Cardiovascular effects of neuropeptide Y in the nucleus tractus solitarius of rats: relationship with noradrenaline and vasopressin

The central haemodynamic effects of neuropeptide Y (NPY), both alone and together with either noradrenaline (NA) or vasopressin (AVP), have been investigated by microinjecting synthetic peptide into the nucleus tractus solitarius (NTS) of anaesthetized rats. NPY alone elicited dose-dependent changes in blood pressure (BP) and heart rate (HR); 470 fmol inducing a pressor response, and 4.7 pmol a fall in BP. The hypotensive response to 20 nmol NA was significantly modified by both simultaneous and prior injection of an ineffective dose (47 fmol) of NPY. Prior injection of a similar dose of NPY also modified the NTS pressor effect of 10 ng AVP. A relationship between the action of AVP and NPY in the NTS was further indicated by the finding that prior injection of an ineffective dose of AVP (1 ng) reduced the hypotensive response to 4.7 pmol NPY, and by the demonstration of contrasting effects of 4.7 pmol NPY in AVP-deficient Brattleboro rats compared to parent strain LE rats. These results, taken together with the recent localization of NPY-like immunoreactivity in the NTS, suggest a role for NPY in central cardiovascular control. In addition, NPY has been shown to exhibit functional interactions with both an amine neurotransmitter and a neuropeptide present in the NTS of rats.     

-MSH exhibits opioid antagonist-like effects in the brainstem of rats

Unilateral microinjections of -MSH (0.3, 1.2 and 12 pmol) into the nucleus tractus solitarius (NTS) of urethane-anaesthetized rats did not modify blood pressure or heart rate (HR). Using a dual microinjection technique, it has been shown that prior injection of -MSH (0.3 pmol) attenuated the pressor effect of a similar injection of dynorphin 1–9 (18 pmol) but did not modify the cardiovascular effects of [Met]enkephalin (14 pmol). Since -MSH has been localized in the NTS, the results indicate that this peptide may play a role in central cardiovascular control, possibly acting in an antagonistic manner to the endogenous opioid peptides.     

Differential cardiovascular effects mediated by mu and kappa opiate receptors in hindbrain nuclei

To further investigate the role of opioid peptides and specific opiate receptor subtypes in central cardiovascular regulation by hindbrain nuclei, mu (D-Ala²,MePhe⁴,Gly-ol⁵ enkephalin, DAGO), delta (D-Ala²,D-Leu⁵ enkephalin, DADL) or kappa (MRZ 2549) agonists were microinjected into hindbrain nuclei of spontaneously or artificially respired, pentobarbital-anesthetized rats. In the nucleus tractus solitarius (NTS), DAGO and DADL (0.3 nmol) elicited pressor responses and tachycardia. MRZ (3.0–16 nmol) depressed blood pressure in spontaneously breathing rats, but accelerated heart rate in artificially ventilated animals. Blood pressure and heart rate of spontaneously breathing animals were not altered following nucleus ambiguus (NA) injection of DAGO or DADL (0.3 nmol), but were elevated in artificially respired animals; MRZ (3.0–10 nmol) injected into the NA depressed blood pressure in both groups. These data suggest that in the absence of respiratory depression, NTS and NA mu receptors mediate pressor responses and tachycardia; kappa receptors in the NA mediate a decrease in blood pressure but cardioacceleration in the NTS.     

