| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Department of Physiology, West Virginia University, Morgantown, West Virginia 26506 - 9229; and 2 Michigan State University, Department of Physiology, B-340 Life Sciences, East Lansing, Michigan 48824
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Considering the coexistence of neuropeptide Y (NPY) and norepinephrine in perivascular sympathetic nerves and the known vasoconstrictor cooperation of NPY with norepinephrine, we investigated the involvement of NPY in long-term control of cardiovascular functions using NPY transgenic (NPY-tg) rats. These rats were developed by injection of the rat (Sprague-Dawley) pronuclei with a 14.5-kb clone of the rat structural NPY gene. When compared with nontransgenic littermates, NPY concentrations were significantly increased in a number of cardiovascular tissues of NPY-tg hemizygotes. Direct basal mean arterial pressure and heart rate were not changed, but calculated total vascular resistance was significantly increased in NPY-tg subjects. Arterial pressure increases, in response to norepinephrine injection, were greater in the NPY-tg rats. Also, the hypotension and bradycardia in response to hemorrhage were significantly reduced in NPY-tg subjects. These results indicate that NPY, when expressed in increased amounts, potentiates the pressor effects of norepinephrine and contributes to maintaining blood pressure during hemorrhage, but it does not alter resting blood pressure. These transgenic rats will facilitate studies of the role of NPY signaling in cardiovascular regulation, particularly regarding its functional cooperation with norepinephrine.
vasoconstriction; cardiac output; adrenoceptors; hemorrhage; hypersensitivity
| |
INTRODUCTION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
NEUROPEPTIDE Y (NPY) is one the most abundantly expressed neuropeptides in the mammalian nervous system and the most highly conserved one discovered to date (46). Together with its receptors, this peptide is expressed in the group of neurons of the central and peripheral nervous systems involved in cardiovascular regulation, particularly in the sympathetic nerve fibers innervating arteries, veins, and the heart (15, 46). Characteristic features of this peptide are its presence along with norepinephrine in the same neuronal secretory vesicle of the sympathetic nerve terminals and its joint release with this neurotransmitter on nerve stimulation (15, 24). Exogenous NPY elicits strong endothelium-independent vasoconstriction and long-lasting pressor responses (4, 46). In addition to direct vasoconstriction, NPY potentiates the action of other vasoconstrictors, including norepinephrine, angiotensin, and vasopressin (4, 42, 43, 46). Compared with normotensive rats, the concentrations of NPY in the cardiovascular tissues and the responsiveness of blood vessels to NPY are increased in the spontaneously hypertensive rat (44). In humans, higher circulating levels of NPY were reported in situations of increased blood pressure, including exercise or some forms of hypertension (40).
Strong genetic evidence for the role of NPY in cardiovascular pathophysiology has also come to light, such as the observations that the NPY locus on chromosome four cosegregates with high blood pressure in the spontaneously hypertensive rat (18) and that the NPY gene polymorphism is associated with higher blood levels of cholesterol and low-density lipoprotein cholesterol in humans (17). Further evidence for the role of this peptide in the control of vascular tone and its potential implication in the pathogenesis of hypertension and heart diseases have been reviewed at great length (46). Nonetheless, the role of endogenous NPY in the long-term regulation of blood pressure and the mechanism of its interaction with norepinephrine in the vascular sympathetic nerves are not completely understood.
The cardiovascular consequences of chronic upregulation of the NPY gene have not been reported. With the increasing availability of a large number of cloned genes relevant to cardiovascular physiology, the transgenic animal approach is emerging as a very powerful tool that yields insights into cardiovascular physiology that could not otherwise be obtained (33).
In the present study, we aimed to determine the role of endogenous NPY in the control of blood pressure regulation by the autonomic nervous system using a gene addition mutation approach in the rat. In particular, we intended to examine whether the apparent upregulation of NPY signaling in NPY transgenic (NPY-tg) rats would affect resting blood pressure as well as responsiveness to pressor doses of norepinephrine or to reflex sympathoexcitation via hemorrhage. Hemorrhage was chosen because it is a typical sympathoexcitation, during which peripheral sympathetic outflow is known to contribute significantly in maintaining blood pressure (14, 38) and during which NPY release from sympathetic nerve terminals is increased (30).
We have chosen the rat species for in vivo gene addition mutation because the rat has been the species of choice for cardiovascular research and research on the biology of NPY and also because of the availability of diverse genetic and experimental models of hypertensive rats (33). Therefore, the results obtained from these transgenic rats can be readily compared with the existing data obtained from the experiments in which rat models were used.
Our hypothesis was that endogenous NPY is a physiological neurotransmitter involved in maintaining vascular tone, enhancing pressor responsiveness to norepinephrine, and sustaining blood pressure during hypovolemic hemorrhage. Here, we report that increasing NPY signaling through genetic manipulation results in 1) increased total vascular resistance, 2) increased pressor responsiveness to norepinephrine, and 3) reduced hemorrhagic hypotension.
| |
MATERIALS AND METHODS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
This study was approved by the West Virginia University Committee on Animal Care and Use before the transgenic rats were developed. The rats were housed in a temperature (20-23°C)- and a light-dark cycle (12:12-h)-controlled room. They were provided Purina laboratory chow and water ad libitum.
NPY-tg rats were generated as previously described (27,
41). The major goal of this experiment was to target the
transgenic NPY expression to the physiological sites and at the same
time ensure that the transgene expression was subjected to the same developmental and physiological regulation as its endogenous
counterpart. For this reason, a 14.5-kb locus of the rat NPY gene
(21) was used as a transgene (Fig.
