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Increased plasma levels of neuropeptide Y-like immunoreactivity and catecholamines in severe hypertension remain after treatment to normotension in man
279
Regulatory Peptides, 32 (1991) 279-287 Elsevier
REGPEP
01002
Increased plasma levels of neuropeptide Y-like immunoreactivity and catecholamines in severe hypertension remain after treatment to normotension in man Lars Edvinsson ‘, Rolf Ekman’ and Thomas Thulin ’ ‘Department of Internal Medicine and ‘Department of Neurochemistry and Psychiatry, Lund University, Lund (Sweden) (Received
1 February
1990; revised
Key words: Neuropeptide
Version received
23 October
Y; Adrenaline;
1990; accepted
Noradrenaline;
15 November
1990)
Hypertension
Summary Circulating levels of neuropeptide Y (NPY)-like immunoreactivity (-LI), adrenaline and noradrenaline (NA) were analysed in 17 patients admitted to the emergency ward due to severe hypertension; blood pressure mean 204/127 mmHg. The levels of NPY-LI and NA were significantly higher (P < 0.001) in the hypertensives as compared to a normotensive control group. HPLC analysis revealed that the plasma contained besides NPY-LI also several NPY-LI fragments of low hydrophobicity. Following 2 to 3 weeks treatment the blood pressure had decreased to a mean of 150/89 mmHg. However, circulating levels of NPY-LI (P < 0.001) and NA (P < 0.01) were still significantly higher than in controls in spite of the marked reduction in blood pressure. Simultaneous measurements of adrenaline did not reveal any significant changes and these values did not differ compared with those in the normotensive subjects. The findings suggest that peripheral markers of the sympathetic system (NPY-LI and NA) in severe hypertension is not directly related to the blood pressure level.
Correspondence:
L. Edvinsson,
Department
of Internal
Medicine,
University
Hospital,
Sweden.
0167-0115/91/$03.50
0
1991 Elsevier
Science
Publishers
B.V. (Biomedical
Division)
S-221 85 Lund,
280 Introduction
Neuropeptide Y (NPY) is a 36 amino acid peptide which is widely distributed both in the central and peripheral nervous system [ 1-2], and thought to be involved in the regulation of cardiovascular function [ 3]. At present, the attention of NPY is primarily focused on its direct effects on various parts of the circulation [4]. Marked interspecies and regional differences exist in the responses to NPY. NPY has direct vasoconstrictor effects, can potentiate vasoconstriction elicited by amines [3,5], and may suppress noradrenaline release from sympathetic nerve terminals [6]. The functional role of NPY in the peripheral circulation appears to differ depending on stimulation frequency. At low intensities it may increase the efficacy and the economy of noradrenaline at sympathetic nerve terminals, whereas NPY per se possesses strong direct vasoconstriction at higher stimulation intensities [4]. In the present study, we have first examined the levels of NPY-like immunoreactivity (-LI) and plasma catecholamines in patients referred to our emergency ward with severe hypertension, e.g., diastolic blood pressure above 120 mmHg, and later NPY-LI and catecholamines have been reexamined after reduction of blood pressure.
Material and Methods Patients
Hypertensive patients were obtained after arriving at the emergency ward due to severe hypertension. Inclusion criteria were: advanced hypertension, e.g., still diastolic blood pressure above 120 mmHg after 1 h rest, no signs of present cardio- or cerebrovascular disease, no antihypertensive medication during the last month and below 75 years of age. The study population consisted of 13 men and 4 females. Their age distribution was between 24 and 75 years of age. They had a previously unknown hypertension and had not previously received antihypertensive medication. The patients received an intravenous catheter and were allowed to rest in the recumbant position for 30-60 min before sampling for NPY-LI, peptide Y (PYY)-LI and catecholamines. During this time frequent blood pressures were recorded in a calm environment and a consistently high blood pressure was recorded. Following this the patients entered the study. After examination and collection of blood samples the patients were transferred to one of the wards at the department of Internal Medicine for further investigation and treatment of their hypertension. During this period ECG, chest X-ray, neurological examination, blood and urine sampling for analysis of kidney malfunction, liver disease, pheochromocytoma or other hormone producing tumors were screened. No evidence for any such disorder was seen. Treatment was initiated in all patients with a selective beta-adrenoceptor blocker (metoprolol) and a dihydropyridine type of calcium antagonist (nifedipine or felodipine). In a few subjects (n = 3) a mild diuretic (bendroflumetiazid) was given in addition to control the blood pressure. Within 2 to 3 weeks all patients had reached clinically acceptable blood pressure levels (Table I), and after another analysis of plasma NPY-LI and catecholamines they were discharged for
