Effects of neuropeptide Y on canine cerebral circulation

European Journal of Pharmacology, 146 (1988) 271-277

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Elsevier EJP 50126

Effects of neuropeptide Y on canine cerebral circulation Y o s h i o Suzuki *, M a s a t o Shibuya, I c h i r o Ikegaki, Shin-ichi Satoh, M a s a k a z u T a k a y a s u and Toshio Asano ~ Department of Neurosurgery, Nagoya University, Nagoya 466 and ~ Pharmaceutical Research and Development Laboratory, Asahi Chemical Industry, Nobeoka 822, Japan

Received 22 July 1987, revised MS received 20 October 1987, accepted 17 November 1987

The effects of neuropeptide Y (NPY) on the vascular tone of isolated cerebral arteries and vertebral blood flow (VBF) were studied in dogs. NPY elicited a dose-dependent contraction of arteries derived from the brain with EDs0 values of 2 nM for the middle cerebral and basilar arteries. Arteries from the neck did not respond to NPY. Intra-arterial administration of NPY as a bolus reduced the VBF dose dependently, with no significant alteration of mean arterial blood pressure and heart rate. The decrease in VBF developed slowly and had a long duration, which was consistent with the observations made in vitro. NPY suppressed the contractile effect of noradrenaline (NA) on isolated cerebral arteries and pretreatment with NPY suppressed the effect of NA on VBF, indicating that NPY contributes to the inhibitory modulation of postsynaptic adrenergic mechanisms. These findings suggest that NPY could have a role in the regulation of cerebral circulation. Neuropeptide Y; Cerebral artery (canine); Blood flow (vertebral); Noradrenaline

1. Introduction The development of immunohistochemical techniques has provided progressively more reliable and informative evidence of the neurogenic regulation of cerebral circulation. Recent studies suggest that peptide-containing nerve fibers richly innervate cerebral blood vessels and that various peptides can function as neurotransmitters. The major candidates are vasoactive intestinal peptide (VIP) (Larsson et al., 1976), neuropeptide Y (NPY) (Lundberg et al., 1983), substance P (Chan-Palay, 1977) and calcitonin gene-related peptide (CGRP) (Hanko et al., 1985). Possible roles for these peptides as non-adrenergic and non-cholinergic vasoactive mediators have been suggested by * To whom all correspondence should be addressed: Department of Neurosurgery, Nagoya University School of Medicine, 65-Tsurumai, Showa, Nagoya 466, Japan.

numerous observations both in vitro (Edvinsson et al., 1985; Lee et al., 1984; Suzuki et al., 1985) and in vivo (Allen et al., 1984; Fisher et al., 1983; Wilson et al., 1981). NPY, originally isolated from porcine brain (Tatemoto et al., 1982), is a 36-amino acid peptide with a characteristic C terminal tyrosine amide. This peptide has been demonstrated to be present in neurons of the central and peripheral nervous systems (Lundberg et al., 1983), and to belong to a family of brain-gut neuroregulatory peptides. NPY appears to be costored with noradrenaline (NA) in the sympathetic nerves. In addition to the similar distribution patterns of N P Y and N A (Allen et al., 1984; Suzuki et al., 1983), a dense network of NPY-containing nerve fibers in cerebral arteries can be eliminated by prior removal of the superior cervical ganglion (Edvinsson et al., 1984). Exogerious administration of N P Y causes long-lasting vasoconstriction in cat preparations both in vitro

0014-2999/88/$03.50 ~3 1988 Elsevier Science Publishers B.V. (Biomedical Division)

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(Edvinsson et al., 1983) and in vivo (Edvinsson et al., 1984) and reduces regional cerebral blood flow in the rat cortex (Allen et al., 1984). However, little has been published on the effects of N P Y on the brain arteries or on the blood flow of dogs. In the present study, we investigated the effects of NPY on the vascular tone of isolated cerebral arteries and on the vertebral blood flow of dogs. The interaction between NPY and adrenergic mechanisms was also studied to elucidate the functional role of the sympathetic innervation of cerebral blood vessels,

2. Materials and methods Synthetic NPY, identical in structure to porcine NPY, was purchased from Peninsula Laboratories (San Carlos, CA). Noradrenaline and propranolol were purchased from Sigma Chemical Co. (St. Louis, MO). Phentolamine was obtained from Ciba Pharmaceutical Co. (Summit, N J). All other chemicals were of reagent grade or were the best cornmercially available,

