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Neuropeptide Y-induced constriction in small resistance vessels of skeletal muscle
Peptides, Vol. 12, pp. 37-41. ©Pergamon Press plc, 1991. Printed in the U.S.A.
0196-9781/91 $3.00 + .00
Neuropeptide Y-Induced Constriction in Small Resistance Vessels of Skeletal Muscle I R V I N G G. J O S H U A
Department of Physiology and Biophysics, School of Medicine 1115A Health Sciences Center, University of Louisville, Louisville, KY 40292 R e c e i v e d 6 M a r c h 1990
JOSHUA, I. G. Neuropeptide Y-induced constriction in small resistance vessels of skeletal muscle. PEPTIDES 12(1) 37-41, 1991. --The in vivo responsiveness of small arterioles and venules in the rat cremaster muscle to topical administration of neuropeptide Y was assessed using closed-circuit television microscopy. Male Sprague-Dawley rats were anesthetized with sodium pentobarbital (50 mg/kg) and the cremaster muscle was exposed to increasing bath concentrations of neuropeptide Y (10-1°-10-7 M). Neuropeptide Y produced dose-dependent constrictions in fhst (90 __.8 ~m), second (50-+ 6 p,m) and third (21 ---4 p,m) order arterioles. Arteriolar reactivity to the peptide was inversely related to vessel diameters. Venules were relatively unresponsive to neuropeptide Y. Exposure to the alpha-adrenergic receptor antagonist, phentolamine (10 -6 M), failed to modify the arteriolar constrictor responses to neuropeptide Y, while pretreatment with the sympathetic neuronal blocking agent, guanethidine (10 -5 M), produced a small, but significant, reduction in sensitivity. These data suggest that neuropeptide Y causes constriction of arterioles of skeletal muscle, primarily by acting directly on vascular smooth muscle to induce contraction, and not via release of endogenous norepinephrine. Neuropeptide Y
Arterioles
Venules
Phentolamine
Guanethidine
NUMEROUS studies have emphasized the importance of the autonomic nervous system in the control of the cardiovascular system. A number of studies have suggested the existence of nonadrenergic and noncholinergic nervous control mechanisms. Several peptides have emerged as likely candidates; however, few peptides have been conclusively demonstrated to exist in perivascular nerve fibers. Neuropeptide Y is a 36 amino acid peptide which has been found to coexist with norepinephrine in many neurons of the sympathetic system supplying the cardiovascular bed (4,11). Generally, neuropeptide Y fibers are numerous around arteries and fewer are found around veins (24). Coronary arteries and small arteries in the respiratory tract and gastrointestinal tract are richly innervated with neuropeptide Y-containing fibers (24). Several recent findings suggest that norepinephrine is not the only neurotransmitter that mediates constriction of blood vessels. Electrical stimulation of pial vessels or isolated rabbit basilar arteries caused constriction which was only partially blocked by alpha-adrenergic receptor blockade (1). Similarly, electrical stimulation of splanchnic and lumbar sympathetic nerve fibers in the cat induced marked vasoconstriction in the cat colon which was only partially abolished by alpha-adrenergic receptor blockade (8). However, this constriction was completely abolished by administration of guanethidine, an agent which blocks both norepinephrine and neuropeptide Y release (12). Only a few studies have investigated the effects of exogenous
Rat
administration of neuropeptide Y on the cardiovascular system. Intravenous administration of neuropeptide Y causes an increase in arterial blood pressure, a fall in heart rate and an increase in local resistance of selected vascular beds (11). Local application of neuropeptide Y to cerebral arteries and arterioles produces a long-lasting concentration-dependent constriction (2). Pernow et al. (20) have recently obtained evidence that neuropeptide Y causes a marked constriction of the vasculature in the human forearm, suggesting a possibility that this peptide plays an important role in the control of skeletal muscle vasculature. The major objectives of the present study were 1) to determine if small arterioles and venules in the microcirculation of skeletal muscle (cremaster muscle) are responsive to exogenously administered neuropeptide Y, and 2) to determine if constrictor responses to this peptide are mediated via local neuronal release of norepinephrine. METHOD
Surgical Procedure Male, five- to six-week-old Sprague-Dawley rats weighing 150-210 g were studied in these experiments. Each rat was anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg). Rectal temperature was maintained at 37°(2 with a heating pad placed under the animal. The trachea was intubated to ensure a patent airway, and the animal was allowed to breath room air spontaneously. The left femoral artery was cannulated
37
38
for measurement of arterial pressure. The cremaster muscle was prepared for microvascular observations using a technique previously described by Joshua et al. (9). A small incision was made in the scrotum and the cremaster muscle surrounding the right testicle was exposed. The cremaster was gently dissected free from the testicle. Care was taken to maintain the vascular and neural connections to the cremaster. Throughout this surgery, the cremaster muscle was frequently irrigated with a physiological salt solution to prevent drying of the tissue. The rat was transferred to a board that was designed so that the hindlegs of the rat straddled a tissue bath (50 ml volume). The cremaster muscle was suspended with silk sutures in a flat position over an optical port in the tissue bath, and the bath was filled with a physiological salt solution (in mM): 25.5, NaHCO3; 112.9, NaC1; 4.7, KC1; 1.19, KH2PO4; 1.19, MgSO4.7 H20; 2.55, CaC12; 11.6, dextrose. Bath temperature was maintained at 34.5°C (the in situ scrotal sac temperature of conscious rats) by means of an immersed insulated heating coil. Bath PO2 (25--40 mmHg), PCO 2 (34-45 mmHg) and pH (7.40 +-0.05) were controlled by varying the amounts of carbon dioxide and nitrogen that were bubbled through the bath. The bubbling action of these gases also ensured complete mixing of drugs that were added to the bath. Bath pH was continuously monitored via an indwelling pH electrode, and bath gases were periodically checked with a blood gas analyzer (IL 213). The preparation was positioned on the stage of a trinocular microscope for observation of the cremaster microcirculation via closed-circuit television microscopy. The image of the cremaster was recorded on videotape and displayed on a calibrated monitor at 1000-1500 x magnification. The microvessels were categorized according to their branching pattern in the muscle. The single largest arteriole that entered the muscle was designated as the first-order arteriole (1A). Branches from this 1A were designated as second-order arterioles (2A). Venules adjacent and parallel to 1A and 2A vessels were labeled the first- (1V) and second-order (2V) venules, respectively. Segments of 1A, 2A, 3A, 1V, and 2V in each cremaster muscle were selected for continuous measurement of vessel diameters. In the first series of experiments, the constrictor response to increasing concentrations of neuropeptide Y (10-11--10-7 M) were observed for arterioles and venules in the cremaster of 8 anesthetized rats. Each experiment began with a 5-minute control period, followed by additions of increasing bath concentration of neuropeptide Y at 10-minute intervals. Vascular diameters were measured at 1-minute intervals. In two additional series of experiments, the ability of the alpha-adrenergic antagonist, phentolamine (10-6), and the adrenergic nerve blocker, guanethidine (10 - 5 M), to block intrinsic tone was investigated at three levels of arterioles and two levels of venules. Following a 5-minute control period, vessels were exposed to phentolamine for 30 minutes and diameters recorded at 1-minute intervals. In a separate experimental group, the cremaster was exposed to two concentrations of guanethidine (10-6 and 10 -5 M) at 30-minute intervals. Maximal microvascular diameters were assessed using the nonspecific vasodilator, nitroprusside (10 - 5 M). In the final two series of experiments, the role of local adrenergic receptor stimulation in the microvascular response to neuropeptide Y was assessed by exposing small arterioles (A3) to phentolamine (bath concentration of 10 - 6 M) or to guanethidine for 30 minutes prior to exposure to increasing concentrations of the peptide. The effectiveness of alpha-adrenergic blockade was tested in each study by exposing the tissue to a high bath concentration of norepinephrine (10 - 7 M) before and after treatment with phentolamine.