281 regular follow ups. Since a subgroup analysis of the three subjects receiving bendroflumetiazid did not differ in NPY-LI and noradrenaline levels from those that did not receive this drug all hypertensives were grouped together. Control subjects (n = 31) were of similar age and sex distribution as the patient material and collected during the same time interval as the hypertensives: they were normotensive, did not receive any antihypertensive medication and samples were taken after a period of rest (30-60 min) (Table I). This material revealed no difference among sex and age for NPY-LI (unpublished data), however, as expected the levels of NA and adrenaline, a slight increase with age was seen [7]
Measurement of catecholamines Blood was collected in a solution of 1 mg/ml EGTA and glutation, transported on ice to the laboratory for immediate centrifugation, the plasma decanted and frozen to - 70 °C for later analysis of catecholamines using H P L C with electrochemical detection [9]. Radioimmunoassay of circulating NP Y Plasma levels of NPY-LI were analysed using a method described in detail [8]. Blood was collected into prechilled viaIJs containing an EDTA solution (1 mg/ml), transported on ice to the laboratory, centrifuged at + 4 °C 2000g for 10 min. The plasma was removed and stored at - 20 ° C for later analysis of NPY-LI and PYY-LI content. For the radioimmunoassay (RIA) of NPY-LI a rabbit antiserum raised against synthetic porcine NPY (gift from Dr. P. C.! Emson, Cambridge, U.K.) conjugated to bovine serum albumin with carbodiimide was used. Porcine 125I-NPY used as tracer was purified by high performance liquid chromatography (HPLC). The antiserum cross-reacted with human NPY to 100 ~o, NPY (2-36) 33 ~o, NPY (4-36) 29~o, PYY to 33 ~o, but not with C-terminal fragments of NPY and PYY (NPY 13-36 and PYY 13-36) nor with bovine pancreatic polypeptide, gastrin iinhibiting peptide, peptide histidine isoleucine, vasoactive intestinal peptide or secretin. Antiserum 200/~1 (diluted 1 • 40,000) was incubated first with 100 #1 of blood samples or standard (synthetic NPY: Peninsula, Belmont, CA, U.S.A.) and with 200 #1 (about 2500 cpm) of the HPLC purified tracer for another 24 h. Bound and free ~zSI-NPY were separated using dextran-coated charcoal. Each sample was assayed in duplicate and corrected for unspecific binding. The detection limit was 11.7 pmol/1. Intraassay variation was 6.5~o while the interassay variation was 7~o. Because of the cross-reactivity with PYY, each sample was also assayed for PYY-LI (K 8413) [ 17 ] and 33 ~o of the values obtained were subtracted from the corresponding values for NPY-LI (i.e., about 10~o). The circulating levels of PYY-LI in control was 28 + 5 pmol/l and in the hypertensives 30 + 3 pmol/1 (P > 0.05). These values were low and from a practical point of view they did not interfere with the NPY-LI value encountered in the study population. Plasma extraction Extracted peptide material from human plasma 0.5 ml plasma/1.0 ml ethanol + 4 ° C, 10 min, centrifugation at 2000g 10 min; the supernatants collected, lyophilized and dissolved in 200/~1 (CH3CN/0.08?/o TFA (5/95)) prior to the H P L C separation.
282
High-performance liquid chromatography (HPL C) NPY-immunoreactive material in extracts from plasma was analyzed by reversephase H P L C on a Waters system using a u B o n d a p a k C18 column (Waters, U.S.A.). A linear gradient of acetonitrile (28-58 %) was applied during the first 60 min, following by 10 min of isocratic elution at 58% acetonitrile (flow rate 1.0 ml/min). Fractions of 0.5 ml were collected and lyophilized. The dry residues were redissolved in the R I A buffer and assayed for NPY-like immunoreactivity.
Statistical methods All the data were recorded on a preposed form. Standard statistical methods were used to determine mean values, standard errors and evaluation of differences between groups. For this purpose Student's t-test with Bonferroni correction for multiple group comparison was used.
Results
The blood pressure values are given in Table I. Both systolic and diastolic blood pressures were significantly (P < 0.001) higher when the patients arrived at the emergency ward as c o m p a r e d to the corresponding blood pressure levels in the control group. A slight increase in heart rate was noted. In the hypertensives the level of N P Y - L I was 145 + 9 pmol/1; while the level of N P Y - L I in the control group was 106 + 3 pmol/1 (P < 0.001 ; Fig. 1). The H P L C elution profile of N P Y - L I from patients with treated hypertension demonstrates a complex pattern with an increase of several N P Y - L I fragments o f low hydrophobicity compared to control plasma (Fig. 2). Corresponding values of N A (P < 0.001) but not adrenaline ( P > 0.05) were increased in the hypertensives as compared to the normotensive subjects (Fig. 3). After treatment blood pressure decreased significantly (P < 0.001) from the mean of 204/127 m m H g to 150/89 m m H g at the discharge from the hospital (Table I). At this
TABLE I Patient characteristics in control, in severe hypertension and following treatment (2-3 weeks) Values given represent means _+S.E.M., n = number of individuals.
Control Acute hypertension After treatment (2-3 weeks)
Diastolic blood pressure (mmHg)
Systolic blood pressure (mmHg)
Heart rate (per min)
31 17
84 + 5 127 + 7*
142 + 10 204 + 13"
71 + 5 93 _+ 12
17
89 + 4
150 + 12
73 + 9
Statistical analyses were performed using Student's t-test with Bonferroni correction for multiple group comparison: * P < 0.001.