Cardiorespiratory effects of μ and δ opiate agonists following third or fourth ventricular injections

The cardiorespiratory effects of prototype μ (morphine and β-casomorphine 1–4) and δ (D-Ala²-D-Leu⁵Enkephalin—DADLE) opioid ligands were compared following microinjection into third and fourth ventricular spaces in conscious and anesthetized rats. The direction of change in arterial pressure produced by ventricular opioid injections varied according to ligand, site of administration, and state of consciousness of the animal. In general, pentobarbital anesthesia blocked or reversed the pressor response to these opiate agonists; depressor responses became magnified following pentobarbital. Qualitatively, the predominant effect of third ventricular DADLE in anesthetized rats was to produce a depression of arterial pressure and pulse pressure, suggesting an involvement of hypothalamic δ opioid receptors in decreasing sympathetic outflow. By contrast, morphine exerted pronounced bradycardic effects following fourth ventricular administration, suggesting an action at μ opioid receptors which influence vagal parasympathetic activity. Both ligands lowered respiratory rates upon fourth ventricular injection, indicating a possible involvement of either opioid receptor subtype in the depression of brainstem respiratory centers. These depressant effects of opioids upon cardiorespiratory function were readily reversed by naloxone. The qualitative similarity between the cardiovascular effects of third ventricular DADLE administration and various forms of circulatory shock may indicate that both phenomena involve delta opioid receptors at hypothalamic sites.     

Effects of Chronic Morphine Treatment on β-Endorphin-Related Peptides in the Caudal Medulla and Spinal Cord

Abstract: The effects of chronic morphine treatment on β-endorphin (βE)-immunoreactive (βE-ir) peptide levels were determined in the rat caudal medulla and different areas of the spinal cord. Seven days of morphine pelleting had no effect on total βE-ir peptides in the caudal medulla. In contrast, it significantly increased βE-ir peptide concentrations in the cervical and thoracic regions of the spinal cord compared with placebo-pelleted controls, whereas in the lumbosacral region this trend did not reach statistical significance. Injections of the opiate receptor antagonist naloxone 1 h before the rats were killed had no effect on the morphine-induced increases in the cord. Chromatographic analyses revealed that enzymatic processing of βE-related peptides in the spinal cord seemed unaffected by the morphine and/or naloxone treatments. In light of previous data showing that morphine down-regulates βE biosynthesis in the hypothalamus, the present results suggest that the regulation of βE-ir peptides in the spinal cord is distinct from that found in other CNS areas. These data provide support for previous results suggesting that βE-expressing neurons may be intrinsic to the spinal cord.     

Comparative effects on blood pressure and heart rate of dynorphin A(1–13) in anterior hypothalamic area, posterior hypothalamic area, nucleus tractus solitarius, and lateral cerebral ventricle in the rat

The objective of this study was to explore the effects of the endogenous opioid peptide dynorphin A(1–13) on the CNS regulation of blood pressure and heart rate. Wistar rats, anesthetized with pentobarbital and halothane, received dynorphin A(1–13) microinjected into the anterior hypothalamus area (AHA), the posterior hypothalamic area (PHA), the nucleus tractus solitarius (NTS), or the lateral cerebral ventricle (ICV). Dynorphin A(1–13), 20 (12 nmol) or 30 μg ICV, produced significant (p < 0.05) reductions in blood pressure and heart rate. Naloxone, 50 μg/kg ICV, completely prevented the blood pressure response and significantly (p < 0.05) blunted the heart rate response to the highest dynorphin concentration, 30 μg ICV (18 nmol). Dynorphin A(1–13), 5 μg, in the NTS significantly (p < 0.05) decreased systolic and diastolic blood pressure and heart rate with the response being evident 10 min and persisting for 30 min after injection. In contrast, the same dose of dynorphin A(1–13) in the AHA produced an immediate, marked, and significant (p < 0.05) decrease in systolic and diastolic blood pressure and heart rate that attained its maximum 1–3 min and returned rapidly towards baseline levels. Dynorphin A(1–13), 5 or 10 μg in the posterior hypothalamic area, was not associated with any change in blood pressure or heart rate. Injection of the diluent at any site was not associated with any changes in blood pressure or heart rate. The maximum change in blood pressure with dynorphin was greater in the AHA than NTS, and the maximum change in heart rate was greater in the NTS than AHA. These data indicate a potential role for dynorphin as a modulator of the CNS regulation of blood pressure and cardiac rate, and this is mediated in part through different areas in the brain that maybe localized to the anterior hypothalamic area and nucleus tractus solitarius but not the posterior hypothalamic area.     