JOSHUA
200-
E
Control
::1.
WT;t Nitroprusside 150-
E ~5 -6 CD
100-
50.
lN i* 1A
2A
3A
2V
V
FIG. 1. Resting diameters and maximal diameters of arterioles (1A, 2A, 3A) and venules (IV, 2V). Data for each group represent the mean _ SEM for 8 vessels with the exception of the 1V group which represents the average of 6 observations. An asterisk indicates a significant (p<0.05) dilation from resting diameters following exposure to nitroprusside (10 5 M).
Statistical Analysis The raw data for arteriolar and venular diameters were normalized by expressing each data point as a percent of the average value during the control period that preceded application of neuropeptide Y. The maximal responses for each dose were used to construct concentration-response curves. The concentration (EDso) that produced 50% of maximal constriction was graphically determined for each of the vessels in each animal. These values were converted to pD 2 values (pD 2 = - log EDso) as an indicator of reactivity to neuropeptide Y. Data were statistically analyzed using paired Student's t-test. Group differences were considered statistically significant at the p < 0 . 0 5 level.
Preparation of Drugs Neuropeptide Y was dissolved in distilled water and frozen in aliquots that were thawed and used daily. Phentolamine (Ciba) and guanethidine (Sigma) were dissolved in distilled water. Sodium nitroprusside (Mallinckrodt) was prepared daily in distilled water and stored in a dark cabinet until use. A norepinephrine stock solution (10-5 M) was prepared daily by dissolving norepinephrine bitartrate (Sigma) in distilled water with ascorbic acid (1 mg/ml) to prevent inactivation. RESULTS
The mean arterial blood pressure for the 32 animals used in this study was 124+-3 mmHg. Blood pressure was not affected by topical administration of either neuropeptide Y, norepinephfine, atropine or guanethidine to the cremaster. Resting diameters of 1A, 2A and 3A arterioles, and 1V and 2V venules are illustrated in Fig. 1. In addition, the diameters of these vessels following exposure to nitroprusside (10 -5 M) are also presented, This nonspecific vasodilator produced significant dilation of 2A (37%) and 3A (136%). Large arterioles (1A) and venules (IV and 2V) were not significantly affected by the vasodilator. Figure 2 shows the response of arterioles and venules to a 10- 7 M bath concentration of norepinephrine in the presence or absence of the alpha-adrenergic antagonist, phentolamine. Norepinephrine produced significant constriction at all arteriolar lev-
IN VIVO RESPONSES TO NEUROPEPTIDE Y
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els with the greatest response being observed for 3A (70 ± 7% constriction). Venules show a small (10%) but significant constriction. Pretreatment with phentolamine for 30 minutes prior to administration of norepinepbxine abolished the constrictor responses in arterioles and venules. Figure 3 illustrates the effect of the alpha-adrenergic antagonist phentolamine or the neuronal blocking agent guanethidine on resting diameters of arterioles and venules. Only the 3A arterioles exhibited dilation ( 1 1 4 ± 9 % of control) in response to the 30minute exposure to phentolamine. Exposure to 10 - 6 M guanethidine produced small but significant dilations of 2A (12 ± 7%) and 3A (33 ± 10%) arterioles. These arterioles dilated by 21 ± 9% and 5 5 - 12%, respectively, when exposed to the 10 - 5 M concentration of guanethidine (Fig. 3). In response to nitroprusside (10 -5 M), 2A and 3A arterioles dilated by 4 4 -+ 12% and 140_+ 12%, respectively. Larger arterioles (IA) and venules (1V and 2V) failed to dilate significantly in response to either concentration of guanethidine. Arteriolar sensitivity to neuropeptide Y is presented in Fig. 4. Microvascular sensitivity is expressed as a pD 2 value ( - log of the EDso ). Sensitivity to neuropeptide Y tended to be inversely related to vessel diameter with the highest degree of sensi-
.
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.
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,
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1A
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FIG. 2. Maximal constriction to norepinephrine (NE) 10 -7 M in the absence or presence of phentolamine (10 -6 M). Each group represents responses (mean -+SEM) of 8 vessels. Responses of arterioles and venules are expressed as percents of the resting vessel diameters. Significant (p<0.05) constrictions in response to NE are indicated with an asterisk.
, 200 ] oE
(J >
I--1 NP-Y mm NP-Y + Phen NP-Y + Guaneth
2A
3A
FIG. 4. Arteriolar sensitivity to neuropeptide Y (NPY) expressed as a pD2 value ( - log EDso). Responses of 3A were also observed in the presence of phentolamine (NP-Y + Phen; 10 -4 M) or guanethidine (NP-Y + Guaneth; 10 -2 M). Values are expressed as mean- SEM for 1A (n=5), 2A (n=6) and 3A (N'P-Y, n=7; NP-Y + Phen, n=8; NP-Y + Guaneth, n = 8). Pretreatment with guanethidine significantly (p<0.05) decreased sensitivity to neuropeptide Y. tivity being exhibited by the smaller 3A arterioles (pD2= 10.10 ± 0.18). In a separate study, we observed the sensitivity of the arterioles to neuropeptide Y in the presence of alpha-adrenergic blockade (3A + Phen). Sensitivity tended to be slightly reduced (PD2=9.69_+0.17), however, the reduction was not significant (p>0.05). Preexposure to guanethidine (3A + Guaneth) significantly attenuated the sensitivity to neuropeptide Y (pD 2 = 9.25±0.25). Figure 5 shows the maximal responses to neuropeptide Y for arterioles and venules. As was the case with microvascular sensitivity, the degree of constriction produced by neuropeptide Y was inversely related to arteriolar diameters with 3A arterioles constricting by 83 ± 6%. In contrast, venules were relatively unresponsive to neuropeptide Y with 1V and 2V venules constricting a maximum of 12 ± 8% and 11 ± 3%, respectively. DISCUSSION The vasoactive effects of neuropeptide Y have been investigated using isolated vessel segments and in vivo perfusion stud-
100.