283
l°° 1 z 501 0 -C A T Fig. 1 Circulating concentrations of NpY-LI in control (C, open bar) (n = 31) and in patients with high blood pressure (A, filled bar) (n = 17) (for details see Table I). The concentration of NPY-LI was significantly raised as compared to 2-3 weeks~ of treatment (T, hatched bar) the levels were still raised (P < 0.001). Values represent means + S.E.M. NPY h
90-
~
60-
A -60
~
-40
30-
-20
E v 0 >o. 90-
i
i
I
g
0 -60
60-
-40
3O-
-20
0 0
/
215 510 ii Elution volume (ml)
7i5
"1"
~
z
0
Fig. 2. HPLO-charaeterization of NP'~-LI in plasma extracts from control A and hypertensive patient B. The arrow indicates the elution volume 0fsynthetic human NPY. The depicted line indicates the acetonitrile gradient.
284 NA (nM) [ ]
A (nM) []
5-
0.5
~
4-
-0.4
e
:.:
0.3
!ii 1OControl
Acute
!ii
.o.t
°J
'0
Treated
Fig. 3. Circulating concentrations of catecholamines in control and in patients with high blood pressure. The level of noradrenaline (NA) was significantly increased (***P < 0.001). Despite normalization of blood pressure circulating N A was still raised 2 - 3 weeks after admission of the patients (**P < 0.01) (for details on blood pressure see Table I). There were no significant changes in adrenaline (A) levels. Values represent means + S.E.M.
occasion new blood samples were collected. The plasma levels of NPY-LI (158 + 10 pmol/1; P < 0.001) and NA (4.0 _+ 0.75 nM; P < 0.01) were still significantly increased in the treated hypertensives as compared to the normotensive controls (Figs. 1 and 3).
Discussion Patients in the control material had diastolic blood pressure well below 90 mmHg. There was no sex or age relation for plasma concentrations of circulating NPY-LI [7]. This is different as compared to that of plasma NA and A which consistently tend to be increased with age [7], concomitant with this there is a reduction in cardiovascular beta-adrenoceptors [ 10]. NPY-LI is co-localized with NA in perivascular sympathetic fibres [ 3,11]. There is much evidence demonstrating a co-storage of NPY-LI with NA in most parts of the circulation [11-13]. At the ultrastructural level NPY-LI appears to occur in the large vesicle population, whereas NA is seen preferentially in the small dense cored vesicles [ 14]. The localization of NPY-LI and NA in two different compartments within the same nerve terminal provides the structural basis for the frequency-dependent differential release of NPY-LI and NA reported in other studies [ 15]. In a recent study, we observed a richer supply of NPY-LI nerve fibres in the distal (resistance) part of the superior mesenteric artery (SMA) as compared to the proximal region near aorta [ 16]. The NPY-LI terminals were with immunoelectron microscopy observed i~ close apposition to the medial layer, hence, a direct physiological control by NPY-LI nerve terminals might be anticipated [16]. The effects of NPY on blood vessds are at least three-fold: (a) direct postjunctional constriction, (b) potentiation of NA responses, and (c) inhibition of released NA [4]. Since NPY-LI and NA are co-stored in various parts of the sympathetic system it
285 is not surprising that plasma levels, reflecting the spill-over from the vascular synapses, showed increases in conjunction with severe hypertension. Although the levels observed were not dramatically high (increased by 40 ~o), they are signs of an increased activity at the sympathetic nerve terminals, also evidenced by the raised levels of circulating NA but not of circulating adrenalirle. Although plasma NPY-LI and NA may be poor indicators of sympathetic nerve l~erminal activity, increasing evidence suggests that also mild hypertension is characterized by augmented sympathetic activity both at rest and in response to stress [ 17-20]. Direct intraneural recordings have recently revealed that efferent muscle sympathetic nerue activity (peroneal nerve) was elevated in hypertensive subjects [21], thus supporting the hypothesis of elevated central sympathetic neural outflow in hypertension. A remarkable observation in our study was the finding that despite marked lowering of blood pressure there was no reduction in the elevated circulating levels of NPY-LI and NA. This suggests that the sympathetic system continues to fire at a higher level despite the reduction in blood pressure. Previous studies have examined NPY-LI in subjects following various degrees of sympathetic activation [22]. Systemic NPY-LI increased progressively from the resting level of 18 to 81 pmol/1 in parallel with a 10,fold increase in NA concentration [22]. Such a relation between increases in circulating NPY-LI and NA was seen in the present severe hypertensives; a more pronoun¢ed increase in NA than in NPY-LI (Figs. 1 and 3). The circulating levels are in the present study higher than those of the above material [22]. The reason for this is two-fold; firstly, we used unextracted plasma, whereas others extracted large molecules from their plasma, and thereby also a considerable portion of the NPY-LI. Secondly, antibodies used have different antigenic