revealed by accumulation above a ligation of the sciatic nerve was reduced by 27% 2 days after vinblastine 3 mg/kg. The vinblastine-evoked depletion of NPY-LI and catecholamines in the kidney as well as in the adrenal was largely prevented by chlorisondamine, a nicotinic ganglionic blocking agent, suggesting that preganglionic neuronal activity was a key factor for this effect, in contrast to the influence on the coeliac ganglion cells and the megakaryocytes in the bone marrow. Furthermore, the delayed vinblastine depletion of NPY-LI in the kidney resembled the influence of surgical axotomy while reserpine caused a more rapid and complete depletion of both NPY-LI and NA. It is concluded that the multiple effects of vinblastine on sympathetic nerves, adrenal gland and megakaryocytes/thrombocytes can be monitored by analysis of NPY and be related to interference with microbutuli function and/or neuronal activation.     

Increase of plasma neuropeptide Y-like immunoreactivity following chronic hypoxia in the rat

In an attempt to understand the changes of circulating neuropeptide Y (NPY) during hypoxia, the plasma level of NPY was investigated by radioimmunoassay. Exposure of rats to hypobaric hypoxia at an altitude of 18,000 ft for 4 weeks causes an increase of pulmonary pressure and an elevation of plasma NPY-like immunoreactivity (NPY-LI). However, the systemic blood pressure was not elevated by this chronic hypoxia. Also, plasma noradrenaline (NA) estimated by chromatographic analysis (HPLC-ECD) was not markedly raised. Failure of bretylium and guanethidine, sympathetic neuron blockers, in reducing the plasma NPY-LI level of these rats ruled out the participation of adrenergic nervous terminals. Adrenal medulla seems responsible for this elevation of plasma NPY-LI because this magnitude disappeared in adrenalectomized rats. These data suggest that chronic hypoxia induced an elevation of circulating NPY from the adrenal gland of rats.     

Mechanisms underlying pre- and postjunctional effects of neuropeptide Y in sympathetic vascular control

The effects of porcine neuropeptide Y (NPY) regarding sympathetic vascular control were studied in vitro on isolated rat blood vessels. The 10(-9)M NPY enhanced (about two-fold) the contractile responses to transmural nerve stimulation (TNS), noradrenaline (NA) and adrenaline (about two-fold) in the femoral artery. Higher concentrations of NPY (greater than 10(-8)M) caused an adrenoceptor-resistant contraction per se. The TNS-evoked [3H]NA efflux was significantly reduced by NPY in a concentration-dependent manner (threshold 10(-9)M). The calcium antagonist, nifedipine, abolished the contractile effects of NPY and the NPY-induced enhancement of NA contractions but did not influence the prejunctional inhibition of [3H]NA release. Receptor-binding studies showed that the ratio of alpha 1-to alpha 2-adrenoceptors in the femoral artery was 30:1. The NPY did not cause any detectable change in the number of alpha 1-or alpha 2-adrenoceptor binding sites or in the affinity of alpha 2-binding sites, as revealed by prazosin- and clonidine-binding, respectively. The NPY also inhibited the TNS-evoked [3H]NA release (by 42-86%) in the superior mesenteric and basilar arteries and in femoral and portal veins. The NPY still depressed TNS-evoked [3H]NA secretion from the portal vein in the presence of phentolamine. The NPY caused a clear-cut contraction in the basilar artery, increased the contractile force of spontaneous contractions in the portal vein, while only weak responses were observed in the superior mesenteric artery and femoral vein. The NA-induced contraction was markedly enhanced by NPY in the superior mesenteric artery, only slightly enhanced in the portal vein and uninfluenced in the femoral vein. In conclusion, in all blood vessels tested, NPY depresses the TNS-evoked [3H]NA secretion via a nifedipine-resistant action. Furthermore, NPY exerts a variable, Ca2+-dependent vasoconstrictor effect and enhancement of NA and TNS contractions.