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2A
3A
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FIG. 3. Maximal responses to phentolamine (10 -6 M) and gnanethidine (10 -5 M) by arterioles and venules. Data for each group represent mean -+SEM for 8 vessels with the exception of the IV group which represents the average of 6 observations. Asterisk indicates significant (p<0.05) dilation from resting diameters.
o._
0 1A
2A
3A
FIG. 5. Maximal constriction of arterioles and. venules to neuropeptide Y (NPY) expressed as a percent of resting vessel diameters. Values are expressed as mean-+SEM for 1A (n=5), 2A (n=6), 3A (n=7), 1V (n-8) and 2V (n = 8). NPY produces a significant (p<0.05) constriction of all vessel levels, except 1V venules.
40
JOSHUA
ies. Neuropeptide Y appears to cause a weak to moderate contractile response in in vitro isolated blood vessel segments (4, 13, 14, 18). Several in vitro studies (19,20) have shown that small artery segments taken from human gluteus maximus muscle were more reactive to neuropeptide Y than mesentery artery segments. In vivo infusion studies show a marked vasoconstriction to neuropeptide Y in human forearm (21), canine gracilis muscle (22) and in cat mesenteric circulation (8). In the present study, neuropeptide Y was found to be a potent vasoconstrictor in the rat cremaster microcirculation, causing dosedependent constriction of all arterioles. Venules, on the other hand, were relatively unresponsive to the vasoconstrictor peptide. Thus our observation confirms a high degree of sensitivity to neuropeptide Y by small resistance vessels in the microcirculation. Cerebral arterioles are also highly reactive to topical application of neuropeptide Y (2). In addition, our results suggest that within the arteriolar microcirculation there is an inverse relationship between vessel diameter and neuropeptide Y sensitivity (Fig. 4). The observed lack of responsiveness by cremaster venules is in contrast to findings by Pernow et al. (21), indicating that human mesenteric veins are responsive to neuropeptide Y. We speculate, however, that the lack of venular response to neuropeptide in this study may reflect a low degree of vascular smooth muscle content in these small venules (23). The small reductions in venular diameter may also reflect passive responses to decreased luminal pressure associated with upstream constriction of small arterioles. In vitro studies (2, 4, 19) suggest that contractile responses to electrical stimulation or exogenous norepinephrine are enhanced in the presence of neuropeptide Y. Edvisson et al. (3) postulated that neuropeptide Y may induce contraction by potentiating the constrictor effects of endogenously released norepinephrine. In vivo studies utilizing the submandibular gland (10) or the spleen (15,16) suggest that the constriction to neuropeptide Y is independent of adrenoceptor stimulation. In the current study, pretreatment with the adrenoceptor antagonist, phentolamine, failed to significantly modify the constrictor response to neuropeptide Y by small cremaster arterioles (Fig. 4). The concentration of phentolamine used (10 - 6 M) was found to completely inhibit constriction to exogenously administered norepinephrine, which normally produces near maximal constriction of third-order arterioles. However, exposure to both phentolamine and the adrenergic neuronal blocker, guanethidine, significantly attenuated the sensitivity to neuropeptide Y. The small reduction in sensitivity suggests that part of the constrictor response to topically applied neuropeptide Y involves release of an endogenous neurotransmitter. While exposure to phentolamine alone did not produce a significant reduction in the sensitivity of neuropeptide Y, there was a trend toward a reduction (Fig. 4). We speculate that the phentolamine may be able to block receptors stimulated by exogenously applied norepinephrine, but is not able to block all of the receptors w h i c h are stimulated by n e u r o n a l l y released norepinephrine. Regardless, neuropeptide Y-induced constriction of arterioles of skeletal muscle appears to be primarily due to mechanisms other than adrenoceptor stimulation. Neuropeptide Y has been identified as a peptide that is stored and released together with norepinephrine from sympathetic perivascular nerves (4, 11, 24). In addition, investigators using adrenergic blocking agents have shown that in some vascular beds
neurally mediated vasoconstriction is only partially mediated by release of norepinephrine (1,8). However, guanethidine, which blocks release of both norepinephrine and neuropeptide Y (12), blocks the sympathetic vasoconstriction that is resistant to alphaadrenergic blockade (3). In a separate series of studies, we compared the effects of alpha-adrenergic blockade and adrenergic neuron blockade on resting tone of small arterioles in the cremaster muscle. Exposure to the alpha-adrenergic antagonist, phentolamine (10 - 6 M), for 30 minutes produced no significant changes in diameters of large arterioles or venules in the cremasters. Third-order arterioles dilated by approximately 14% (Fig. 3). Previously, we (7) reported a small dilation (15%) of small arterioles (4A) in the cremaster of decerebrated rats following exposure to phentolamine. These studies suggest that intrinsic tone in small arterioles of the cremaster is not mediated either by neuronally released or circulating catecholamines. Recent studies utilizing an acutely denervated cremaster preparation (5,6) support the absence of a constrictor influence of circulation catecholamines on microvascular tone of 3A arterioles. Adrenergic neuron blockade with guanethidine (10 -5 M) caused significant dilation of both 2A and 3A arterioles (21% and 55%, respectively). This dilation was significantly greater than the dilation observed in response to phentolamine and represented 48% and 40% of the maximal dilation to nitroprusside by 2A and 3A arterioles, respectively. The dilation observed for 3A arterioles was also similar in magnitude to the dilation observed in response to acute surgical denervation of the cremaster (25). Guanethidine has been shown to block both norepinephrine and neuropeptide Y release (12) and neuropeptide Y has been proposed to be responsible for the neuronal sympathetic vasoconstriction which is resistant to alpha-adrenergic blockade. The current results might be explained by the presence of a phentolamine-resistant neurogenic constrictor tone in small arterioles of the cremaster. Owen et al. (17) have observed arteries in the rabbit ear and reported that the ability of phentolamine to block contractions induced by transmural nerve stimulation was decreased as artery diameters decreased. They concluded, however, that this contraction is small in magnitude, occurs only at high nerve frequencies and is probably physiologically insignificant. Alternately, our data would support the hypothesis that a substantial degree of the neurogenically mediated tone in cremaster muscle is mediated by neuronally released neuropeptide Y. In summary, small arterioles in an in vivo skeletal muscle preparation (rat cremaster muscle) are highly responsive to exogenous neuropeptide Y. A small portion of the constrictor responses appear to be related to stimulation of the released endogenous catecholamines. The data presented are also consistent with a physiological role for neuropeptide Y in the maintenance of neurogenic tone in small arterioles in skeletal muscle. ACKNOWLEDGEMENTS We thank Mary Englebert for her excellent technical assistance. This work was supported by grants from the American Heart Association and the University of Louisville Graduate School Research Council. Dr. Irving G. Joshua is the recipient of an Established Investigator Award from the American Heart Association.