sites and therefore recognize different part of the NPY molecule. Thus, the discrepancy in resting levels of NPY-LI could be explained in terms of a difference in specificity of the respective antibodies used. Although PYY-LI may interfere with the NPY-LI RIA, we did not observe any change in PYY-LI in the hypertensives as compared to normotensives. An intriguing finding in this preliminary study was the HPLC profile of treated patients with hypertension demonstrating increased concentration of NPY-LI eluting earlier than that of synthetic human NPY. Fragments of NPY have recently been found to have effects by themselves on blood pressure (Ref. 23 and unpublished). The patients were upon arrival unmedicated. In order to exclude any influence of stress on our measurements thelsamples were removed after 30-60 min of rest in a calm environment, the venous catheter was inserted before the resting period and blood pressure was recorded every 5 min with an automated system. Furthermore, in a subsample of subjects plasma :cortisol was taken at the same time and found to be unaltered as compared to conffol (not shown). Thus, we feel confident that our data represent an enhanced activity iat the peripheral sympathetic system and not due to a stress response. The values given probably represent a spill-over of the sympathetic neuroeffector system as has been discussed previously for circulating catecholamines. The unaltered level of circulating adrenaline is in support of selective activation of sympathetic fibres rather than ~ generalised effect involving also the adrenal medulla. The remaining high levels o~ NPY-LI and NA after treatment of the hypertensive episode and in the follow up period suggests that the peripheral sympathetic system was not modified by treatment of the high blood pressure with the conventional therapy of /
286 b l o o d pressure used. It m a y be argued that b e t a - a d r e n o c e p t o r b l o c k a d e might modify the circulating levels o f N P Y - L I and N A . A t the sympathetic neuroeffector betab l o c k a d e rather reduces N P Y - L I and N A [24,25], and hence the observations m a d e might thus be underestimated. Vasodilator treatment, calcium entry blockade, might initially cause sympathetic activation by triggering the baroreflex. However, there is no available d a t a that chronic calcium b l o c k a d e changes N P Y - L I in man. In contrast, another v a s o d i l a t o r mechanism, angiotensin converting enzyme inhibition, caused reduction o f circulating levels o f N P Y - L I in an animal m o d e l [26]. Clearly, much w o r k is to be c o n d u c t e d in the future to examine variations and long-term effects o f p h a r m a cological intervention on the two m a r k e r s o f the sympathetic activity, N P Y - L I a n d N A , in severe hypertension. The observations point t o w a r d s a centrally m e d i a t e d activation o f the sympathetic system in severe hypertension. In o r d e r to u n d e r s t a n d m o r e about central sympathetic involvement studies with agents acting at such sites will be important tools in the future.
Acknowledgements S u p p o r t e d by the Swedish M e d i c a l R e s e a r c h Council (05958, 07517) and the M e d i c a l F a c u l t y o f L u n d University, Sweden.
References 1 Tatemoto, K., Carlquist, M. and Mutt, V., Neuropeptide Y: a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide, Nature, 296 (1982) 659-660. 2 Adrian, T.E., Allen, J.M., Bloom, S.R., Ghatei, M.A., Rossor, M.N., Roberts, G.W., Crow, T.J., Tatemoto, K. and Polak, J.M., Neuropeptide Y distribution in human brain, Nature, 306 (1983) 584-585. 3 Ekblad, E., Edvinsson, L., Wahlestedt, C., Uddman, R., H~tkanson, R. and Sunder, F., Neuropeptide Y coexists and co-operates with noradrenaline in perivascular nerve fibres, Regul. Pept., 8 (1984) 225-235. 4 Edvinsson, L., H~tkanson, R., Wahlestedt, C. and Uddman, R., Effects of neuropeptide Y on the cardiovascular system, Trends Pharmacol. Sci., 8 (1987) 231-235. 5 Edvinsson, L., Ekblad, E., H~tkanson, R. and Wahlestedt, C., Neuropeptide Y potentiates the effect of various vasoconstrictor agents on rabbit blood vessels, Br. J. Pharmacol., 83 (1984) 519-525. 6 Pernow, J., Saria, A. and Lundberg, J.M., Mechanisms underlying pre- and postjunctional effects of neuropeptide Y in sympathetic vascular control, Acta. Physiol. Scand., 126 (1986) 239-249. 7 Lake, S. R., Ziegler, M. G., Coleman, M. D. and Kopin, I. J., Age-adjusted plasma norepinephrine levels are similar in normotensive and hypertensive subjects, N. Engl. J. Med., 296 (1977) 208-209. 8 Widerl6v, E., Heilig, M., Ekman, R. and Wahlestedt, C., Possible relationship between neuropeptide Y (NPY) and major depression - evidence from human and animal studies, Nord. Psykiatr. Tidsskr., 42 (1988) 131-137. 9 Hjemdahl, P., Daleskog, M. and Kahan T., Determination of plasma catecholamines by highperformance liquid chromatography with electrochemical detection: comparison with a radioenzymatic method, Life. Sci., 15 (1979) 131-138. 10 Sachs, Ch., Hamberger, B. and Kaijser, L., Cardiovascular responses and plasma catecholamines in old age, Clin. Physiol., 5 (1985) 553-565. 11 Lundberg, J.M., Terenius, L., HOkfelt, T., Martling, C., Tatemoto, K., Mutt, V., Polak, J.M., Bloom,