Central cardiovascular effects of mammalian neurohypophyseal peptides in conscious rats

To confirm and extend the results of previous studies which demonstrated central cardiovascular effects of vasopressin in anesthetized rats, we determined blood pressure and heart rate changes for 30 minutes after intracerebroventricular injections of arginine vasopressin, arginine vasotocin and oxytocin in conscious rats. As compared to sham injections, significantly greater increases in either systolic or diastolic blood pressure were noted over the 30 minutes which followed the injection of 0.15, 1.0 or 10.0 nM of either vasopressin or vasotocin. In animals given vasopressin, plasma levels of the peptide were determined. There was a substantial increase in plasma vasopressin only after the highest dose. Overall blood pressure responses to doses of oxytocin as high as 100 nM were not significantly different than sham injections. Heart rate following both vasopressin and vasotocin was increased at 0.15 nM, was initially decreased then increased at 1.0 nM and was substantially decreased after the 10.0 nM dose. There was a significant increase in heart rate at the 10.0 nM and 100 nM doses of oxytocin. Dose response curves for systolic blood pressure and heart rate 20 minutes after injection were similar for vasopressin and vasotocin. We conclude that arginine vasopressin has significant central pressor and tachycardic effects in conscious rats, and it is related, at least in part, to the tail structure of the peptide, which is shared with arginine vasotocin.     

Substance P binding sites in the nucleus tractus solitarius of the cat

Substance P binding sites in the nucleus tractus solitarius were visualized with receptor autoradiography using Bolton-Hunter [¹²⁵I]substance P. Substance P binding sites were found to have distinct patterns within the cat nucleus tractus solitarius. The majority of substance P binding sites were present in the medial, intermediate and the peripheral rim of the parvocellular subdivisions. Lower amounts of substance P binding sites were present in the commissural, ventrolateral, interstitial and dorsolateral subdivisions. No substance P binding sites were present in the central region of the parvocellular subdivision or the solitary tract. The localization of substance P binding sites in the nucleus tractus solitarius is very similar to the patterns of substance P immunoreactive fibers previously described for this region. Results of this study add further support for a functional role of substance P in synaptic circuits of the nucleus tractus solitarius.     

Substance P as a baro- and chemoreceptor afferent neurotransmitter: immunocytochemical and neurochemical evidence in the rat

The possibility that substances P (SP) is a neurotransmitter of baro- and chemoreceptor afferents in the rat was investigated. SP-like immunoreactivity (SP-I) was analyzed quantitatively by radioimmunoassay in various levels of the nucleus tractus solitarius (NTS), the site of termination of these afferents while SP-containing afferent neurons were studied in various portions of the peripheral pathways by immunocytochemistry. It was found that the NTS contained significant amounts of SP-I and that unilateral removal of the nodose ganglia reduces the SP-I content of those portions of the NTS known to receive vagal afferents. In addition, SP-I was visualized in discrete fibers in the tunica adventitia of the aortic arch and carotid sinus regions, the vagus nerve and nodose ganglia. These results in the rat are consistent with our previous studies in the cat and provide further evidence that SP is contained within baro- and chemoreceptor afferent nerves.     

Regional interactions of opioid peptides at μ and δ sites in rat brain

Opiate binding sites in five brain regions were labeled with the μ and δ markers, ³H-morphine and ³H-[D-Ala²,D-leu⁵]enkephalin, respectively. The highest densities of both ³H-morphine and ³H-DADLE labeled sites are found in striatum and frontal cortex. Hypothalamus and midbrain contain predominantly ³H-morphine labeled sites. The selectivity of the opioid peptides [D-Ala²,D-leu⁵]enkephalin, β-endorphin and dynorphin(1–13) for the two opiate sites was investigated by comparing the potency of these unlabeled compounds against the μ and δ markers in different brain regions. This determination has the effect of controlling for the breakdown of peptides within each region. While the enkephalin analogue shows a preference for the δ binding site and β-endorphin is more nearly equipotent towards the two binding sites, dynorphin(1–13) shows a high affinity and selective preference for the μ binding site over the δ site. The potency of the opioid peptides in displacing the μ and δ markers varies from region to region according to the relative densities of the two opiate binding site populations.     