REFERENCES 1. Auer, L. M.; Edvinsson, L.; Johansson, B. B. Effect of sympathetic nerve stimulation and adrenoceptor blockade on pial arterial and venous calibre and on intracranial pressure in the cat. Acta Physiol. Scand. 119:213-217; 1983. 2. Edvinsson, L.; Ekblad, E.; Hakanson, R.; Wahlestedt, C. Neuropep-
tide-Y potentiates the effect of various vasoconstrictor agents on rabbit blood vessels. Br. J. Pharmacol. 83:519-524; 1984. 3. Edvissson, L.; Hakanson, R.; Wahlestedt, C.; Uddman, R. Effects of neuropeptide-Y on the cardiovascular system. Trends Pharmacol. Sci. 8:231-235; 1987.
IN VIVO RESPONSES TO NEUROPEPTIDE Y
4. Ekblad, E.; Edvinsson, L.; Wahlestedt, C.; Uddman, R.; Hakanson, R.; Sundler, F. Neuropeptide-Y co-exists and co-operates with noradrenaline in perivascular nerve fibres. Regul. Pept. 8:225-235; 1984. 5. Faber, J. E. In situ analysis of alpha-adrenoceptors on arteriolar and venular smooth muscle in rat skeletal muscle microcirculation. Circ. Res. 62:37-50; 1988. 6. Faber, J. E. Effect of local tissue cooling on microvascular smooth muscle and postjunctional ct2-adrenoceptors. Am. J. Physiol. 255: H121-H130; 1988. 7. Faber, J. E.; Harris, P. D.; Joshua, I. G. Microvascular responses to blockade of prostaglandin synthesis in rat skeletal muscle. Am. J. Physiol. 243:HS1-H60; 1982. 8. Hellstrom, P.; Olerup, O.; Tatemoto, K. Neuropeptide-Y may mediate effects of sympathetic nerve stimulations on colonic motility and blood flow in the cat. Acta Physiol. Scand. 124:613-624; 1985. 9. Joshua, I. G.; Wiegman, D. L.; Harris, P. D.; Miller, F. N. Progressive microvascular alterations with the development of renovascular hypertension. Hypertension 6:61-67; 1984. 10. Lundberg, J. M.; Tatemoto, K. Pancreatic polypeptide family (APP, BPP, NPY and PYY) in relation to sympathetic vasoconstriction resistant to alpha-adrenoceptor blockade. Acta Physiol. Scand. 116: 393-402; 1982. 11. Lundberg, J. M.; Terenius, L.; Hokfelt, T.; Martling, C. R.; Tatemoto, K.; Mutt, V.; Polak, J.; Bloom, S. R.; Goldstein, M. Neuropeptide-Y (NPY)-like immunoreactivity in peripheral noradrenergic neurons and effects of NPY on sympathetic function. Acta Physiol. Scand. 116:477-480; 1982. 12. Lundberg, J. M.; Anggard, A.; Theodorsson-Norheim, E.; Pernow, J. Guanethidine-sensitive release of NPY-like immunoreactivity by sympathetic nerve stimulation. Neurosci. Lett. 52:175-180; 1984. 13. Lundberg, J. M.; Pernow, J.; Tatemoto, K.; Dahlof, C. Pre- and postjunctional effects of neuropeptide-Y on sympathetic vascular control in rat femoral artery. Acta Physiol. Scand. 123:511-513; 1985. 14. Lundberg, J. M.; Torsell, J.; Sollevi, A.; Pernow, J.; TheodorssonNorheim, E.; Anggard, A.; Hamberger, B. Neuropeptide-Y and sympathetic vascular control in man. Regul. Pept. 13:41-52; 1985.
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15. Lundberg, J. M.; Anggald, A.; Pernow, J.; Hokfelt, T. Neuropeptide-Y-, substance P- and VIP-immunoreactive nerves in cat spleen in relation to autonomic vascular and volume control. Cell Tissue Res. 239:9; 1985. 16. Lundberg, J. M.; Fried, G.; Pernow, J.; Theodorsson-Norheim, E.; Anggard, A. NPY--a mediator of reserpine-resistant, non-adrenergic vasoconstriction in cat spleen after preganglionic denervation? Acta Physiol. Scand. 126:151-152; 1986. 17. Owen, M. P.; Quinn, C.; Bevans, J. A. Phentolamine-resistant neurogenic constriction occurs in small arteries at high frequencies. Am. J. Physiol. 249:H404-H414; 1985. 18. Pernow, J.; Saria, A.; Lundberg, J. M. Mechanisms underlying preand postjunctional effects of neuropeptide-Y in sympathetic vascular control. Acta Physiol. Scand. 126:239-249; 1986. 19. Pernow, J.; Lundberg, J. M. Neuropeptide-Y constricts human skeletal muscle arteries via a nifedipine-sensitive mechanism independent of extracellular calcium? Acta Physiol. Scand. 128:655--656; 1986. 20. Pemow, J.; Svenberg, T.; Lundberg, J. M. Action of calcium antagonists on pre- and postjunction effects of neuropeptide-Y on human peripheral blood vessels in vitro. Eur. J. Pharmacol. 136:207-218; 1987. 21. Pernow, J.; Lundberg, J. M.; Kaijser, L. Vasoconstrictor effects in vivo and plasma disappearance rate of neuropeptide-Y in man. Life Sci. 40:47-54; 1987. 22. Pernow, J.; Kahan, T.; Hemdahl, P.; Lundberg, J. M. Possible involvement of neuropeptide-Y in sympathetic vascular control of canine skeletal muscle. Acta Physiol. Scand. 132:43-50; 1988. 23. Rhodin, J. A. G. Ultrastructure of mammalian venous capillaries, venules and small collecting veins. J. Ultrastruct. Res. 25:452-500; 1968. 24. Uddman, R.; Ekblad, E.; Edvinsson, L.; Hakanson, R.; Sundier, F. Neuropeptide-Y-like immunoreactivity in perivascular nerve fibers of the guinea-pig. Regul. Pept. 10:243-257; 1985. 25. Yu, J.; Roberts, A. M.; Joshua, I. G. Lung inflation evokes reflex dilation of microvessel in rat skeletal muscle. Am. J. Physiol. 258: H939-H945; 1990.
