Cardiac enkephalins interrupt vagal bradycardia via delta 2-opioid receptors in sinoatrial node

Local cardiac opioids appear to be important in determining the quality of vagal control of heart rate. Introduction of the endogenous opioid methionine-enkephalin-arginine-phenylalanine (MEAP) into the interstitium of the canine sinoatrial node by microdialysis attenuates vagally mediated bradycardia through a delta-opioid receptor mechanism. The following studies were conducted to test the hypothesis that a delta(2)-opiate receptor subtype mediates the interruption of vagal transmission. Twenty mongrel dogs were anesthetized and instrumented with microdialysis probes inserted into the sinoatrial node. Vagal frequency responses were performed at 1, 2, and 3 Hz during vehicle infusion and during treatment with the native agonist MEAP, the delta(1)-opioids 2-methyl-4aa-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12aalpha-octahydroquinolino[2,3,3- g]isoquinoline (TAN-67) and [d-pen(2,5)]-enkephalin (DPDPE), and the delta(2) opioid deltorphin II. The vagolytic effects of intranodal MEAP and deltorphin were then challenged with the delta(1)- and delta(2)-opioid receptor antagonists 7-benzylidenenaltrexone (BNTX) and naltriben, respectively. Although the positive control deltorphin II was clearly vagolytic in each experimental group, TAN-67 and DPDPE were vagolytically ineffective in the same animals. In contrast, TAN-67 improved vagal bradycardia by 30-35%. Naltriben completely reversed the vagolytic effects of MEAP and deltorphin. BNTX was ineffective in this regard but did reverse the vagal improvement observed with TAN-67. These data support the hypothesis that the vagolytic effect of the endogenous opioid MEAP was mediated by delta(2)-opioid receptors located in the sinoatrial node. These data also support the existence of vagotonic delta(1)-opioid receptors also in the sinoatrial node.     

The monosialosyl ganglioside GM-1 reduces the vagolytic efficacy of delta2-opioid receptor stimulation

The cardiac enkephalin, methionine-enkephalin-arginine-phenylalanine (MEAP), alters vagally induced bradycardia when introduced by microdialysis into the sinoatrial (SA) node. The responses to MEAP are bimodal; lower doses enhance bradycardia and higher doses suppress bradycardia. The opposing vagotonic and vagolytic effects are mediated, respectively, by delta(1) and delta(2) phenotypes of the same receptor. Stimulation of the delta(1) receptor reduced the subsequent delta(2) responses. Experiments were conducted to test the hypothesis that the delta-receptor interactions were mediated by the monosialosyl ganglioside GM-1. When the mixed agonist MEAP was evaluated after nodal GM-1 treatment, delta(1)-mediated vagotonic responses were enhanced, and delta(2)-mediated vagolytic responses were reduced. Prior treatment with the delta(1)-selective antagonist 7-benzylidenaltrexone (BNTX) failed to prevent attrition of the delta(2)-vagolytic response or restore it when added afterward. Thus the GM-1-mediated attrition was not mediated by delta(1) receptors or increased competition from delta(1)-mediated vagotonic responses. When GM-1 was omitted, deltorphin produced a similar but less robust loss in the vagolytic response. In contrast, however, to GM-1, the deltorphin-mediated attrition was prevented by pretreatment with BNTX, indicating that the decline in response after deltorphin alone was mediated by delta(1) receptors and that GM-1 effectively bypassed the receptor. Whether deltorphin has intrinsic delta(1) activity or causes the release of an endogenous delta(1)-agonist is unclear. When both GM-1 and deltorphin were omitted, the subsequent vagolytic response was more intense. Thus GM-1, deltorphin, and time all interact to modify subsequent delta(2)-mediated vagolytic responses. The data support the hypothesis that delta(1)-receptor stimulation may reduce delta(2)-vagolytic responses by stimulating the GM-1 synthesis.     