Evidence for Presynaptic Adenosine A2a Receptors Associated with Norepinephrine Release and Their Desensitization in the Rat Nucleus Tractus Solitarius

Abstract: Rat medullary brain segments containing primarily nucleus tractus solitarius (NTS) were used for superfusion studies of evoked transmitter release and for isotherm receptor binding assays. Isotherm binding assays with [³H]CGS-21680 on membranes prepared from NTS tissue blocks indicated a single high-affinity binding site with a K_D of 5.1 ± 1.4 nM and a B_max of 20.6 ± 2.4 fmol/mg of protein. The binding density for [³H]CGS-21680 on NTS membranes was 23 times less than comparable binding on membranes from striatal tissue. Electrically stimulated (1 min at 25 mA, 2 ms, 3 Hz) release of [³H]norepinephrine ([³H]NE) from 400-µm-thick NTS tissue slices resulted in an S₂/S₁ ratio of 0.96 ± 0.02. Superfusion of single tissue slices with 0.1–100 nM CGS-21680, a selective adenosine A_2a receptor agonist, for 5 min before the S₂ stimulus produced a significant concentration-dependent increase in the S₂/S₁ fractional release ratio that was maximal (31.3% increase) at 1.0 nM. However, superfusion of tissue slices with CGS-21680 over the same concentration range for 20 min before the S₂ stimulus did not alter the S₂/S₁ ratio significantly from control release ratios. The augmented release of [³H]NE mediated by 1.0 nM CGS-21680 with a 5-min tissue exposure was abolished by 1.0 and 10 nM CGS-15943 as well as by 100 nM 8-(3-chlorostyryl)caffeine, both A_2a receptor antagonists, but not by 1.0 nM 8-cyclopentyl-1,3-dipropylxanthine, the A₁ receptor antagonist. Taken together, these results suggest that CGS-21680 augmented the evoked release of [³H]NE in the NTS via activation of presynaptic A_2a receptors within the same concentration range as the binding affinity observed for [³H]CGS-21680. It was also apparent that this population of presynaptic adenosine A_2a receptors in the NTS desensitized within 20 min because the augmenting action of CGS-21680 on evoked transmitter release was not evident at the longer interval.     

Increases in heart rate and blood pressure produced by microinjections of atrial natriuretic factor into the AV3V region of rat brain

Recent studies have provided evidence for the dense localization of atrial natriuretic factor (ANF) in the anteroventral third ventricle (AV3V) region of the rat brain. This area is currently thought to be involved in the regulation of blood pressure and fluid and electrolyte balance. To investigate whether ANF may play a role in central cardiovascular regulation, the effects of microinjection of ANF into the preoptic suprachiasmatic nucleus (POSC), which is located in the AV3V region of the brain, were examined in the present study. Low doses of ANF (2–4 pmol) produced modest elevations in systolic and diastolic pressures, approximately 10–14%, and a small rise in HR of roughly 7%. Higher doses of ANF (20–40 pmol) produced significant increases in systolic (15–19%), mean arterial (12–14%) and pulse (25–36%) pressures. In addition, much larger increases in HR, approximately 20%, were produced by these higher doses of ANF. The onset of effects produced by ANF on BP and HR was seen 15–45 min after injection. Peak effects were usually observed approximately 60–150 min after onset, and the duration of the effect was 2–4 hours, after which time values usually returned to baseline. These studies indicate that ANF produces significant increases in BP and HR when injected at pmol doses into the POSC, and lends support to the idea that this peptide may play an important role in central cardiovascular regulatory mechanisms.     