1A). It contained the rat NPY
gene promoter, the coding sequences with four exons and introns as well
as 5- and 1.5-kb fragments of the 5' and 3' regions of this gene,
respectively. The identity of the cloned fragment was verified by
restriction enzyme mapping, PCR amplification of several known
fragments, and partial dideoxy-sequencing (Amersham) using primers
located at three different locations of the known sequences of the NPY gene.
|
The egg donors were 6- to 8-wk-old Sprague-Dawley females mated with adult Sprague-Dawley males (Hilltop Laboratory Animals, Scottdale, PA). After the injection of pronuclei with the DNA solution, the eggs were transferred to the oviducts of pseudopregnant Sprague-Dawley females. Transgenic founders and their progeny were identified by Southern blot analysis (Fig. 1B) of tail DNA digested with the restriction enzyme EcoR I. The hybridization probe, a 0.97-kb fragment of the rat NPY sequence, was amplified by PCR. The primer-binding sites flanked portions of the first exon and of the 5'-untranslated region (377-1348 bp) of the rat NPY sequences (21). The PCR product was labeled with psoralen-biotin for chemiluminescence detection using the Schleicher & Schuell kit (Schleicher & Schuell, Keene, NH). The hybridization signal from the endogenous NPY (13 kb) served as an internal control and was used to estimate the number of copies of the transgene in each line.
In this study, second generation (F2) of 3- to 5-mo-old NPY transgenic
hemizygote males carrying five copies of the transgene (line no. 400)
were used, except for the tail blood pressure experiment, in which
males of the F1 generation from all six of the transgenic lines
generated were tested. Similarly, the F2 generation of NPY-tg rats was
used for analysis of concentrations of NPY in the tissues examined (see
Fig. 2), in plasma (see Fig.
6C), in the hypothalamus, and in the posterior pituitary.
Skeletal muscles, adrenal glands, segments of the gastrointestinal
tract, lungs, salivary gland, and spleen were only tested in animals
from the F1 generation.
|
In each experiment, nontransgenic littermates were used as controls. Because siblings have a very similar genetic background, the utilization of nontransgenic littermates as controls significantly enhances the precision of the observations.
Analysis of transgene expression.
Tissue concentrations of NPY were measured using a specific antiserum
and radioimmunoassays as previously described (5, 25, 26).
For dissection of tissues, rats were anesthetized with pentobarbital
sodium injection and perfused with saline via the left cardiac
ventricle. For dissection of the hypothalamic areas, the brains were
quickly removed, frozen on dry ice in isopentane, and stored at
80°C. Serial sections of 300 µm were cut, and discrete brain
areas were microdissected. The landmarks for this dissection were
derived from an atlas of the rat brain (34). Total protein content in brain samples was determined by the bicinchoninic acid protein assay (Pierce, Rockford, IL). NPY distribution in the coronal
sections of the brain was determined by immunocytochemistry using the
indirect peroxidase-antiperoxidase technique as described in detail
(39). Blood samples were collected from conscious rats
using chronic vascular catheters dispensing to ice-cold tubes containing 0.5 trypsin-inhibitory unit of aprotinin and 10 IU of
heparin. Plasma was immediately separated by centrifugation (14,000 g, 4°C, 15 min) and stored at 70°C until assayed. This protocol has been reported to provide platelet-free plasma and to
prevent NPY release from the platelets (46).
Direct blood pressure measurement. Direct systolic and diastolic arterial blood pressures and heart rates were measured in resting, unrestrained, and conscious rats using a chronic arterial catheter. For implantation of a catheter, the rat was anesthetized with pentobarbital sodium (50 mg/kg ip). With the use of a sterile technique, polyurethane tubing (0.033 in. OD and 0.014 in. ID, Micro-Renathane, Braintree Scientific, Braintree, MA) was inserted into the lower abdominal aorta through the femoral artery. At the same time, another catheter was inserted into the inferior vena cava via the femoral vein for the administration of drugs. Both catheters were then exteriorized at the back of the neck, filled with a mixture of heparin (100 U/ml), dextrose (50%), and penicillin G (1,000 U/ml) in saline, secured with a silk suture, and plugged with a piece of fish line. During the 3- to 4-day recovery period, the animals were housed individually with food and water ad libitum. To adapt the animals to the experimental conditions, the rats were brought to the laboratory in their home cages for 3-4 h every day. At that time, their vascular catheters were flushed with heparinized dextrose solution. All rats appeared to be in good health and were moving freely at the time of study. On the day of experiment, 2 h before recordings, the rats were moved into the laboratory in their plastic home cages. The catheters were connected to tubing extensions that permitted the recording of arterial blood pressures, blood sample withdrawal, or drug infusion without restraining or otherwise disturbing the rat. Each animal was allowed an additional 30-min adaptation period between the time the catheter extensions were connected and data collection began. Food and water were not available while the rat was in the recording cage. The arterial catheter was connected to a pressure transducer coupled to a computerized cardiovascular continuous monitoring system (BPA-100, Micro-Med, Lexington, KY). This system allowed measurement and storage of systolic, diastolic, mean arterial blood pressures, and heart rates from two rats simultaneously. Transgenic and nontransgenic littermates were measured at the same time.
Cardiac output and total peripheral vascular resistance.
Cardiac output was determined in anesthetized rats by the
indicator-dilution method with a thermodilution technique. The change in temperature detected by the thermocouple probe was recorded continuously against time, and cardiac output was determined using a
computerized Cardiomax III (Columbus Instruments, Columbus, OH). The
animals were anesthetized with pentobarbital sodium (50 mg/kg ip).
During the cardiac-output measurements, the depth of anesthesia was
controlled by checking the pedal spinal reflex. If necessary, the
animals received an additional injection (intraperitoneally) of
pentobarbital sodium. To maintain airway patency, a PE-200 cannula was
placed in the trachea through which the animals were breathing
spontaneously. A polyurethane tubing (0.033 in. OD and 0.014 in. ID,
Micro-Renathane, Braintree Scientific) was inserted into the right
jugular vein, and the catheter was advanced into the right atrium for
rapid injection of saline to produce thermodilution curves. The
thermodilution microprobe (Columbus Instruments, Columbus, OH) was
inserted into the left carotid artery, and its tip was advanced to the
aortic arch to come into contact with free blood flow and to record the
temperature of the blood at that site. At the same time, a 0.033 in. OD
and 0.014 in. ID Micro-Renathane catheter was inserted into the
abdominal aorta through the femoral artery for measurements of arterial
blood pressure and heart rate. The microprobe location was inspected by
the analysis of the shapes of thermodilution curves. Once the correct
placement of the thermodilution probe was ascertained and when the
aortic blood temperature of the rat was at least 36°C, a bolus of 140 µl of physiological saline of a known temperature (18-19°C)
was rapidly injected into the right atrium via the jugular catheter.