Repeated delta1-opioid receptor stimulation reduces delta2-opioid receptor responses in the SA node

Ultra-low-dose methionine-enkephalin-arginine-phenylalanine improves vagal transmission (vagotonic) and decreases heart rate via delta(1)-opioid receptors within the sinoatrial (SA) node. Higher doses activate delta(2)-opioid receptors, interrupt vagal transmission (vagolytic), and reduce the bradycardia. Preconditioning-like occlusion of the nodal artery produced a vagotonic response that was reversed by the delta(1)-antagonist 7-benzylidenaltrexone (BNTX). The following study tested the hypothesis that extended delta(1)-opioid receptor stimulation reduces subsequent delta(2)-receptor responses. The delta(2)-agonist deltorphin II was introduced in the SA node by microdialysis to evaluate delta(2) responses before and after infusion of the delta(1)-agonist TAN-67. TAN-67 reduced the vagolytic effect of deltorphin by two-thirds. When the delta(1)-antagonist BNTX was combined with TAN-67, the deltorphin response was preserved, suggesting that attrition of the prior response was mediated by delta(1) activity. When TAN-67 was omitted in time control studies, some loss of delta(2) responses was apparent in the absence of the delta(1) treatment. This loss was also eliminated by BNTX, suggesting that the attenuation of the response after deltorphin alone was also the result of delta(1) activity. Additional studies tested TAN-67 alone in the absence of prior deltorphin. When time controls were conducted without the initial deltorphin treatment, a robust vagolytic response was observed. When TAN-67 preceded the delayed deltorphin, the vagolytic response was eroded, indicating an independent effect of TAN-67. BNTX infused afterward was unable to restore the delta(2) response. These data support the conclusion that the loss of the delta(2) response resulted from reduced delta(2) activity mediated by continued delta(1)-receptor stimulation and not the arithmetic consequence of increased competition from that same delta(1) receptor.     

Leucine-enkephalin interrupts sympathetically mediated tachycardia prejunctionally in the canine sinoatrial node

This study examined the role of leucine-enkephalin (LE) in the sympathetic regulation of the cardiac pacemaker. LE was administered by microdialysis into the interstitium of the canine sinoatrial node during either sympathetic nerve stimulation or norepinephrine infusion. In study one, the right cardiac sympathetic nerves were isolated as they exit the stellate ganglion and were stimulated to produce graded (low, 20-30 bpm; high 40-50 bpm) increases in heart rate (HR). LE (1.5 nmoles/min) was added to the dialysis inflow and the sympathetic stimulations were repeated after 5 and 20 min of LE infusion. After 5 min, LE reduced the tachycardia during sympathetic stimulation at both low (18.2 +/- 1.3 bpm to 11.4 +/- 1.4 bpm) and high (45 +/- 1.5 bpm to 22.8 +/- 1.5 bpm) frequency stimulations. The inhibition was maintained during 20 min of continuous LE exposure with no evidence of opioid desensitization. The delta-opioid antagonist, naltrindole (1.1 nmoles/min), restored only 30% of the sympathetic tachycardia. Nodal delta-receptors are vagolytic and vagal stimulations were included in the protocol as positive controls. LE reduced vagal bradycardia by 50% and naltrindole completely restored the vagal bradycardia. In Study 2, additional opioid antagonists were used to determine if alternative opioid receptors might be implicated in the sympatholytic response. Increasing doses of the kappa-antagonist, norbinaltorphimine (norBNI), were combined with LE during sympathetic stimulation. NorBNI completely restored the sympathetic tachycardia with an ED50 of 0.01 nmoles/min. A single dose of the micro -antagonist, CTAP (1.0 nmoles/min), failed to alter the sympatholytic effect of LE. Study 3 was conducted to determine if the sympatholytic effect was prejunctional or postjunctional in character. Norepinephrine was added to the dialysis inflow at a rate (30-45 pmoles/min) sufficient to produce intermediate increases (35.2 +/- 1.8 bpm) in HR. LE was then combined with norepinephrine and responses were recorded at 5-min intervals for 20 min. The tachycardia mediated by added norepinephrine was unaltered by LE or LE plus naltrindole. At the same 5-min intervals, LE reduced vagal bradycardia by more than 50%. This vagolytic effect was again completely reversed by naltrindole. Collectively, these observations support the hypothesis that the local nodal sympatholytic effect of LE was mediated by kappa-opioid receptors that reduced the effective interstitial concentration of norepinephrine and not the result of a postjunctional interaction between LE and norepinephrine.     