287 S. and Goldstein, M., Neuropeptid~ Y (NPY)-like immunoreactivity in peripheral noradrenergic neurons and effects of NPY on sympatheti+ function, Acta. Physiol. Scand., 116 (1982) 477-480. 12 Edvinsson, L., Emson, P., McCulloch, J., Tatemoto, K. and Uddman, R., Neuropeptide Y: Cerebrovascular innervation and vasomotor effects in the cat, Neurosci. Lett. 43 (1983) 79-84. 13 Uddman, R., Ekblad, E., Edvinsson, L., H~kanson, R. and Sandier, F., Neuropeptide Y-like immunoreactivity in perivascular nerve fibres of the guinea-pig, Regul. Pept., 10 (1985) 243-257. 14 Fried, G., Terenius, L., H/)kfelt, T. and Goldstein, M., Evidence for differential localization of noradrenaline and neuropeptide Y in neural storage vesicles isolated from rat was defence, J. Neurosci., 5 (1985) 450-458. 15 Lundberg, J.M. and HSkfelt, T., Coexistence of peptides and classical neurotransmitters, Trends •Neurosci., 6 (1983) 325-328. 16 Edvinsson, L., Gulbenkian, S., Jansen, I., Wharton, J., Cervantes, C. and Polak, J.M., Comparison of peptidergic mechanisms in different parts of the guinea-pig superior mesenteric artery: Immunocytochemistry at the light and ultrastructural levels and responses in vitro of large and small arteries, J. Auton. Nerv. Syst., 28 (1989) 141-154. 17 Julius, S., The emerging field of borderline hypertension, J Cardiovasc. Pharmacol. 8 (suppl 5) (1986) s4-s7. 18 Egan, B., Panis, R., Hinderliter, A., Schork, N. and Julius, S., Mechanism of increased alpha adrenergic vasoconstriction in human essential hypertension, J. Clin. Invest., 80 (1987) 812-817. 19 Esler, M., Jennings, G., Biviano, B., Lamber, G. and Hasking, G., Mechanism of elevated plasma noradrenaline in the course of essential hypertension, J. Cardiovasc. Pharmacol., 8 (suppl 5) (1986) s39-s43. 20 Goldstein, D.S., Plasma catecholamines and essential hypertension. An analytical review, Hypertension, 5 (1983) 86-99. 21 Anderson, E.A., Sinkey, C.A., Lawton, W.J., and Mark, A.L., Elevated sympathetic nerve activity in borderline hypertensive humans. Evidence from direct intraneural recordings, Hypertension, 14 (1989) 177-183. 22 Pernow, J., Lundberg, J.M., Kaijser, L., Hjemdahl, P., Theodorsson-Norheim, E., Martinsson, A. and Pernow, B., Plasma neuropeptide Y-like immunoreactivity and catecholamines during various degrees of sympathetic activation in man, Clin. Physiol., 6 (1986) 561-578. 23 Boulik, J., Scott, N., Taulane, J., Goodman, M., Brown, M. and Rivier, J., Neuropeptide Y and neuropeptide Y18-36, Int. J. Pept. Prot. Res., 33 (1989) 11-15. 24 Dahltif, C., Studies on ]~-adrenoc¢ptor mediated faciliation of sympathetic neurotransmission, Acta. Physiol. Scand. Suppl., 500 (1981) 1-147. 25 Dahl6f, P., Lundberg, J. M. and DahlSf, C., c~-and/~-adrenoceptor mediated effects of nerve stimulation evoked release ofneuropeptide Y (NPY)-like immunoreactivity in the pithed guinea-pig, J. Auton. Nerv. Syst., (1991), in press. 26 Dahl/)f, P., Lundberg, J.M. and DahlSf, C., Effect of angiotensin II and captopril on plasma levels of neuropeptide Y (NPY)-like immunoreactivity in the pithed guinea-pig, Naunyn-Schmiederbergs Arch. Phamacol., (1990), in press.
Regulatory Peptides, 32 (1991) 279-287 Elsevier
REGPEP
01002
Increased plasma levels of neuropeptide Y-like immunoreactivity and catecholamines in severe hypertension remain after treatment to normotension in man Lars Edvinsson ‘, Rolf Ekman’ and Thomas Thulin ’ ‘Department of Internal Medicine and ‘Department of Neurochemistry and Psychiatry, Lund University, Lund (Sweden) (Received
1 February
1990; revised
Key words: Neuropeptide
Version received
23 October
Y; Adrenaline;
1990; accepted
Noradrenaline;
15 November
1990)
Hypertension
Summary Circulating levels of neuropeptide Y (NPY)-like immunoreactivity (-LI), adrenaline and noradrenaline (NA) were analysed in 17 patients admitted to the emergency ward due to severe hypertension; blood pressure mean 204/127 mmHg. The levels of NPY-LI and NA were significantly higher (P < 0.001) in the hypertensives as compared to a normotensive control group. HPLC analysis revealed that the plasma contained besides NPY-LI also several NPY-LI fragments of low hydrophobicity. Following 2 to 3 weeks treatment the blood pressure had decreased to a mean of 150/89 mmHg. However, circulating levels of NPY-LI (P < 0.001) and NA (P < 0.01) were still significantly higher than in controls in spite of the marked reduction in blood pressure. Simultaneous measurements of adrenaline did not reveal any significant changes and these values did not differ compared with those in the normotensive subjects. The findings suggest that peripheral markers of the sympathetic system (NPY-LI and NA) in severe hypertension is not directly related to the blood pressure level.
Correspondence:
L. Edvinsson,
Department
of Internal
Medicine,
University
Hospital,
Sweden.