Six thermodilution curves were obtained at 2-min intervals. The results
from the first injection were discarded to allow for possible artifacts from temperature transfer across the catheter, and then the mean value
of the five subsequent injections was taken as the measure of cardiac
output. Coefficient of variation of the repeated cardiac measures was
0.034 ± 0.004 (SE, n = 12). Cardiac output was
evaluated as the cardiac index, which is the cardiac output divided by
the body mass
(ml · min
1 · kg1). Stroke
volume index was calculated by dividing the stroke volume by the
body mass of the rat
(ml · beat1 · kg1). Total
vascular resistance index was calculated by dividing mean blood
pressure (obtained at the same time) by cardiac output and by
body mass
(mmHg · g1 · min1 · ml1).
Tail-cuff measurement of systolic blood pressure in conscious rats. Caudal artery systolic blood pressure was measured in conscious, restrained rats by the tail-cuff method using a computerized system (model 631, IITC, Woodland Hills, CA). Three transgenic and three control rats restrained in the holders were placed in the temperature-controlled chamber at 28.5°C at the same time. Blood pressure readings were obtained by averaging values from five consecutive readings taken on the fourth day after the 3-day training.
Pressor responsiveness to norepinephrine. Conscious, freely moving males with chronic arterial and femoral catheters were used in this experiment. Treatment and blood pressure recordings were performed on an NPY-tg and nontransgenic littermate at the same time using the blood pressure monitoring system described earlier. After 40 min of basal blood pressure recording, boluses of increasing doses of norepinephrine (Bitartrate salt, Sigma Chemical, St. Louis, MO) in 0.2 ml/kg saline containing citric acid (mg/ml, to prevent oxidation of norepinephrine) were administered into the femoral vein catheter at 20- to 30-min intervals, allowing complete dissipation of the preceding response. The increase in mean arterial pressure from the prestimulatory values was calculated for each dose of norepinephrine and expressed as a percentage of the maximal pressor response. The maximal pressor response was defined for each rat as the first highest mean arterial pressure response at the beginning of the plateau portion of the response curve. The dose-response curves for both groups of animals were constructed by plotting the mean arterial pressure increases against the logarithm of incremental norepinephrine doses.
The norepinephrine concentration values that produced the half-maximal blood pressure response (ED50) for each rat were calculated from the regression function between the logarithm of the molar concentration of norepinephrine and the mean arterial pressure response. The values between 20 and 80% of maximum response were used for this calculation.Hemorrhage. Experiments were performed in conscious rats at least 3 days after the catheter implantation. All experiments began in the afternoon, with the first blood pressure recording started between 1400 and 1500. After 40 min of basal blood pressure and heart rate recordings, the rats were hemorrhaged by removing 1.3% body weight of blood over a period of 5-7 min from the chronic femoral artery catheter. The first and last portions of 1.5 ml of blood were saved as blood samples for NPY assays at basal and posthemorrhage states, respectively. Thereafter, blood pressure and heart rate were continuously recorded for 20 min.
| |
RESULTS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
General appearance of NPY-tg rats and transgene overexpression pattern. The six transgenic lines generated had approximately 1, 2, 3, 5, 10, and 24 copies of the NPY transgene. Male offspring of a male founder (designated no. 400) that carried five copies of the transgene were selected for this study (Fig. 1B). Transgenic animals with two to five copies of the transgene are optimal for most purposes (35). When crossed with wild-type (Sprague-Dawley) females (Hilltop Laboratory Animals), this male passed the transgene to 50.5% (109 of 216) of its offspring. Southern analysis (Fig. 1B) of the DNA from the animals of this transgenic line digested with the Cla I restriction enzyme (a single transgene cutter) demonstrated the expected 14.5-kb band (not present in wild-type animals), indicating that the incorporated transgene was intact. Transgenic rats of this line reproduced normally, giving 12-15 pups per litter. No developmental abnormalities or reduced longevity were observed in these mutant rats. Altogether, these results indicate that in this line, the entire molecule of the transgene was stably integrated within the rat genome, the transgene was transmitted to the next generation in a Mendelian pattern, and there was no embryonic death among the transgenics.
At 16-36 wk of age, the body weights of the F1 generation of these NPY-tg males were ~7.6 ± 0.1-9.7 ± 0.3% higher than in nontransgenic littermates (P < 0.05). However, the F2 generation of these transgenic males did not differ from the nontransgenic littermates (Table 1). This discrepancy could be due to the particular genetic makeup of the founder not specifically related to the transgene. Nonetheless, the increased vascular resistance, enhanced pressure responsiveness to norepinephrine, reduced hemorrhagic hypotension, or the higher concentrations of NPY in a number of examined tissues reported here were consistently observed in the F2 generations of these NPY-tg males.
|
Direct arterial and tail blood pressures, cardiac output, and total
peripheral resistance in NPY-tg rats.
Direct systolic, diastolic, and mean arterial blood pressures, heart
rates, and cardiac outputs did not differ between NPY-tg and
nontransgenic littermates (Table 1 and Fig.
3). On the other hand, the calculated
total vascular resistance was significantly higher in NPY-tg rats than
in nontransgenic littermates (Fig. 3).
|
|
Pressor response to norepinephrine.
Intravenous administration of increasing doses of norepinephrine
resulted in elevated mean arterial pressure both in NPY-tg and
nontransgenics (Fig. 5A).