Participation of capsaicin-sensitive neurons in the cardiovascular effects of neurotensin in guinea pigs

Intravenous (IV) infusions of neurotensin (NT) in anesthetized guinea pigs elicited dose-dependent pressor effects and tachycardia. Both effects were significantly reduced or abolished in guinea pigs given a chronic treatment with the neurotoxin capsaicin. In guinea pig isolated atria NT evoked a positive inotropic and chronotropic effect. Both effects were completely abolished in atria derived from capsaicin-treated guinea pigs. The positive inotropic and chronotropic effects of NT in guinea pig atria were mimicked by capsaicin and calcitonin gene-related peptide (CGRP). These results were interpreted as an indication that NT produces its cardiovascular effects in guinea pigs by activating capsaicin-sensitive sensory neurons.     

In vivo measurements of the cerebral perfusion and cardiovascular effects of the novel guanidine ME10092 in the non-human primate, Papio ursinus

The novel guanidines N-(3,4-dimethoxy-2-chlorobenzylideneamino)-guanidine (ME 10092) and N-(3,4-dimethoxy-2-chlorobenzylideneamino)-N1-hydroxyguanidine (PR5) were recently reported to exhibit promising cardioprotective activities in myocardial ischaemia and reperfusion in rats. The current study investigated for the first time pharmacological effects of ME10092 in the primate, viz. the Cape baboon Papio ursinus. The effects of ME10092 (1 and 2 mg/kg doses) on the cerebral blood flow, heart rates and the systolic and diastolic blood pressure were investigated after intravenous injection to the baboon under anaesthesia. The cerebral perfusion effects of ME10092 were assessed using Single Photon Emission Computed Tomography according to the split-dose approach and 99mTc-hexamethyl-propylene amine oxime as brain perfusion tracer. The observation that the recovery times from the anaesthesia were unacceptably prolonged excluded doses beyond 2 mg/kg. The data indicate that no cerebral perfusion changes were induced at both the 1 and 2 mg/kg doses of ME10092. Both these doses of ME10092 showed blood pressure and heart rate effects, with the latter being more significant. Decreases in heart rate were seen directly after ME10092 administration reaching levels of about 20% for the 2 mg/kg dose and about 15% for the 1 mg/kg dose at around 6 min post drug administration. A transient decrease in both systolic and diastolic blood pressure was observed for the higher dose. The blood pressure data further suggest an attenuation of the anaesthesia induced increase in pressure usually present in non-intervention studies. ME10092 clearly exhibits mycocardial effects in the non-human primate, similar to the effects previously observed in the ischaemia-reperfusion rat model, where ME10092 showed strong protection.     

N-α-biotinylated-neuropeptide Y analogs: Syntheses, cardiovascular properties, and application to cardiac NPY receptor visualization

Two monobiotinylated analogs of neuropeptide Y (NPY) were synthesized by coupling the N-hydroxysuccinimidyl esters of biotin and (6-biotinylamido)-hexanoic acid, respectively, to the free alpha-NH2 group of the side chain protected NPY peptide resin. Crude peptides obtained by HF cleavage were purified by RPLC and their integrities were confirmed by amino acid and mass spectral analysis. As with NPY, both biotinylated analogs inhibited 125I-NPY binding and adenylate cyclase activity of rat cardiac ventricular membranes in a dose-dependent manner. N-alpha-[(6-biotinylamido)-hexanoyl]-NPY exhibited potencies comparable to that of NPY whereas N-alpha-biotinyl-NPY was slightly less potent. In the in vivo experiments, however, both the biotinylated analogs exhibited responses comparable to NPY in increasing arterial blood pressure and decreasing heart rate in anesthetized rats. The responses of the biotinyl analogs were longer lasting than those of NPY. Histochemical studies revealed that N-alpha-[(6-biotinylamido)-hexanoyl]-NPY could label the NPY receptors in rat cardiac ventricular tissues. This labeling was specific since intact NPY inhibited the staining. These studies show that biotinyl-NPY analogs exhibit biological potencies comparable to intact NPY and can therefore be used to further probe the NPY-receptor interaction.     