0167-0115/91/$03.50
0
1991 Elsevier
Science
Publishers
B.V. (Biomedical
Division)
S-221 85 Lund,
280 Introduction
Neuropeptide Y (NPY) is a 36 amino acid peptide which is widely distributed both in the central and peripheral nervous system [ 1-2], and thought to be involved in the regulation of cardiovascular function [ 3]. At present, the attention of NPY is primarily focused on its direct effects on various parts of the circulation [4]. Marked interspecies and regional differences exist in the responses to NPY. NPY has direct vasoconstrictor effects, can potentiate vasoconstriction elicited by amines [3,5], and may suppress noradrenaline release from sympathetic nerve terminals [6]. The functional role of NPY in the peripheral circulation appears to differ depending on stimulation frequency. At low intensities it may increase the efficacy and the economy of noradrenaline at sympathetic nerve terminals, whereas NPY per se possesses strong direct vasoconstriction at higher stimulation intensities [4]. In the present study, we have first examined the levels of NPY-like immunoreactivity (-LI) and plasma catecholamines in patients referred to our emergency ward with severe hypertension, e.g., diastolic blood pressure above 120 mmHg, and later NPY-LI and catecholamines have been reexamined after reduction of blood pressure.
Material and Methods Patients
Hypertensive patients were obtained after arriving at the emergency ward due to severe hypertension. Inclusion criteria were: advanced hypertension, e.g., still diastolic blood pressure above 120 mmHg after 1 h rest, no signs of present cardio- or cerebrovascular disease, no antihypertensive medication during the last month and below 75 years of age. The study population consisted of 13 men and 4 females. Their age distribution was between 24 and 75 years of age. They had a previously unknown hypertension and had not previously received antihypertensive medication. The patients received an intravenous catheter and were allowed to rest in the recumbant position for 30-60 min before sampling for NPY-LI, peptide Y (PYY)-LI and catecholamines. During this time frequent blood pressures were recorded in a calm environment and a consistently high blood pressure was recorded. Following this the patients entered the study. After examination and collection of blood samples the patients were transferred to one of the wards at the department of Internal Medicine for further investigation and treatment of their hypertension. During this period ECG, chest X-ray, neurological examination, blood and urine sampling for analysis of kidney malfunction, liver disease, pheochromocytoma or other hormone producing tumors were screened. No evidence for any such disorder was seen. Treatment was initiated in all patients with a selective beta-adrenoceptor blocker (metoprolol) and a dihydropyridine type of calcium antagonist (nifedipine or felodipine). In a few subjects (n = 3) a mild diuretic (bendroflumetiazid) was given in addition to control the blood pressure. Within 2 to 3 weeks all patients had reached clinically acceptable blood pressure levels (Table I), and after another analysis of plasma NPY-LI and catecholamines they were discharged for
281 regular follow ups. Since a subgroup analysis of the three subjects receiving bendroflumetiazid did not differ in NPY-LI and noradrenaline levels from those that did not receive this drug all hypertensives were grouped together. Control subjects (n = 31) were of similar age and sex distribution as the patient material and collected during the same time interval as the hypertensives: they were normotensive, did not receive any antihypertensive medication and samples were taken after a period of rest (30-60 min) (Table I). This material revealed no difference among sex and age for NPY-LI (unpublished data), however, as expected the levels of NA and adrenaline, a slight increase with age was seen [7]
Measurement of catecholamines Blood was collected in a solution of 1 mg/ml EGTA and glutation, transported on ice to the laboratory for immediate centrifugation, the plasma decanted and frozen to - 70 °C for later analysis of catecholamines using H P L C with electrochemical detection [9]. Radioimmunoassay of circulating NP Y Plasma levels of NPY-LI were analysed using a method described in detail [8]. Blood was collected into prechilled viaIJs containing an EDTA solution (1 mg/ml), transported on ice to the laboratory, centrifuged at + 4 °C 2000g for 10 min. The plasma was removed and stored at - 20 ° C for later analysis of NPY-LI and PYY-LI content. For the radioimmunoassay (RIA) of NPY-LI a rabbit antiserum raised against synthetic porcine NPY (gift from Dr. P. C.! Emson, Cambridge, U.K.) conjugated to bovine serum albumin with carbodiimide was used. Porcine 125I-NPY used as tracer was purified by high performance liquid chromatography (HPLC). The antiserum cross-reacted with human NPY to 100 ~o, NPY (2-36) 33 ~o, NPY (4-36) 29~o, PYY to 33 ~o, but not with C-terminal fragments of NPY and PYY (NPY 13-36 and PYY 13-36) nor with bovine pancreatic polypeptide, gastrin iinhibiting peptide, peptide histidine isoleucine, vasoactive intestinal peptide or secretin. Antiserum 200/~1 (diluted 1 • 40,000) was incubated first with 100 #1 of blood samples or standard (synthetic NPY: Peninsula, Belmont, CA, U.S.A.) and with 200 #1 (about 2500 cpm) of the HPLC purified tracer for another 24 h. Bound and free ~zSI-NPY were separated using dextran-coated charcoal. Each sample was assayed in duplicate and corrected for unspecific binding. The detection limit was 11.7 pmol/1. Intraassay variation was 6.5~o while the interassay variation was 7~o. Because of the cross-reactivity with PYY, each sample was also assayed for PYY-LI (K 8413) [ 17 ] and 33 ~o of the values obtained were subtracted from the corresponding values for NPY-LI (i.e., about 10~o). The circulating levels of PYY-LI in control was 28 + 5 pmol/l and in the hypertensives 30 + 3 pmol/1 (P > 0.05). These values were low and from a practical point of view they did not interfere with the NPY-LI value encountered in the study population. Plasma extraction Extracted peptide material from human plasma 0.5 ml plasma/1.0 ml ethanol + 4 ° C, 10 min, centrifugation at 2000g 10 min; the supernatants collected, lyophilized and dissolved in 200/~1 (CH3CN/0.08?/o TFA (5/95)) prior to the H P L C separation.