However, there was a significant leftward shift in mean arterial
pressure responses to increasing doses of norepinephrine in NPY-tg
animals (P < 0.05). Accordingly, the norepinephrine
dose that produced ED50 was significantly lower (P < 0.05) in NPY-tg than in nontransgenic littermates
(1.8 vs. 4.0 nmol/kg, respectively; Fig. 5B). The maximal
mean arterial pressure response to the highest dose of norepinephrine
used in this experiment did not differ between the groups (183.6 ± 3.4 vs. 184.6 ± 2.6 mmHg in control vs. NPY-tg, respectively).
Heart rate responses to increasing doses of norepinephrine were not different between the genotypes (Fig. 5C). This experiment
was performed separately on the F1 and F2 generations of NPY-tg rats with very similar results. The results from the experiment using the F2
generation of animals are presented here. The norepinephrine ED50 values of the first generation were strikingly similar
to those of the second (2.5 ± 0.2 and 4.6 ± 0.6 nmol/kg,
P < 0.05, for transgenic and wild-type genotypes,
respectively).
|
1 · s1). This dose
produced approximately a half-maximal pressor response. An increase in
cardiac output of nearly 10% occurred in both groups of animals during
norepinephrine infusion with no significant difference between the
genotypes (415.5 ± 14.7 vs. 402.4 ± 14.4 mmHg · ml · min1 · g1,
n = 14, in control vs. NPY-tg rats, respectively).
Cardiovascular responses to hemorrhage.
Experimental bleeding (1.3% of body wt) caused a significant reduction
in mean arterial pressure in both groups of animals (Fig.
6A). However, the hemorrhagic
hypotension was more profound (P < 0.05) in the
nontransgenic littermates. The blood pressure in this group decreased
by 62.2 ± 3.8 mmHg compared with 33.6 ± 9.3 mmHg reduction
in NPY-tg rats. In both groups, the maximum decrease in mean arterial
pressure occurred ~1-2 min after cessation of the blood
withdrawal and then started to return to basal levels. However, the
slopes of blood pressure recovery curves were significantly (P < 0.05) higher in control rats (2.1 ± 0.4 vs.
1.1 ± 0.2 mmHg/min), indicating that the wild-type animals
recovered at a faster rate than the transgenics. Similar to the
observed decrease in blood pressures, the decrease in heart rates after
the blood loss was significantly reduced (P < 0.05) in
NPY-tg compared with nontransgenic controls (Fig. 6B). This
experiment was performed separately on the F1 and F2 generations of
NPY-tg rats with very similar results. The results from the experiment
using the F2 generation animals are presented here. In the F1
generation, blood pressures after hemorrhage decreased by 69.4 ± 8.9 and 46.4 ± 6.4 mmHg (P < 0.05) in wild-type
and transgenic rats, respectively.
|
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
General appearance of NPY-tg rats and transgene expression pattern. Body weights of the NPY-tg rats were similar to those of the nontransgenic littermates. Thus, in the light of well-substantiated evidence of NPY involvement in controlling energy accumulation (7), it was surprising to find that body weights were unaffected in NPY-tg males. This finding may indicate that there are compensatory changes in other transmitter systems that mask the impact of elevations of NPY levels. It is also possible that the transgenic animals did not produce additional NPY at physiologically relevant sites or that chronically elevated NPY does not have the impact on physiological functions as single, large doses of exogenous peptide do. This remains to be studied in more detail, and our observation does not contradict the extensive evidence linking NPY to metabolic functions (7). The lack of profound effects of NPY on body weight and development was also observed in NPY knockout mice, in which there was an absence of NPY expression in physiologically important locations (8).
Our observation of the higher concentrations of NPY in a number of visceral organs, small blood vessels, and brain centers that normally express this peptide (15, 46) suggests that in the NPY-tg subjects, overexpression of NPY was indeed targeted to the NPY neurons involved in controlling blood pressure, including the nerve fibers contacting small arteries, which greatly contribute to peripheral resistance (9, 31). The higher circulating levels of NPY in the transgenic rats after hemorrhage indicate that the transgene expression was regulated in a manner similar to that of endogenous NPY. The lack of NPY overexpression in some tissues, including the skeletal muscles, larger blood vessels, or in the adrenal glands, was surprising. This could be related to the size of the samples from some organs being too small or to a particularly tight regulation of NPY expression in some other organs.Arterial blood pressure. Resting blood pressure was not changed in NPY-tg rats. Thus the present finding suggests that NPY apparently does not contribute significantly to maintaining resting blood pressure. This observation was unexpected in the light of the reported potent vasoconstrictor activity of exogenous NPY (4, 42, 46). On the other hand, this observation is in agreement with the report that administration of the Y1 selective antagonist does not alter resting blood pressure in normotensive animals (6). It could be that the potential pressor action of genetically increased NPY was masked by counteracting adaptive mechanisms involving, for example, the baroreceptor reflex and reduced cardiac output. Exogenous NPY has been reported to increase baroreflex sensitivity (20, 29).
Total vascular resistance was significantly increased in the NPY-tg rats. The increased blood vessel tone in the NPY-tg rats was most likely related to a direct vasoconstrictor effect of transgenic NPY, as evidenced by the higher NPY concentrations in a number of organs and small blood vessels of these transgenic rats. Nonetheless, we cannot exclude the possibility that the higher vascular resistance in the transgenic rats was due to a lower cardiac output. NPY has been demonstrated to decrease cardiac contractility through reduction of
-adrenergic signaling in the rat cardiomyocytes (28).
One caveat of this study was that the cardiac outputs were measured in
animals anesthetized with pentobarbital sodium. However, we have no
evidence indicating that pentobarbital anesthesia interacts with NPY to
inhibit cardiac function to a greater degree in the transgenic rats
than it does in wild-type rats. The present finding of increased total
vascular resistance in NPY-tg rats is consistent with the reported
pressor activity of exogenous NPY (4, 42-46), with
the reduced hemorrhagic hypotension in these subjects, as well as with
our previous reports of alternations in blood flows in a number of
organs after treatment with NPY or with NPY antiserum (5,
26). In those experiments, blood flows were also measured in
pentobarbital-anesthetized rats. Altogether, the increased vascular
resistance in NPY-tg rats with genetic upregulation of NPY in the
cardiovascular system supports the view of the physiological importance
of endogenous NPY in the regulation of the blood vessel tone.