2.1. In vitro studies 2.1.1. Preparation of arterial strip Mongrel dogs of both sexes weighing 7-15 kg were anesthetized with sodium pentobarbital (35 m g / k g i.v.) and exsanguinated from the femoral artery. Basilar and middle cerebral arteries from the brain, and the common carotid and vertebral arteries from the neck, were removed quickly and kept on crushed ice in oxygenated Krebs-Henseleit solution (mM: NaC1 115, KC1 4.7, CaC12 2.5, MgC12 1.2, N a H C O 3 2.5, KH2PO4 1.2 and dextrose 10.0). Each artery was helically cut into equal strips 20-25 m m long and 1-2 m m wide with the aid of a dissecting microscope after removal of the arachnoid membrane and connective tissue, The strips were suspended under a resting tension of 1.0 g in a 20 ml tissue bath containing KrebsHenseleit solution at 37 o C, and were aerated continuously with a mixture of 95% 02 and 5% CO2. The arterial strips were allowed to stabilize at this level for 90 min before the start of the experiments. The changes in arterial tension were re-

corded isometrically through a force-displacement transducer (Sanei, T7-8-240) and a polygraph.

2.1.2. Experimental procedures After 90 min, a submaximally effective concentration of KC1 (30 mM) was added dropwise to the bath two or three times until the successive responses of the tissue remained constant. These contractions were used as the standard for cornparison with the responses induced by N P Y in the arterial strips from the various regions. Cumulative dose-response curves for N P Y were obtained by a stepwise increase in the concentration of NPY. As cerebral arteries developed tachyphylaxis in response to repeated administration of NPY, paired strips from the same dogs were compared as indicators of changes in tissue sensitivity during the course of the experiments. Some tests were carried out with only one strip in the presence of the ~-adrenoceptor blocker phentolamine or the fl-adrenoceptor blocker propranolol. The effects of N P Y on the dose-response curve for NA were determined in arterial strips from the various regions. Care was taken to ensure that the maximum contractile tensions and EDs0 values are corrected accordingly when changes in tissue sensitivity were noted. 2.2. In vivo studies 2.2.1. Measurement of vertebral blood flow Mongrel dogs were intubated through the trachea during anesthesia with i.v. pentobarbital and were ventilated with room air delivered from a respirator (Harvard, model 607A). The ventilation rate (12-16 cycles/rain) and tidal volume (22-27 m l / k g ) were adjusted so as to maintain arterial blood gases and pH within physiological limits. Body temperature was maintained at 3 7 - 3 8 ° C with a heating pad. Catheters were placed in the right femoral artery for the measurement of pulsative arterial blood pressure (MBP). Heart rate (HR) was monitored with a cardiotachometer (Sanei, 2336A) triggered by a lead II electrocardiogram. To evaluate the effects of NPY on vertebral blood flow (VBF), arterial blood taken from the femoral artery was led to the left vertebral artery

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by a short extracorporeal loop, as described previously (Asano and Hidaka, 1985). Blood flow was measured by means of an electromagnetic flow meter with an adequately sized probe of the extracorporeal type (Nihon Kohden, model MFV1200). N P Y or N A was given by microinjection into a rubber tube connected to the arterial cannula for a period of 5 s. Heparin sodium (400 U / k g i.v.) was administered to prevent clotting. All responses are expressed as m e a n s + S.E. Comparison of the results was made with Student's t-test. Statistical significance was assumed when P < 0.05.

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Fig. 1. Dose-response curves of NPY-induced arterial contraction. Helical strips of arteries obtained from dog brain and neck were made to contract in response to the cumulative addition of NPY. The contraction elicited by 30 m M KCI was used as the standard response. The effects of N P Y are given as

3. Results

3.1. Contractile effect of N P Y on isolated cerebral arteries

NPY elicited a dose-dependent contraction of arteries obtained from the brain in concentrations ranging from 10 10 to 10 - 7 M , ( f i g . 1). The contractile response to N P Y was prolonged, but was relatively slow to start. The middle cerebral and basilar arteries responded to N P Y with similar EDs0 values (2 nM) but the maximal contrac-

a percentage of the standard. Each point represents the mean _+S.E. from the number of experiments (in parentheses). O Middle cerebral artery (10); • basilar artery (11); ~ carotid artery (6); • vertebral artery (5).

tions induced by 10 -7 M N P Y were 49.5 and 29.1%, respectively, of the contraction caused by 30 m M KC1. The contractions induced by NPY were unaffected by the c~- and /3-adrenoceptor blocking drugs, phentolamine and propranolol, at

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a concentration of 1 /.tM. N P Y in concentrations of up to 10 7 M was completely inactive in arteries from the neck, that is, the vertebral and c o m m o n carotid arteries. In order to assess whether N P Y would potentiate or suppress the contractions induced by NA, N P Y (10 -]°, 10 -8 M) was added to the bath

before a N A dose-response study was made. The N A dose-response curve was started once the arterial strips were contracted by N P Y (fig. 2). The contractile response of the cerebral artery to N A was suppressed in the presence of N P Y (10 -8 M) and suppression was seen with all three concentrations in the case of basilar artery (fig. 3).