282
High-performance liquid chromatography (HPL C) NPY-immunoreactive material in extracts from plasma was analyzed by reversephase H P L C on a Waters system using a u B o n d a p a k C18 column (Waters, U.S.A.). A linear gradient of acetonitrile (28-58 %) was applied during the first 60 min, following by 10 min of isocratic elution at 58% acetonitrile (flow rate 1.0 ml/min). Fractions of 0.5 ml were collected and lyophilized. The dry residues were redissolved in the R I A buffer and assayed for NPY-like immunoreactivity.
Statistical methods All the data were recorded on a preposed form. Standard statistical methods were used to determine mean values, standard errors and evaluation of differences between groups. For this purpose Student's t-test with Bonferroni correction for multiple group comparison was used.
Results
The blood pressure values are given in Table I. Both systolic and diastolic blood pressures were significantly (P < 0.001) higher when the patients arrived at the emergency ward as c o m p a r e d to the corresponding blood pressure levels in the control group. A slight increase in heart rate was noted. In the hypertensives the level of N P Y - L I was 145 + 9 pmol/1; while the level of N P Y - L I in the control group was 106 + 3 pmol/1 (P < 0.001 ; Fig. 1). The H P L C elution profile of N P Y - L I from patients with treated hypertension demonstrates a complex pattern with an increase of several N P Y - L I fragments o f low hydrophobicity compared to control plasma (Fig. 2). Corresponding values of N A (P < 0.001) but not adrenaline ( P > 0.05) were increased in the hypertensives as compared to the normotensive subjects (Fig. 3). After treatment blood pressure decreased significantly (P < 0.001) from the mean of 204/127 m m H g to 150/89 m m H g at the discharge from the hospital (Table I). At this
TABLE I Patient characteristics in control, in severe hypertension and following treatment (2-3 weeks) Values given represent means _+S.E.M., n = number of individuals.
Control Acute hypertension After treatment (2-3 weeks)
Diastolic blood pressure (mmHg)
Systolic blood pressure (mmHg)
Heart rate (per min)
31 17
84 + 5 127 + 7*
142 + 10 204 + 13"
71 + 5 93 _+ 12
17
89 + 4
150 + 12
73 + 9
Statistical analyses were performed using Student's t-test with Bonferroni correction for multiple group comparison: * P < 0.001.
283
l°° 1 z 501 0 -C A T Fig. 1 Circulating concentrations of NpY-LI in control (C, open bar) (n = 31) and in patients with high blood pressure (A, filled bar) (n = 17) (for details see Table I). The concentration of NPY-LI was significantly raised as compared to 2-3 weeks~ of treatment (T, hatched bar) the levels were still raised (P < 0.001). Values represent means + S.E.M. NPY h
90-
~
60-
A -60
~
-40
30-
-20
E v 0 >o. 90-
i
i
I
g
0 -60
60-
-40
3O-
-20
0 0
/
215 510 ii Elution volume (ml)
7i5
"1"
~
z
0
Fig. 2. HPLO-charaeterization of NP'~-LI in plasma extracts from control A and hypertensive patient B. The arrow indicates the elution volume 0fsynthetic human NPY. The depicted line indicates the acetonitrile gradient.
284 NA (nM) [ ]
A (nM) []
5-
0.5
~
4-
-0.4
e
:.:
0.3
!ii 1OControl
Acute
!ii
.o.t
°J
'0
Treated
Fig. 3. Circulating concentrations of catecholamines in control and in patients with high blood pressure. The level of noradrenaline (NA) was significantly increased (***P < 0.001). Despite normalization of blood pressure circulating N A was still raised 2 - 3 weeks after admission of the patients (**P < 0.01) (for details on blood pressure see Table I). There were no significant changes in adrenaline (A) levels. Values represent means + S.E.M.
occasion new blood samples were collected. The plasma levels of NPY-LI (158 + 10 pmol/1; P < 0.001) and NA (4.0 _+ 0.75 nM; P < 0.01) were still significantly increased in the treated hypertensives as compared to the normotensive controls (Figs. 1 and 3).