In the light of unchanged direct systolic arterial pressure, the higher
tail blood pressures in the NPY-tg may reflect an increased pressor
responsiveness of these rats to some forms of stress. Tail blood
pressure determination involves restraining the animals, increased
ambient temperature, as well as probable pain induced by sudden tail
occlusion by the cuff. The higher tail blood pressure in NPY-tg rats is
consistent with the increased total vascular resistance and the
enhanced pressor responsiveness to norepinephrine observed in these
rats. Others have also reported higher tail blood pressures in the
spontaneously hypertensive rats and diabetic rats compared with direct
blood pressures (3, 10, 19). In the experiment reported
here, the fact that the higher tail blood pressures were observed in
more than one NPY transgenic line of rats and actually only in the
lines having more than two copies of the transgene, is important for
the validation of this model because it confirms that the increased
tail blood pressure in NPY-tg rats was specific to the transgene
product and not due to transgene integration site artifacts.
Increased pressor response to norepinephrine.
Pressor responsiveness to norepinephrine was significantly increased in
NPY-tg rats. Most likely, increased cardiac output did not contribute
to this effect, because these two genotypes clearly did not differ
regarding cardiac output and bradycardia responses to norepinephrine.
This suggestion is further supported by the findings of others
demonstrating that cardiac output does not seem to contribute
significantly to norepinephrine-induced blood pressure increases, at
least when moderate doses of the agonist are administered (13,
23). Thus our result suggests that genetic upregulation of NPY
enhances 
-adrenoceptor sensitivity occurring most likely at the
neurovascular synapse. These in vivo genetic data corroborate the
previous in vitro (isolated blood vessels) and in vivo (rat) findings,
where acutely administered NPY, by interacting with -adrenergic
transmission, enhanced vasoconstriction induced by sympathetic nerve
stimulation or by exogenous norepinephrine (4, 42-44,
46). A similar increase in vasoconstrictor responsiveness to
norepinephrine has been reported in a number of situations including
sympathetic denervation or treatment with adrenergic antagonists, in
spontaneously hypertensive rats, diabetes, or in hypertrophy of
vascular smooth muscles (1, 11, 32).
-adrenergic signaling
by increasing the accumulation of transducer proteins in the vascular
smooth muscles. This is a very likely possibility, because, as members
of the G protein-coupled receptor family, the vascular NPY receptors
share postreceptor signaling pathways with -adrenoceptors
(42-44, 46). Importantly, a similar postreceptor mechanism has been suggested to explain the enhanced vascular responsiveness to norepinephrine in diabetic rats (1).
Reduced hemorrhage-induced hypotension. NPY has been suggested to be involved in the compensatory response of the cardiovascular system to hemorrhagic hypotension (30, 37, 46). In NPY-tg rats, together with increased concentrations of NPY in a number of small blood vessels and organs, plasma levels of this peptide after hemorrhage were also higher than in the wild type. At the same time, hemorrhagic hypotension and bradycardia were significantly reduced in the NPY-tg rats. The faster rates of blood pressure recovery in the control animals could be related to the more dramatic drop in blood pressures in these animals. The more severe hemorrhagic hypotension in control animals could have triggered some additional compensatory mechanism that was not engaged in the NPY-tg rats. The present data suggest that the reduced hemorrhagic hypotension in NPY-tg rats was due to the increased NPY release and, thereby, provide direct evidence that endogenous NPY participates in the physiological reflex mechanism maintaining blood pressure in an acute hypovolemic state.
The mechanism producing this effect could involve the action of transgenic NPY both peripherally and in the central nervous system itself. Acting peripherally, NPY could have stimulated vasoconstriction directly via its own vascular receptors or indirectly by enhancing vascular responsiveness to other endogenous vasoconstrictors including norepinephrine, vasopressin, and angiotensin II (42-46). The reduced hemorrhagic hypotension in NPY-tg rats could also be attributed to the action of the excess NPY in the central nervous system. Hemorrhage has been shown to activate groups of neurons in several cardiovascular regions of the brain, which, at the same time, are either the sources or the targets of brain NPY-ergic innervation (2, 15). Because the transgene presumably contained all the regulatory elements of the rat NPY gene and because hemorrhage-induced plasma levels of NPY were significantly higher in the NPY-tg subjects, it is very likely that NPY overexpression had also occurred in the centers important in cardiovascular adaptation to blood loss. In view of the higher concentrations of NPY in the "vasopressinergic areas" of these NPY-tg rats, including the paraventricular nucleus, supraoptic nucleus, and the posterior pituitary, it is conceivable that central hemodynamic effects of transgenic NPY involved the interaction of NPY with the vasopressinergic signaling system. Vasopressin is known to contribute to maintaining arterial blood pressure after hemorrhage (12), and NPY has been demonstrated to have anatomical connections and stimulatory functional interactions with this neurohormone (16, 22, 43, 44, 46). The less pronounced drop in the heart rates together with the reduced hypotension after hemorrhage observed in NPY-tg rats suggest that upregulation of NPY resulted in increased baroreflex sensitivity. Arterial baroreceptor reflexes elicit short-term control over blood pressure after hemorrhage in the rat by increasing peripheral vascular tone and cardiac performance via autonomic effectors (38). NPY is expressed in the groups of neurons of the baroreflex arc (15). Likewise, administration of NPY either systemically or locally (into the nucleus tractus solitarius) increases the sensitivity of the aortic baroreceptor reflex in the rat (20, 29, 36). The present finding suggests that, by means of modifying baroreflex function, endogenous NPY is