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Time (min) Fig. 4. Time course of the hemodynamic response to intravertebral arterial administration of NPY in dogs. Vehicle (saline solution) or NPY (1 and 5 nmol) was injected into the vertebral artery as a bolus at time 0. Changes in blood flow are expressed as percentage of control. Each data point represents the mean+_S.E, from the number of experiments shown in parentheses.

Neither of the neck arteries showed any change in the N A dose-response curve in the presence of 10 ~ M NPY. 3.2. Vasoconstrictive action of N P Y on vertebral vascular bed

The effect of a bolus injection of N P Y into the vertebral artery is summarized in fig. 4. The changes after a single injection of vehicle (saline solution) or N P Y (1 and 5 nmol) were measured continuously for 30 min after initial base-line values for VBF (13.2 _+ 3.0 m l / m i n ; 100%) were obtained as a control. Bolus administration of N P Y into the vertebral artery of dogs caused a dose-dependent decrease in VBF. For example, the decrease in VBF produced by N P Y (5 nmol) was maximal at 3 min, with a value of 8.8 _+ 1.9 m l / m i n (68.4 + 2.2% of the control) and the VBF remained depressed for the entire 30 min period. The decreases caused by 1 and 5 nmol N P Y were statistically significant from 1 to 5 min

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Fig. 5. The influence of NPY on exogenous NA in VBF of dogs. The effect of NA (open bar) in decreasing VBF was compared before and after administration of NPY (hatched bar). The effect of 0.1/~g NA was significantly suppressed after 2 min and was slightly suppressed even 20 min after the administration of 1 nmol NPY. Each bar represents the mean _+S.E. of 4 experiments.

and from 1 to 20 min, respectively. MBP and H R were not significantly altered by the administration of N P Y (fig. 4). The influence of N P Y on the effect of exogenous N A was also studied in this in vivo system. A bolus injection of N A (0.1 and 1.0 /~g) into the vertebral artery caused a short-lasting decrease of VBF. The decrease in the VBF induced by 0.1 /~g N A was significantly suppressed 2 min after administration of 1 nmol NPY. Thus the influence of N A after N P Y was less than the expected additive values. This suppression by N P Y could be observed even 20 min after the administration of 1 nmol NPY. The results obtained in four dogs are summarized in fig. 5. The interaction between N P Y and N A in this study in vivo appeared to be quite consistent with our observations in vitro.

4. Discussion Potent long-lasting contractile responses to N P Y were observed in vitro in helical strips of

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arteries from the brain of dogs. However, N P Y in concentrations up to 10 7 M had no effect on arteries from the neck. Such a regional difference in the arterial response to NPY is consistent with the results of previous studies on N P Y vasoconstriction performed in rings of cat cerebral and peripheral blood vessels in vitro (Edvinsson et al., 1983; Ekblad et al., 1984). The ED~0 and the maximum (% of maximum for KC1) of the NPYinduced contraction of the cat middle cerebral artery (Edvinsson et al., 1983) are of a comparable order of magnitude to those observed in the present study with dog middle cerebral arteries. However, NPY has only a slight contractile effect on most isolated peripheral blood vessels such as mesenteric and femoral arteries (Ekblad et al., 1984), and has none whatsoever on arteries from the neck. Direct application of NPY to cat pial arterioles and veins in situ by means of the cranial window technique resulted in strong vasoconstriction which was more marked in the arterioles than in the veins (Edvinsson et al., 1984). These results suggest that NPY, as a neuropeptide that mediates vasoconstriction, possesses a relatively selective cerebrovascular action, Administration of N P Y by the intravertebral route has been shown to produce a dose-dependent long-lasting decrease in VBF in dogs, without significantly changing the MBP and HR. This effect is possibly explained as a reflection of the blood flow in intracranial vascular beds supplied by the vertebral artery, since evidence from studies in vitro showed that arteries from intracranial portions, but not from the neck, respond potently to NPY. Allen et al. (1984) have reported that intracarotid administration of N P Y to rats reduced the mean cerebral cortical blood flow, and that this decrease was slow to start and had a long duration. Our findings extended these observations by the demonstration of a persistent reduction of VBF in dogs after intravertebral administration of NPY. Since N P Y is costored with N A in sympathetic nerves around the blood vessels, it would be important to clarify whether NPY contributes to the modulation of presynaptic and postsynaptic adrenergic mechanisms in the cerebral blood vessels. The vasocontractile effect of N P Y is not