0196-9781/91 $3.00 + .00
Neuropeptide Y-Induced Constriction in Small Resistance Vessels of Skeletal Muscle I R V I N G G. J O S H U A
Department of Physiology and Biophysics, School of Medicine 1115A Health Sciences Center, University of Louisville, Louisville, KY 40292 R e c e i v e d 6 M a r c h 1990
JOSHUA, I. G. Neuropeptide Y-induced constriction in small resistance vessels of skeletal muscle. PEPTIDES 12(1) 37-41, 1991. --The in vivo responsiveness of small arterioles and venules in the rat cremaster muscle to topical administration of neuropeptide Y was assessed using closed-circuit television microscopy. Male Sprague-Dawley rats were anesthetized with sodium pentobarbital (50 mg/kg) and the cremaster muscle was exposed to increasing bath concentrations of neuropeptide Y (10-1°-10-7 M). Neuropeptide Y produced dose-dependent constrictions in fhst (90 __.8 ~m), second (50-+ 6 p,m) and third (21 ---4 p,m) order arterioles. Arteriolar reactivity to the peptide was inversely related to vessel diameters. Venules were relatively unresponsive to neuropeptide Y. Exposure to the alpha-adrenergic receptor antagonist, phentolamine (10 -6 M), failed to modify the arteriolar constrictor responses to neuropeptide Y, while pretreatment with the sympathetic neuronal blocking agent, guanethidine (10 -5 M), produced a small, but significant, reduction in sensitivity. These data suggest that neuropeptide Y causes constriction of arterioles of skeletal muscle, primarily by acting directly on vascular smooth muscle to induce contraction, and not via release of endogenous norepinephrine. Neuropeptide Y
Arterioles
Venules
Phentolamine
Guanethidine
NUMEROUS studies have emphasized the importance of the autonomic nervous system in the control of the cardiovascular system. A number of studies have suggested the existence of nonadrenergic and noncholinergic nervous control mechanisms. Several peptides have emerged as likely candidates; however, few peptides have been conclusively demonstrated to exist in perivascular nerve fibers. Neuropeptide Y is a 36 amino acid peptide which has been found to coexist with norepinephrine in many neurons of the sympathetic system supplying the cardiovascular bed (4,11). Generally, neuropeptide Y fibers are numerous around arteries and fewer are found around veins (24). Coronary arteries and small arteries in the respiratory tract and gastrointestinal tract are richly innervated with neuropeptide Y-containing fibers (24). Several recent findings suggest that norepinephrine is not the only neurotransmitter that mediates constriction of blood vessels. Electrical stimulation of pial vessels or isolated rabbit basilar arteries caused constriction which was only partially blocked by alpha-adrenergic receptor blockade (1). Similarly, electrical stimulation of splanchnic and lumbar sympathetic nerve fibers in the cat induced marked vasoconstriction in the cat colon which was only partially abolished by alpha-adrenergic receptor blockade (8). However, this constriction was completely abolished by administration of guanethidine, an agent which blocks both norepinephrine and neuropeptide Y release (12). Only a few studies have investigated the effects of exogenous
Rat
administration of neuropeptide Y on the cardiovascular system. Intravenous administration of neuropeptide Y causes an increase in arterial blood pressure, a fall in heart rate and an increase in local resistance of selected vascular beds (11). Local application of neuropeptide Y to cerebral arteries and arterioles produces a long-lasting concentration-dependent constriction (2). Pernow et al. (20) have recently obtained evidence that neuropeptide Y causes a marked constriction of the vasculature in the human forearm, suggesting a possibility that this peptide plays an important role in the control of skeletal muscle vasculature. The major objectives of the present study were 1) to determine if small arterioles and venules in the microcirculation of skeletal muscle (cremaster muscle) are responsive to exogenously administered neuropeptide Y, and 2) to determine if constrictor responses to this peptide are mediated via local neuronal release of norepinephrine. METHOD
Surgical Procedure Male, five- to six-week-old Sprague-Dawley rats weighing 150-210 g were studied in these experiments. Each rat was anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg). Rectal temperature was maintained at 37°(2 with a heating pad placed under the animal. The trachea was intubated to ensure a patent airway, and the animal was allowed to breath room air spontaneously. The left femoral artery was cannulated
37
38
for measurement of arterial pressure. The cremaster muscle was prepared for microvascular observations using a technique previously described by Joshua et al. (9). A small incision was made in the scrotum and the cremaster muscle surrounding the right testicle was exposed. The cremaster was gently dissected free from the testicle. Care was taken to maintain the vascular and neural connections to the cremaster. Throughout this surgery, the cremaster muscle was frequently irrigated with a physiological salt solution to prevent drying of the tissue. The rat was transferred to a board that was designed so that the hindlegs of the rat straddled a tissue bath (50 ml volume). The cremaster muscle was suspended with silk sutures in a flat position over an optical port in the tissue bath, and the bath was filled with a physiological salt solution (in mM): 25.5, NaHCO3; 112.9, NaC1; 4.7, KC1; 1.19, KH2PO4; 1.19, MgSO4.7 H20; 2.55, CaC12; 11.6, dextrose. Bath temperature was maintained at 34.5°C (the in situ scrotal sac temperature of conscious rats) by means of an immersed insulated heating coil. Bath PO2 (25--40 mmHg), PCO 2 (34-45 mmHg) and pH (7.40 +-0.05) were controlled by varying the amounts of carbon dioxide and nitrogen that were bubbled through the bath. The bubbling action of these gases also ensured complete mixing of drugs that were added to the bath. Bath pH was continuously monitored via an indwelling pH electrode, and bath gases were periodically checked with a blood gas analyzer (IL 213). The preparation was positioned on the stage of a trinocular microscope for observation of the cremaster microcirculation via closed-circuit television microscopy. The image of the cremaster was recorded on videotape and displayed on a calibrated monitor at 1000-1500 x magnification. The microvessels were categorized according to their branching pattern in the muscle. The single largest arteriole that entered the muscle was designated as the first-order arteriole (1A). Branches from this 1A were designated as second-order arterioles (2A). Venules adjacent and parallel to 1A and 2A vessels were labeled the first- (1V) and second-order (2V) venules, respectively. Segments of 1A, 2A, 3A, 1V, and 2V in each cremaster muscle were selected for continuous measurement of vessel diameters. In the first series of experiments, the constrictor response to increasing concentrations of neuropeptide Y (10-11--10-7 M) were observed for arterioles and venules in the cremaster of 8 anesthetized rats. Each experiment began with a 5-minute control period, followed by additions of increasing bath concentration of neuropeptide Y at 10-minute intervals. Vascular diameters were measured at 1-minute intervals. In two additional series of experiments, the ability of the alpha-adrenergic antagonist, phentolamine (10-6), and the adrenergic nerve blocker, guanethidine (10 - 5 M), to block intrinsic tone was investigated at three levels of arterioles and two levels of venules. Following a 5-minute control period, vessels were exposed to phentolamine for 30 minutes and diameters recorded at 1-minute intervals. In a separate experimental group, the cremaster was exposed to two concentrations of guanethidine (10-6 and 10 -5 M) at 30-minute intervals. Maximal microvascular diameters were assessed using the nonspecific vasodilator, nitroprusside (10 - 5 M). In the final two series of experiments, the role of local adrenergic receptor stimulation in the microvascular response to neuropeptide Y was assessed by exposing small arterioles (A3) to phentolamine (bath concentration of 10 - 6 M) or to guanethidine for 30 minutes prior to exposure to increasing concentrations of the peptide. The effectiveness of alpha-adrenergic blockade was tested in each study by exposing the tissue to a high bath concentration of norepinephrine (10 - 7 M) before and after treatment with phentolamine.