involved in cardiovascular responses that preserve cardiac output and blood pressure in hypovolemic states. Similar mechanisms to those observed after hemorrhage may operate during situations of vascular insensitivity to norepinephrine, e.g., in toxemia, fainting, vaso-vagal, or neurocardiogenic syncope. If so, specific NPY-receptor agonists may prove useful for the management of traumas accompanied by blood loss or in the treatment of those who suffer recurrent syncopal episodes of this type. In conclusion, we report two major findings in the present study. First, a genetically caused increase in expression of NPY in transgenic rats did not affect resting blood pressure, but it increased total vascular resistance and enhanced blood pressure responsiveness to norepinephrine. Second, genetically upregulated NPY played an important role in the spontaneous recovery of blood pressure after acute hypotensive hemorrhage, which is a specific hypovolemic state known to involve sympathetic nervous system activity. Altogether, these results provide direct genetic evidence that endogenous NPY is an important regulator of blood vessel tone and shed new light on the functional cooperation between NPY and other neurotransmitters in signal transmission to the cardiovascular system. The NPY-tg rats described here provide a good opportunity to study the involvement of endogenous NPY in long-term regulation of cardiovascular functions. Furthermore, when combined with specific NPY-receptor antagonists, this model will be particularly useful for the study of the physiological functions of the novel NPY receptors.Perspectives
It has been difficult to establish a physiological role for endogenous NPY in cardiovascular regulation, because this peptide is expressed in numerous areas of the nervous system and it acts through many types of receptors. Transgenic approaches offer an attractive model for such studies. The transgene in the present model is designed to assure excess NPY release in the locations and at the times the body requires it. Years of study of NPY indicate that this peptide is poised to regulate excitability in extreme physiological circumstances such as chronic stress, heart failure, epileptogenesis, or stroke. For this reason and also because of the model's feature described above, the NPY-overexpressing rats may provide a useful model for the longitudinal studies of the interaction between NPY and other signaling molecules in a number of physiological and disease models.The observed cardiovascular phenotypes in the NPY-tg rats are suggestive of reduced norepinephrine release. This indicates that coexisting with norepinephrine in the nerve terminals NPY may function as a buffering system for catecholamine release. This action could be particularly important in situations of chronic excessive release of norepinephrine. Such buffering action may protect cardiovascular and neuronal tissues from the adverse effect of excess norepinephrine. In combination with previously reported anticonvulsant and memory effects, such a buffering action of NPY could be expected to be involved in glutamatergic signaling. This gives rise to an attractive and verifiable hypothesis that genetic deficiencies in NPY signaling may be underlying causative factors in the etiology of some forms of hypertension, heart failure, stroke, or epilepsy and that targeting NPY receptors with specific agonists or antagonists can offer new possibilities for therapeutic interventions to treat these diseases.
| |
ACKNOWLEDGEMENTS |
|---|
We thank D. Larhammar for the NPY clones, S. Lilleberg for subcloning, J. Yun and K. Mohr for sharing experiences with pronuclear injections, M. Wilson, R. Keith, K. Knestaut, and S. Lee for technical help, A. Vezzani for the immunocytochemistry on the brain sections, J. E. Dumont, H. Lubon, G. Konat, M. Evans, and E. Bytchkova for helpful discussions, and J. Stewart for editing the manuscript.
| |
FOOTNOTES |
|---|
This research was supported by a grant from the American Heart Association West Virginia affiliate (to M. Puskar) and National Heart, Lung, and Blood Institute Grants HL-57921 (to M. Michalkiewicz) and HL-27781 and HL-59189 (to D. L. Kreulen).
Address for reprint requests and other correspondence: M. Michalkiewicz, Dept. of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Rd., PO Box 26509, Milwaukee, WI 53226-0509 (E-mail: mmichalk{at}mcw.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 14 June 2000; accepted in final form 23 March 2001.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
1.
Abebe, W,
and
MacLeod KM.
Enhanced arterial contractility to noradrenaline in diabetic rats is associated with increased phosphinositide metabolism.
Can J Physiol Pharmacol
69:
355,
1991[Web of Science][Medline].
2.
Badoer, E,
McKinley BJ,
Oldfield MJ,
and
McAllen RM.
Distribution of hypothalamic, medullary and lamina terminalis neurons expressing Fos after hemorrhage in conscious rats.
Brain Res
582:
323-328,
1992[Web of Science][Medline].
3.
Bunag, RD,
and
Teravainen TL.
Tail-cuff detection of systolic hypertension in different strains of ageing rats.
Mech Ageing Dev
59:
197-213,
1991[Web of Science][Medline].
4.
Dahlof, C,
Dahlof P,
and
Lundberg JM.
Neuropeptide Y enhancement of blood pressure increase upon -adrenoceptor activation and direct pressor effects in pithed rats.
Eur J Pharmacol
109:
289-292,
1985[Web of Science][Medline].
5.
Dey, M,
Michalkiewicz M,
Huffman LJ,
and
Hedge GA.
Sympathetic thyroidal vasoconstriction is not blocked by a neuropeptide Y antagonist or antiserum.
Peptides
14:
1179-1186,
1993[Web of Science][Medline].
6.
Doods, HN,
Wienen W,
Entzeroth M,
Rudolf K,
Eberlein W,
Engel W,
and
Wieland HA.
Pharmacological characterization of the selective nonpeptide neuropeptide Y Y1 receptor antagonist BIBP 3226.
J Pharmacol Exp Ther
275:
136-142,
1995
7.
Dube, MG,
Xu B,
Crowley WR,
Kalra PS,
and
Kalra SP.
Evidence that neuropeptide Y is a physiological signal for normal food intake.
Brain Res
646:
341-344,
1994[Web of Science][Medline].
8.
Erickson, JC,
Clegg KE,
and
Palmiter RD.
Sensitivity to leptin and susceptibility to seizures of mice lacking neuropeptide Y.
Nature
381:
415-418,
1996[Medline].
9.
Fenger-Gron, J,
Mulvany MJ,
and
Christensen KL.
Mesenteric blood pressure profile of conscious, freely moving rats.
J Physiol (Lond)
488:
753-760,
1995
10.