Integration of autonomic and local mechanisms in regulating cardiovascular responses to heating and cooling in a reptile (<Emphasis Type="Italic">Crocodylus porosus</Emphasis>)

Reptiles change heart rate and blood flow patterns in response to heating and cooling, thereby decreasing the behavioural cost of thermoregulation. We tested the hypothesis that locally produced vasoactive substances, nitric oxide and prostaglandins, mediate the cardiovascular response of reptiles to heat. Heart rate and blood pressure were measured in eight crocodiles (Crocodylus porosus) during heating and cooling and while sequentially inhibiting nitric-oxide synthase and cyclooxygenase enzymes. Heart rate and blood pressure were significantly higher during heating than during cooling in all treatments. Power spectral density of heart rate and blood pressure increased significantly during heating and cooling compared to the preceding period of thermal equilibrium. Spectral density of heart rate in the high frequency band (0.19–0.70 Hz) was significantly greater during cooling in the saline treatment compared to when nitric-oxide synthase and cyclooxygenase enzymes were inhibited. Cross spectral analysis showed that changes in blood pressure preceded heart rate changes at low frequencies (<0.1 Hz) only. We conclude that the autonomic nervous system controls heart rate independently from blood pressure at higher frequencies while blood pressure changes determine heart rate at lower frequencies. Nitric oxide and prostaglandins do not control the characteristic heart rate hysteresis response to heat in C. porosus, although nitric oxide was important in buffering blood pressure against changes in heart rate during cooling, and inhibition caused a compensatory decrease in parasympathetic stimulation of the heart.     

Speaking from the heart: cardiovascular components of stress rating changes and the relative reactivity of physiological and psychological variables

The first part of the experiment compared the relative reactivity of various cardiovascular measurements and self-reported assessment of mental arithmetic, an active coping stress task. It was found that the self-reported stres rating was more responsive (student's t=9.4) than the physiological measurements. Among the cardiovascular indices, heart rate was the most responsive (student's t=6.5), followed by the interval between R-wave and the maximal systolic pressure (student's t=5.0). The second part of the study used a multiple regression to examine the cardiovascular components of change in stres rating following mental arithmetic. Both resting (psychophysiological trait varibles) and change under stress (psychophysiological state variables) were used to predict change in stress rating. Stress rating increase were associated with greater maximal systolic pressure rate of rise (finger dP/dt) at baseline and less heart rate increase during mental arithmetic, yielding a multiple r of 0.67. The dual autonomic nature of active coping stress response is discussed. The shortcomings of using a correlate of a correlate are noted.     

TRH: Cardiovascular and sympathetic modulation in brain nuclei of the rat

The cardiovascular and sympathetic effects of TRH in discrete cardiovascular-related brain nuclei were studied. Microinjections of TRH were made into the nucleus preopticus medialis (POM) of conscious rats and the nucleus tractus solitarius (NTS) of pentobarbitone-anesthetized, artificially respired rats. POM injections (1 μl, 0.8–80 nM) elicited dose dependent pressor and tachycardic responses which were accompanied by increased levels of norepinephrine (NE) and epinephrine (EPI) in the plasma. These pressor/tachycardic effects of TRH were also elicited in adrenal demedullated (ADM-x) rats, but completely abolished in ADM-x rats pretreated with bretylium (30 mg/kg, IA). NTS injections (0.1 μl, 30 and 150 nM) had a short depressor effect on blood pressure (BP) and a delayed increase in heart rate (HR). From these findings we suggest that the POM, a central nucleus in the AV3V region, may be an important forebrain site for autonomic regulation by TRH, mediated through the sympathetic nervous system.