Discussion Patients in the control material had diastolic blood pressure well below 90 mmHg. There was no sex or age relation for plasma concentrations of circulating NPY-LI [7]. This is different as compared to that of plasma NA and A which consistently tend to be increased with age [7], concomitant with this there is a reduction in cardiovascular beta-adrenoceptors [ 10]. NPY-LI is co-localized with NA in perivascular sympathetic fibres [ 3,11]. There is much evidence demonstrating a co-storage of NPY-LI with NA in most parts of the circulation [11-13]. At the ultrastructural level NPY-LI appears to occur in the large vesicle population, whereas NA is seen preferentially in the small dense cored vesicles [ 14]. The localization of NPY-LI and NA in two different compartments within the same nerve terminal provides the structural basis for the frequency-dependent differential release of NPY-LI and NA reported in other studies [ 15]. In a recent study, we observed a richer supply of NPY-LI nerve fibres in the distal (resistance) part of the superior mesenteric artery (SMA) as compared to the proximal region near aorta [ 16]. The NPY-LI terminals were with immunoelectron microscopy observed i~ close apposition to the medial layer, hence, a direct physiological control by NPY-LI nerve terminals might be anticipated [16]. The effects of NPY on blood vessds are at least three-fold: (a) direct postjunctional constriction, (b) potentiation of NA responses, and (c) inhibition of released NA [4]. Since NPY-LI and NA are co-stored in various parts of the sympathetic system it
285 is not surprising that plasma levels, reflecting the spill-over from the vascular synapses, showed increases in conjunction with severe hypertension. Although the levels observed were not dramatically high (increased by 40 ~o), they are signs of an increased activity at the sympathetic nerve terminals, also evidenced by the raised levels of circulating NA but not of circulating adrenalirle. Although plasma NPY-LI and NA may be poor indicators of sympathetic nerve l~erminal activity, increasing evidence suggests that also mild hypertension is characterized by augmented sympathetic activity both at rest and in response to stress [ 17-20]. Direct intraneural recordings have recently revealed that efferent muscle sympathetic nerue activity (peroneal nerve) was elevated in hypertensive subjects [21], thus supporting the hypothesis of elevated central sympathetic neural outflow in hypertension. A remarkable observation in our study was the finding that despite marked lowering of blood pressure there was no reduction in the elevated circulating levels of NPY-LI and NA. This suggests that the sympathetic system continues to fire at a higher level despite the reduction in blood pressure. Previous studies have examined NPY-LI in subjects following various degrees of sympathetic activation [22]. Systemic NPY-LI increased progressively from the resting level of 18 to 81 pmol/1 in parallel with a 10,fold increase in NA concentration [22]. Such a relation between increases in circulating NPY-LI and NA was seen in the present severe hypertensives; a more pronoun¢ed increase in NA than in NPY-LI (Figs. 1 and 3). The circulating levels are in the present study higher than those of the above material [22]. The reason for this is two-fold; firstly, we used unextracted plasma, whereas others extracted large molecules from their plasma, and thereby also a considerable portion of the NPY-LI. Secondly, antibodies used have different antigenic sites and therefore recognize different part of the NPY molecule. Thus, the discrepancy in resting levels of NPY-LI could be explained in terms of a difference in specificity of the respective antibodies used. Although PYY-LI may interfere with the NPY-LI RIA, we did not observe any change in PYY-LI in the hypertensives as compared to normotensives. An intriguing finding in this preliminary study was the HPLC profile of treated patients with hypertension demonstrating increased concentration of NPY-LI eluting earlier than that of synthetic human NPY. Fragments of NPY have recently been found to have effects by themselves on blood pressure (Ref. 23 and unpublished). The patients were upon arrival unmedicated. In order to exclude any influence of stress on our measurements thelsamples were removed after 30-60 min of rest in a calm environment, the venous catheter was inserted before the resting period and blood pressure was recorded every 5 min with an automated system. Furthermore, in a subsample of subjects plasma :cortisol was taken at the same time and found to be unaltered as compared to conffol (not shown). Thus, we feel confident that our data represent an enhanced activity iat the peripheral sympathetic system and not due to a stress response. The values given probably represent a spill-over of the sympathetic neuroeffector system as has been discussed previously for circulating catecholamines. The unaltered level of circulating adrenaline is in support of selective activation of sympathetic fibres rather than ~ generalised effect involving also the adrenal medulla. The remaining high levels o~ NPY-LI and NA after treatment of the hypertensive episode and in the follow up period suggests that the peripheral sympathetic system was not modified by treatment of the high blood pressure with the conventional therapy of /
286 b l o o d pressure used. It m a y be argued that b e t a - a d r e n o c e p t o r b l o c k a d e might modify the circulating levels o f N P Y - L I and N A . A t the sympathetic neuroeffector betab l o c k a d e rather reduces N P Y - L I and N A [24,25], and hence the observations m a d e might thus be underestimated. Vasodilator treatment, calcium entry blockade, might initially cause sympathetic activation by triggering the baroreflex. However, there is no available d a t a that chronic calcium b l o c k a d e changes N P Y - L I in man. In contrast, another v a s o d i l a t o r mechanism, angiotensin converting enzyme inhibition, caused reduction o f circulating levels o f N P Y - L I in an animal m o d e l [26]. Clearly, much w o r k is to be c o n d u c t e d in the future to examine variations and long-term effects o f p h a r m a cological intervention on the two m a r k e r s o f the sympathetic activity, N P Y - L I a n d N A , in severe hypertension. The observations point t o w a r d s a centrally m e d i a t e d activation o f the sympathetic system in severe hypertension. In o r d e r to u n d e r s t a n d m o r e about central sympathetic involvement studies with agents acting at such sites will be important tools in the future.
Acknowledgements S u p p o r t e d by the Swedish M e d i c a l R e s e a r c h Council (05958, 07517) and the M e d i c a l F a c u l t y o f L u n d University, Sweden.
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