Ferrari, AU,
Daffonchio A,
Albergati F,
Bertoli P,
and
Mancia G.
Intra-arterial pressure alterations during tail-cuff blood pressure measurements in normotensive and hypertensive rats.
J Hypertens
8:
909-911,
1990[Web of Science][Medline].
11.
Friedman, JJ.
Vascular sensitivity and reactivity to norepinephrine in diabetes mellitus.
Am J Physiol Heart Circ Physiol
256:
H1134-H1138,
1989
12.
Fujisawa, Y,
Miyatake A,
Hayashida Y,
Aki Y,
Kimura S,
Tamaki T,
and
Abe Y.
Role of vasopressin on cardiovascular changes during hemorrhage in conscious rats.
Am J Physiol Heart Circ Physiol
267:
H1713-H1718,
1994
13.
Gray, R,
Shah PK,
Singh B,
Conklin C,
and
Matloff JM.
Low cardiac output states after open heart surgery. Comparative hemodynamic effects of dobutamine, dopamine, and norepinephrine plus phentolamine.
Chest
80:
16-22,
1981
14.
Hall, JE,
Schwinghamer JM,
and
Lalone B.
Mechanisms of blood vessel constriction during hemorrhage.
Am J Physiol
230:
569-578,
1976.
15.
Hokfelt, T,
Everitt BJ,
and
Fuxe K.
Transmitter and peptide systems in areas involved in the control of blood pressure.
Clin Exp Hypertens
A6:
23-41,
1984.
16.
Kagotani, Y,
Hisano S,
Tsuruo Y,
Daikoku S,
Okimura Y,
and
Chihara K.
Intragranular co-storage of neuropeptide Y and arginine vasopressin in the paraventricular magnocellular neurons of the rat hypothalamus.
Cell Tissue Res
262:
47-52,
1990[Web of Science][Medline].
17.
Karvonen, MK,
Pesonen U,
Koulu M,
Niskanen L,
Laakso M,
Rissanen A,
Dekker JM,
Hart LM,
Valve R,
and
Uusitupa MI.
Association of a leucine(7)-to-proline(7) polymorphism in the signal peptide of neuropeptide Y with high serum cholesterol and LDL cholesterol levels.
Nat Med
4:
1434-1437,
1998[Web of Science][Medline].
18.
Katsuya, T,
Higaki J,
Zhao Y,
Miki T,
Mikami H,
Serikawa T,
and
Ogihara T.
A neuropeptide Y locus on chromosome 4 cosegregates with blood pressure in the spontaneously hypertensive rat.
Biochem Biophys Res Commun
192:
261-267,
1993[Web of Science][Medline].
19.
Kohler, L,
Boillat N,
Luthi P,
Atkinson J,
and
Peters-Haefeli L.
Influence of streptozotocin-induced diabetes on blood pressure and on renin formation and release.
Naunyn Schmiedebergs Arch Pharmacol
313:
257-261,
1980[Web of Science][Medline].
20.
Kubo, T,
and
Kihara M.
Modulation of the aortic baroreceptor reflex by neuropeptide Y, neurotensin and vasopressin microinjected into the nucleus tractus solitarii of the rat.
Naunyn Schmiedebergs Arch Pharmacol
342:
182-188,
1990[Web of Science][Medline].
21.
Larhammar, D,
Ericsson A,
and
Persson H.
Structure and expression of the rat neuropeptide Y gene.
Proc Natl Acad Sci USA
84:
2068-2072,
1987
22.
Larsen, PJ,
Jukes KE,
Chowdrey HS,
Lightman SL,
and
Jessop DS.
Neuropeptide-Y potentiates the secretion of vasopressin from the neurointermediate lobe of the rat pituitary gland.
Endocrinology
134:
1635-1639,
1994
23.
Lorijn, RH,
and
Longo LD.
Norepinephrine elevation in the fetal lamb: oxygen consumption and cardiac output.
Am J Physiol Regulatory Integrative Comp Physiol
239:
R115-R122,
1980
24.
Lundberg, JM,
Rudehill A,
Sollevi A,
Theodorsson E,
and
Hamberger B.
Frequency and reserpine dependent chemical coding of sympathetic transmission: differential release of noradrenaline and neuropeptide Y from pig spleen.
Neurosci Lett
63:
96-100,
1986[Web of Science][Medline].
25.
Michalkiewicz, M,
Dey M,
Huffman LJ,
and
Hedge GA.
The neuropeptides, VIP and NPY, that are present in the thyroid nerves are not released into the thyroid vein.
Thyroid
8:
1073-1079,
1998.
26.
Michalkiewicz, M,
Huffman LJ,
Dey M,
and
Hedge GA.
Endogenous neuropeptide Y regulates thyroid blood flow.
Am J Physiol Endocrinol Metab
264:
E699-E705,
1993
27.
Michalkiewicz, M,
and
Michalkiewicz T.
Developing transgenic neuropeptide Y rats.
In: Methods in Molecular Biology: Neuropeptide Y Protocols, edited by Balasubramaniam A.. Totowa, NJ: Humana, 2000, p. 73-89.
28.
Millar, BC,
Weis T,
Piper HM,
Weber M,
Borchard U,
McDermott BJ,
and
Balasubramaniam AA.
Positive and negative contractile effects of neuropeptide Y on ventricular cardiomyocytes.
Am J Physiol Heart Circ Physiol
261:
H1727-H1733,
1991
29.
Minson, RB,
McRitchie RJ,
and
Chalmers JP.
Effects of neuropeptide Y on baroreflex control of heart rate and myocardial contractility in conscious rabbits.
Clin Exp Pharmacol Physiol
17:
39-49,
1990[Web of Science][Medline].
30.
Morris, M,
Kapoor V,
and
Chalmers J.
Plasma neuropeptide Y concentration is increased after hemorrhage in conscious rats: relative contributions of sympathetic nerves and the adrenal medulla.
J Cardiovasc Pharmacol
9:
541-545,
1987[Web of Science][Medline].
31.
Mulvany, MJ,
and
Aalkjaer C.