JOSHUA
200-
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Control
::1.
WT;t Nitroprusside 150-
E ~5 -6 CD
100-
50.
lN i* 1A
2A
3A
2V
V
FIG. 1. Resting diameters and maximal diameters of arterioles (1A, 2A, 3A) and venules (IV, 2V). Data for each group represent the mean _ SEM for 8 vessels with the exception of the 1V group which represents the average of 6 observations. An asterisk indicates a significant (p<0.05) dilation from resting diameters following exposure to nitroprusside (10 5 M).
Statistical Analysis The raw data for arteriolar and venular diameters were normalized by expressing each data point as a percent of the average value during the control period that preceded application of neuropeptide Y. The maximal responses for each dose were used to construct concentration-response curves. The concentration (EDso) that produced 50% of maximal constriction was graphically determined for each of the vessels in each animal. These values were converted to pD 2 values (pD 2 = - log EDso) as an indicator of reactivity to neuropeptide Y. Data were statistically analyzed using paired Student's t-test. Group differences were considered statistically significant at the p < 0 . 0 5 level.
Preparation of Drugs Neuropeptide Y was dissolved in distilled water and frozen in aliquots that were thawed and used daily. Phentolamine (Ciba) and guanethidine (Sigma) were dissolved in distilled water. Sodium nitroprusside (Mallinckrodt) was prepared daily in distilled water and stored in a dark cabinet until use. A norepinephrine stock solution (10-5 M) was prepared daily by dissolving norepinephrine bitartrate (Sigma) in distilled water with ascorbic acid (1 mg/ml) to prevent inactivation. RESULTS
The mean arterial blood pressure for the 32 animals used in this study was 124+-3 mmHg. Blood pressure was not affected by topical administration of either neuropeptide Y, norepinephfine, atropine or guanethidine to the cremaster. Resting diameters of 1A, 2A and 3A arterioles, and 1V and 2V venules are illustrated in Fig. 1. In addition, the diameters of these vessels following exposure to nitroprusside (10 -5 M) are also presented, This nonspecific vasodilator produced significant dilation of 2A (37%) and 3A (136%). Large arterioles (1A) and venules (IV and 2V) were not significantly affected by the vasodilator. Figure 2 shows the response of arterioles and venules to a 10- 7 M bath concentration of norepinephrine in the presence or absence of the alpha-adrenergic antagonist, phentolamine. Norepinephrine produced significant constriction at all arteriolar lev-
IN VIVO RESPONSES TO NEUROPEPTIDE Y
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els with the greatest response being observed for 3A (70 ± 7% constriction). Venules show a small (10%) but significant constriction. Pretreatment with phentolamine for 30 minutes prior to administration of norepinepbxine abolished the constrictor responses in arterioles and venules. Figure 3 illustrates the effect of the alpha-adrenergic antagonist phentolamine or the neuronal blocking agent guanethidine on resting diameters of arterioles and venules. Only the 3A arterioles exhibited dilation ( 1 1 4 ± 9 % of control) in response to the 30minute exposure to phentolamine. Exposure to 10 - 6 M guanethidine produced small but significant dilations of 2A (12 ± 7%) and 3A (33 ± 10%) arterioles. These arterioles dilated by 21 ± 9% and 5 5 - 12%, respectively, when exposed to the 10 - 5 M concentration of guanethidine (Fig. 3). In response to nitroprusside (10 -5 M), 2A and 3A arterioles dilated by 4 4 -+ 12% and 140_+ 12%, respectively. Larger arterioles (IA) and venules (1V and 2V) failed to dilate significantly in response to either concentration of guanethidine. Arteriolar sensitivity to neuropeptide Y is presented in Fig. 4. Microvascular sensitivity is expressed as a pD 2 value ( - log of the EDso ). Sensitivity to neuropeptide Y tended to be inversely related to vessel diameter with the highest degree of sensi-
.
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FIG. 2. Maximal constriction to norepinephrine (NE) 10 -7 M in the absence or presence of phentolamine (10 -6 M). Each group represents responses (mean -+SEM) of 8 vessels. Responses of arterioles and venules are expressed as percents of the resting vessel diameters. Significant (p<0.05) constrictions in response to NE are indicated with an asterisk.
, 200 ] oE
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2A
3A
FIG. 4. Arteriolar sensitivity to neuropeptide Y (NPY) expressed as a pD2 value ( - log EDso). Responses of 3A were also observed in the presence of phentolamine (NP-Y + Phen; 10 -4 M) or guanethidine (NP-Y + Guaneth; 10 -2 M). Values are expressed as mean- SEM for 1A (n=5), 2A (n=6) and 3A (N'P-Y, n=7; NP-Y + Phen, n=8; NP-Y + Guaneth, n = 8). Pretreatment with guanethidine significantly (p<0.05) decreased sensitivity to neuropeptide Y. tivity being exhibited by the smaller 3A arterioles (pD2= 10.10 ± 0.18). In a separate study, we observed the sensitivity of the arterioles to neuropeptide Y in the presence of alpha-adrenergic blockade (3A + Phen). Sensitivity tended to be slightly reduced (PD2=9.69_+0.17), however, the reduction was not significant (p>0.05). Preexposure to guanethidine (3A + Guaneth) significantly attenuated the sensitivity to neuropeptide Y (pD 2 = 9.25±0.25). Figure 5 shows the maximal responses to neuropeptide Y for arterioles and venules. As was the case with microvascular sensitivity, the degree of constriction produced by neuropeptide Y was inversely related to arteriolar diameters with 3A arterioles constricting by 83 ± 6%. In contrast, venules were relatively unresponsive to neuropeptide Y with 1V and 2V venules constricting a maximum of 12 ± 8% and 11 ± 3%, respectively. DISCUSSION The vasoactive effects of neuropeptide Y have been investigated using isolated vessel segments and in vivo perfusion stud-
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FIG. 3. Maximal responses to phentolamine (10 -6 M) and gnanethidine (10 -5 M) by arterioles and venules. Data for each group represent mean -+SEM for 8 vessels with the exception of the IV group which represents the average of 6 observations. Asterisk indicates significant (p<0.05) dilation from resting diameters.