influenced by the presence of adrenoceptor blockers in either the cerebral or peripheral arteries (Franco-Cereceda et al., 1985). NPY is known to potentiate the adrenergically mediated contractile response or peripheral blood vessels to electrical stimulation of to the application of N A (DahlSf et al., 1985; Ekblad et al., 1984). Furthermore, NPY inhibits N A release from the sympathetic neurons (Dahl/Sf et al., 1985; Lundberg et al., 1985). However, H a n k o et al. (1986) recently showed that, in isolated blood vessels from humans, cats and rabbits, NPY potentiates the neurally evoked contractile response of peripheral arteries, but not the response to exogenous NA. They therefore concluded that NPY could modulate the release of N A through a presynaptic mechanism rather than by a postsynaptic interaction between the two substances. The ability of NPY to enhance vascular smooth muscle reactivity to N A was not ohserved in either cerebral arteries or arteries from the neck in our present study in vitro. In agreemerit with the recent observations of H a n k o et al. (1986), our experiments with arteries from the neck revealed that N P Y did not cause any potentiation or suppression of the contractile response to NA. However, our results obtained with cerebral arteries were different from those obtained with peripheral arteries, that is, NPY apparently suppressed the postsynaptic effect of NA. The results of our in vivo study confirmed this in vitro evidence that NPY suppressed the effect of N A on VBF as well, indicating that NPY could play an inhibitory role in the regulation of postsynaptic adrenergic mechanisms in cerebral blood vessels. In conclusion, our findings obtained from experiments in vitro and in vivo suggest that NPY, as a neurotransmitter of the sympathetic nerve endings that innervate blood vessels, could participate in the control of cerebral blood flow, not only by direct modulation of the vascular tone through its own specific receptors shown to exist in rat brain membrane (Und6n et al., 1984), but also through adrenergic mechanisms.

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277 circle of Willis and its possible role in cerebral vasospasm, Lancet ii, 550. Asano, T. and H. Hidaka, 1985, Intracellular Ca 2+ antagonist, HA 1004: Pharmacological properties different from those of nicardipine, J. Pharmacol. Exp. Ther. 233, 454. Chan-Palay, V., 1977, Innervation of cerebral blood vessels by norepinephrine, indoleamine, substance P and neurotensin fibres and leptomeningeal indoleamine axons: their roles in vasomotor activity and local alterations of brain blood composition, in: Neurogenic Control of Brain Circulation, eds. C. Owman and L. Edvinsson (Pergamon Press, Oxford) p. 39. Dahl/Sf, C., P. Dahlt~f, K. Tatemoto and J.M. Lundberg, 1985, Neuropeptide Y (NPY) reduces field stimulation-evoked release of noradrenaline and enhances force of contraction in the rat portal vein, Naunyn-Schmiedeb. Arch. Pharmacol. 328, 327. Edvinsson, L., P. Emson, J. McCulloch, K. Tatemoto and R. U d d m a n , 1983, Neuropeptide Y: cerebrovascular innervation and vasomotor effects in the cat, Neurosci. Lett. 43, 79. Edvinsson, L., P. Emson, J. McCulloch, K. Tatemoto and R. U d d m a n , 1984, Neuropeptide Y: Immunocytochemical 1ocalization to and effect upon feline pial arteries and veins in vitro and in situ, Acta Physiol. Scand. 122, 155. Edvinsson, L., B.B. Fredholm, E. Hamel, I. Jansen and C. Verrecchia, 1985, Perivascular peptides relax cerebral arteries concomitant with stimulation of cyclic adenosine monophosphate accumulation or release of an endothelium-derived relaxing factor in the cat, Neurosci. Lett. 58, 213. Ekblad, E., L. Edvinsson, C. Wahlestedt, R. U d d m a n , R. Hgtkanson and F. Sundler, 1984, Neuropeptide Y co-exists and co-operates with noradrenaline in perivascular nerve fibers, Regul. Pept. 8, 225. Fisher, L.A., D.O. Kikkawa, J.E. Rivier, S.G. Amara, R.M. Evans, M.G. Rosenfeld, W.W. Vale and M.R. Brown, 1983, Stimulation of noradrenergic sympathetic out flow by calcitonin gene-related peptide, Nature 305, 534. Franco-Cereceda, A., J.M. Lundberg and C. Dahl/3f, 1985, Neuropeptide Y and sympathetic control of heart contractility and coronary vascular tone, Acta Physiol. Scand. 124, 361.

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