Structure and function of small arteries.
Physiol Rev
70:
921-961,
1990
32.
Oriowo, MA,
Bevan JA,
and
Bevan RD.
Variation in sensitivity of six cat and six rat arteries to norepinephrine can be related to differences in agonist affinity and receptor reserve.
J Pharmacol Exp Ther
251:
16-20,
1989
33.
Paul, M,
Wagner J,
Hoffmann S,
Urata H,
and
Ganten D.
Transgenic rats: new experimental models for the study of candidate genes in hypertension research.
Annu Rev Physiol
56:
811-829,
1994[Web of Science][Medline].
34.
Paxinos, G,
and
King C.
The Rat Brain (4th ed.). New York: Academic, 1998.
35.
Pinkert, CA.
Transgenic Animal Technology. New York: Academic, 1994.
36.
Potter, EK,
and
McCloskey DI.
Peptide YY reduces effects of sympathetic nerves and neuropeptide Y on cardiac vagal action.
Peptides
12:
805-808,
1991[Web of Science][Medline].
37.
Qureshi NU, Dayao EK, Shirali S, Zukowska-Grojec Z, and Hauser GJ.
Endogenous neuropeptide Y mediates vasoconstriction during endotoxic
and hemorrhagic shock. Regul Pept 2: 75-76 and
215-220, 1998.
38.
Schadt, JC,
and
Ludbrook J.
Hemodynamic and neurohumoral responses to acute hypovolemia in conscious mammals.
Am J Physiol Heart Circ Physiol
260:
H305-H318,
1991
39.
Schwarzer, C,
Williamson JM,
Lothman EW,
Vezzani A,
and
Sperk G.
Somatostatin, neuropeptide Y, neurokinin B and cholecystokinin immunoreactivity in two chronic models of temporal lobe epilepsy.
Neuroscience
69:
831-845,
1995[Web of Science][Medline].
40.
Solt, VB,
Brown MR,
Kennedy B,
Kolterman OG,
and
Ziegler MG.
Elevated insulin, norepinephrine, and neuropeptide Y in hypertension.
Am J Hypertens
3:
823-828,
1990[Web of Science][Medline].
41.
Thorsell, A,
Michalkiewicz M,
Dumont Y,
Quirion R,
Caberlotto L,
Rimondini R,
Mathé A,
and
Heilig M.
Behavioral insensitivity to restraint stress, absent fear suppression of behavior and impaired spatial learning in neuropeptide Y transgenic rats.
Proc Natl Acad Sci USA
97:
12852-12857,
2000
42.
Wahlestedt, C,
Hakanson R,
Vaz CA,
and
Zukowska-Grojec Z.
Norepinephrine and neuropeptide Y vasoconstrictor cooperation in vivo and in vitro.
Am J Physiol Regulatory Integrative Comp Physiol
258:
R736-R742,
1990
43.
Westfall, TC,
Carpentier S,
Chen X,
Beinfeld MC,
Naes L,
and
Meldrum MJ.
Prejunctional and postjunctional effects of neuropeptide Y at the noradrenergic neuroeffector junction of the perfused mesenteric arterial bed of the rat.
J Cardiovasc Pharmacol
10:
716-722,
1987[Web of Science][Medline].
44.
Westfall, TC,
Han SP,
Knuepfer M,
Martin J,
Chen X,
Del Valle K,
Ciarleglio A,
and
Naes L.
Neuropeptides in hypertension: role of neuropeptide Y and calcitonin gene related peptide.
Br J Clin Pharmacol
30, Suppl:
75S-82S,
1990.
45.
Zukowska-Grojec, Z,
Dayao EK,
Karwatowska-Prokopczuk E,
Hauser GJ,
and
Doods HN.
Stress-induced mesenteric vasoconstriction in rats is mediated by neuropeptide Y Y1 receptors.
Am J Physiol Heart Circ Physiol
270:
H796-H800,
1996
46.
Zukowska-Grojec, Z,
and
Wahlestedt C.
Origin and actions of NPY in the cardiovascular system.
In: The Biology of Neuropeptide Y and Related Peptides, edited by Colmers WF,
and Wahlestedt C.. Totowa, NJ: Humana, 1993, p. 315-389.
This article has been cited by other articles:
|
|
 |
|
  E. Y. Callanan, E. W. Lee, J. U. Tilan, J. Winaver, A. Haramati, S. E. Mulroney, and Z. Zukowska Renal and cardiac neuropeptide Y and NPY receptors in a rat model of congestive heart failure Am J Physiol Renal Physiol, December 1, 2007; 293(6): F1811 - F1817. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Michalkiewicz, T. Michalkiewicz, A. M. Geurts, R. J. Roman, G. R. Slocum, O. Singer, D. Weihrauch, A. S. Greene, M. Kaldunski, I. M. Verma, et al. Efficient transgenic rat production by a lentiviral vector Am J Physiol Heart Circ Physiol, July 1, 2007; 293(1): H881 - H894. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Michalkiewicz, T. Michalkiewicz, R. A. Ettinger, E. A. Rutledge, J. M. Fuller, D. H. Moralejo, B. Van Yserloo, A. J. MacMurray, A. E. Kwitek, H. J. Jacob, et al. Transgenic rescue demonstrates involvement of the Ian5 gene in T cell development in the rat Physiol Genomics, October 4, 2004; 19(2): 228 - 232. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. M. Berglund, P. A. Hipskind, and D. R. Gehlert Recent Developments in Our Understanding of the Physiological Role of PP-Fold Peptide Receptor Subtypes Experimental Biology and Medicine, March 1, 2003; 228(3): 217 - 244. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. E. Lohmeier Neurohumoral regulation of arterial pressure in hemorrhage and heart failure Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2002; 283(4): R810 - R814. [Full Text] [PDF]
|
|
|
|
|
|
G. F. DiBona Neuropeptide Y Am J Physiol Regulatory Integrative Comp Physiol, March 1, 2002; 282(3): R635 - R636. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