o._
0 1A
2A
3A
FIG. 5. Maximal constriction of arterioles and. venules to neuropeptide Y (NPY) expressed as a percent of resting vessel diameters. Values are expressed as mean-+SEM for 1A (n=5), 2A (n=6), 3A (n=7), 1V (n-8) and 2V (n = 8). NPY produces a significant (p<0.05) constriction of all vessel levels, except 1V venules.
40
JOSHUA
ies. Neuropeptide Y appears to cause a weak to moderate contractile response in in vitro isolated blood vessel segments (4, 13, 14, 18). Several in vitro studies (19,20) have shown that small artery segments taken from human gluteus maximus muscle were more reactive to neuropeptide Y than mesentery artery segments. In vivo infusion studies show a marked vasoconstriction to neuropeptide Y in human forearm (21), canine gracilis muscle (22) and in cat mesenteric circulation (8). In the present study, neuropeptide Y was found to be a potent vasoconstrictor in the rat cremaster microcirculation, causing dosedependent constriction of all arterioles. Venules, on the other hand, were relatively unresponsive to the vasoconstrictor peptide. Thus our observation confirms a high degree of sensitivity to neuropeptide Y by small resistance vessels in the microcirculation. Cerebral arterioles are also highly reactive to topical application of neuropeptide Y (2). In addition, our results suggest that within the arteriolar microcirculation there is an inverse relationship between vessel diameter and neuropeptide Y sensitivity (Fig. 4). The observed lack of responsiveness by cremaster venules is in contrast to findings by Pernow et al. (21), indicating that human mesenteric veins are responsive to neuropeptide Y. We speculate, however, that the lack of venular response to neuropeptide in this study may reflect a low degree of vascular smooth muscle content in these small venules (23). The small reductions in venular diameter may also reflect passive responses to decreased luminal pressure associated with upstream constriction of small arterioles. In vitro studies (2, 4, 19) suggest that contractile responses to electrical stimulation or exogenous norepinephrine are enhanced in the presence of neuropeptide Y. Edvisson et al. (3) postulated that neuropeptide Y may induce contraction by potentiating the constrictor effects of endogenously released norepinephrine. In vivo studies utilizing the submandibular gland (10) or the spleen (15,16) suggest that the constriction to neuropeptide Y is independent of adrenoceptor stimulation. In the current study, pretreatment with the adrenoceptor antagonist, phentolamine, failed to significantly modify the constrictor response to neuropeptide Y by small cremaster arterioles (Fig. 4). The concentration of phentolamine used (10 - 6 M) was found to completely inhibit constriction to exogenously administered norepinephrine, which normally produces near maximal constriction of third-order arterioles. However, exposure to both phentolamine and the adrenergic neuronal blocker, guanethidine, significantly attenuated the sensitivity to neuropeptide Y. The small reduction in sensitivity suggests that part of the constrictor response to topically applied neuropeptide Y involves release of an endogenous neurotransmitter. While exposure to phentolamine alone did not produce a significant reduction in the sensitivity of neuropeptide Y, there was a trend toward a reduction (Fig. 4). We speculate that the phentolamine may be able to block receptors stimulated by exogenously applied norepinephrine, but is not able to block all of the receptors w h i c h are stimulated by n e u r o n a l l y released norepinephrine. Regardless, neuropeptide Y-induced constriction of arterioles of skeletal muscle appears to be primarily due to mechanisms other than adrenoceptor stimulation. Neuropeptide Y has been identified as a peptide that is stored and released together with norepinephrine from sympathetic perivascular nerves (4, 11, 24). In addition, investigators using adrenergic blocking agents have shown that in some vascular beds
neurally mediated vasoconstriction is only partially mediated by release of norepinephrine (1,8). However, guanethidine, which blocks release of both norepinephrine and neuropeptide Y (12), blocks the sympathetic vasoconstriction that is resistant to alphaadrenergic blockade (3). In a separate series of studies, we compared the effects of alpha-adrenergic blockade and adrenergic neuron blockade on resting tone of small arterioles in the cremaster muscle. Exposure to the alpha-adrenergic antagonist, phentolamine (10 - 6 M), for 30 minutes produced no significant changes in diameters of large arterioles or venules in the cremasters. Third-order arterioles dilated by approximately 14% (Fig. 3). Previously, we (7) reported a small dilation (15%) of small arterioles (4A) in the cremaster of decerebrated rats following exposure to phentolamine. These studies suggest that intrinsic tone in small arterioles of the cremaster is not mediated either by neuronally released or circulating catecholamines. Recent studies utilizing an acutely denervated cremaster preparation (5,6) support the absence of a constrictor influence of circulation catecholamines on microvascular tone of 3A arterioles. Adrenergic neuron blockade with guanethidine (10 -5 M) caused significant dilation of both 2A and 3A arterioles (21% and 55%, respectively). This dilation was significantly greater than the dilation observed in response to phentolamine and represented 48% and 40% of the maximal dilation to nitroprusside by 2A and 3A arterioles, respectively. The dilation observed for 3A arterioles was also similar in magnitude to the dilation observed in response to acute surgical denervation of the cremaster (25). Guanethidine has been shown to block both norepinephrine and neuropeptide Y release (12) and neuropeptide Y has been proposed to be responsible for the neuronal sympathetic vasoconstriction which is resistant to alpha-adrenergic blockade. The current results might be explained by the presence of a phentolamine-resistant neurogenic constrictor tone in small arterioles of the cremaster. Owen et al. (17) have observed arteries in the rabbit ear and reported that the ability of phentolamine to block contractions induced by transmural nerve stimulation was decreased as artery diameters decreased. They concluded, however, that this contraction is small in magnitude, occurs only at high nerve frequencies and is probably physiologically insignificant. Alternately, our data would support the hypothesis that a substantial degree of the neurogenically mediated tone in cremaster muscle is mediated by neuronally released neuropeptide Y. In summary, small arterioles in an in vivo skeletal muscle preparation (rat cremaster muscle) are highly responsive to exogenous neuropeptide Y. A small portion of the constrictor responses appear to be related to stimulation of the released endogenous catecholamines. The data presented are also consistent with a physiological role for neuropeptide Y in the maintenance of neurogenic tone in small arterioles in skeletal muscle. ACKNOWLEDGEMENTS We thank Mary Englebert for her excellent technical assistance. This work was supported by grants from the American Heart Association and the University of Louisville Graduate School Research Council. Dr. Irving G. Joshua is the recipient of an Established Investigator Award from the American Heart Association.
REFERENCES 1. Auer, L. M.; Edvinsson, L.; Johansson, B. B. Effect of sympathetic nerve stimulation and adrenoceptor blockade on pial arterial and venous calibre and on intracranial pressure in the cat. Acta Physiol. Scand. 119:213-217; 1983. 2. Edvinsson, L.; Ekblad, E.; Hakanson, R.; Wahlestedt, C. Neuropep-
tide-Y potentiates the effect of various vasoconstrictor agents on rabbit blood vessels. Br. J. Pharmacol. 83:519-524; 1984. 3. Edvissson, L.; Hakanson, R.; Wahlestedt, C.; Uddman, R. Effects of neuropeptide-Y on the cardiovascular system. Trends Pharmacol. Sci. 8:231-235; 1987.
IN VIVO RESPONSES TO NEUROPEPTIDE Y
4. Ekblad, E.; Edvinsson, L.; Wahlestedt, C.; Uddman, R.; Hakanson, R.; Sundler, F. Neuropeptide-Y co-exists and co-operates with noradrenaline in perivascular nerve fibres. Regul. Pept. 8:225-235; 1984. 5. Faber, J. E. In situ analysis of alpha-adrenoceptors on arteriolar and venular smooth muscle in rat skeletal muscle microcirculation. Circ. Res. 62:37-50; 1988. 6. Faber, J. E. Effect of local tissue cooling on microvascular smooth muscle and postjunctional ct2-adrenoceptors. Am. J. Physiol. 255: H121-H130; 1988. 7. Faber, J. E.; Harris, P. D.; Joshua, I. G. Microvascular responses to blockade of prostaglandin synthesis in rat skeletal muscle. Am. J. Physiol. 243:HS1-H60; 1982. 8. Hellstrom, P.; Olerup, O.; Tatemoto, K. Neuropeptide-Y may mediate effects of sympathetic nerve stimulations on colonic motility and blood flow in the cat. Acta Physiol. Scand. 124:613-624; 1985. 9. Joshua, I. G.; Wiegman, D. L.; Harris, P. D.; Miller, F. N. Progressive microvascular alterations with the development of renovascular hypertension. Hypertension 6:61-67; 1984. 10. Lundberg, J. M.; Tatemoto, K. Pancreatic polypeptide family (APP, BPP, NPY and PYY) in relation to sympathetic vasoconstriction resistant to alpha-adrenoceptor blockade. Acta Physiol. Scand. 116: 393-402; 1982. 11. Lundberg, J. M.; Terenius, L.; Hokfelt, T.; Martling, C. R.; Tatemoto, K.; Mutt, V.; Polak, J.; Bloom, S. R.; Goldstein, M. Neuropeptide-Y (NPY)-like immunoreactivity in peripheral noradrenergic neurons and effects of NPY on sympathetic function. Acta Physiol. Scand. 116:477-480; 1982. 12. Lundberg, J. M.; Anggard, A.; Theodorsson-Norheim, E.; Pernow, J. Guanethidine-sensitive release of NPY-like immunoreactivity by sympathetic nerve stimulation. Neurosci. Lett. 52:175-180; 1984. 13. Lundberg, J. M.; Pernow, J.; Tatemoto, K.; Dahlof, C. Pre- and postjunctional effects of neuropeptide-Y on sympathetic vascular control in rat femoral artery. Acta Physiol. Scand. 123:511-513; 1985. 14. Lundberg, J. M.; Torsell, J.; Sollevi, A.; Pernow, J.; TheodorssonNorheim, E.; Anggard, A.; Hamberger, B. Neuropeptide-Y and sympathetic vascular control in man. Regul. Pept. 13:41-52; 1985.
41
15. Lundberg, J. M.; Anggald, A.; Pernow, J.; Hokfelt, T. Neuropeptide-Y-, substance P- and VIP-immunoreactive nerves in cat spleen in relation to autonomic vascular and volume control. Cell Tissue Res. 239:9; 1985. 16. Lundberg, J. M.; Fried, G.; Pernow, J.; Theodorsson-Norheim, E.; Anggard, A. NPY--a mediator of reserpine-resistant, non-adrenergic vasoconstriction in cat spleen after preganglionic denervation? Acta Physiol. Scand. 126:151-152; 1986. 17. Owen, M. P.; Quinn, C.; Bevans, J. A. Phentolamine-resistant neurogenic constriction occurs in small arteries at high frequencies. Am. J. Physiol. 249:H404-H414; 1985. 18. Pernow, J.; Saria, A.; Lundberg, J. M. Mechanisms underlying preand postjunctional effects of neuropeptide-Y in sympathetic vascular control. Acta Physiol. Scand. 126:239-249; 1986. 19. Pernow, J.; Lundberg, J. M. Neuropeptide-Y constricts human skeletal muscle arteries via a nifedipine-sensitive mechanism independent of extracellular calcium? Acta Physiol. Scand. 128:655--656; 1986. 20. Pemow, J.; Svenberg, T.; Lundberg, J. M. Action of calcium antagonists on pre- and postjunction effects of neuropeptide-Y on human peripheral blood vessels in vitro. Eur. J. Pharmacol. 136:207-218; 1987. 21. Pernow, J.; Lundberg, J. M.; Kaijser, L. Vasoconstrictor effects in vivo and plasma disappearance rate of neuropeptide-Y in man. Life Sci. 40:47-54; 1987. 22. Pernow, J.; Kahan, T.; Hemdahl, P.; Lundberg, J. M. Possible involvement of neuropeptide-Y in sympathetic vascular control of canine skeletal muscle. Acta Physiol. Scand. 132:43-50; 1988. 23. Rhodin, J. A. G. Ultrastructure of mammalian venous capillaries, venules and small collecting veins. J. Ultrastruct. Res. 25:452-500; 1968. 24. Uddman, R.; Ekblad, E.; Edvinsson, L.; Hakanson, R.; Sundier, F. Neuropeptide-Y-like immunoreactivity in perivascular nerve fibers of the guinea-pig. Regul. Pept. 10:243-257; 1985. 25. Yu, J.; Roberts, A. M.; Joshua, I. G. Lung inflation evokes reflex dilation of microvessel in rat skeletal muscle. Am. J. Physiol. 258: H939-H945; 1990.