Cardiac enkephalins attenuate vagal bradycardia: interactions with NOS-1-cGMP systems in canine sinoatrial node

Endogenous opioids and nitric oxide (NO) are recognized modulators of cardiac function. Enkephalins and inhibitors of NO synthase (NOS) both produce similar interruptions in the vagal control of heart rate. This study was conducted to test the hypothesis that NO systems within the canine sinoatrial (SA) node facilitate local vagal transmission and that the endogenous enkephalin methionine-enkephalin-arginine-phenylalanine (MEAP) attenuates vagal bradycardia by interrupting the NOS-cGMP pathway. Microdialysis probes were inserted into the SA node, and they were perfused with nonselective (Nomega-nitro-l-arginine methyl ester) and neuronal (7-nitroindazole) NOS inhibitors. The right vagus nerve was stimulated and both inhibitors gradually attenuated the resulting vagal bradycardia. The specificity of these inhibitions was verified by an equally gradual reversal of the inhibition with an excess of the NOS substrate l-arginine. Introduction of MEAP into the nodal interstitium produced a quickly developing but quantitatively similar interruption of vagal bradycardia that was also slowly reversed by the addition of l-arginine and not by d-arginine. Additional support for convergence of opioid and NO pathways was provided when the vagolytic effects of MEAP were also reversed by the addition of the NO donor S-nitroso-N-acetyl-penicillamine, the protein kinase G activator 8-bromo-cGMP, or the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. MEAP and 7-nitroindazole were individually combined with the direct acting muscarinic agonist methacholine to evaluate potential interactions with muscarinic receptors within the SA node. MEAP and 7-nitroindazole were unable to overcome the bradycardia produced by methacholine. These data suggest that NO and enkephalins moderate the vagal control of heart rate via interaction with converging systems that involve the regulation of cAMP within nodal parasympathetic nerve terminals.     

Repeated arterial occlusion, delta-opioid receptor (DOR) plasticity and vagal transmission within the sinoatrial node of the anesthetized dog

Brief interruptions in coronary blood flow precondition the heart, engage delta-opioid receptor (DOR) mechanisms and reduce the damage that typically accompanies subsequent longer coronary occlusions. Repeated short occlusions of the sinoatrial (SA) node artery progressively raised nodal methionine-enkephalin-arginine-phenylalanine (MEAP) and improved vagal transmission during subsequent long occlusions in anesthetized dogs. The DOR type-1 (DOR-1) antagonist, BNTX reversed the vagotonic effect. Higher doses of enkephalin interrupted vagal transmission through a DOR-2 mechanism. The current study tested whether the preconditioning (PC) protocol, the later occlusion or a combination of both was required for the vagotonic effect. The study also tested whether evolving vagotonic effects included withdrawal of competing DOR-2 vagolytic influences. Vagal transmission progressively improved during successive SA nodal artery occlusions. The vagotonic effect was absent in sham animals and after DOR-1 blockade. After completing the PC protocol, exogenously applied vagolytic doses of MEAP reduced vagal transmission under both normal and occluded conditions. The magnitude of these DOR-2 vagolytic effects was small compared to controls and repeated MEAP challenges rapidly eroded vagolytic responses further. Prior DOR-1 blockade did not alter the PC mediated, progressive loss of DOR-2 vagolytic responses. In conclusion, DOR-1 vagotonic responses evolved from signals earlier in the PC protocol and erosion of competing DOR-2 vagolytic responses may have contributed to an unmasking of vagotonic responses. The data support the hypothesis that PC and DOR-2 stimulation promote DOR trafficking, and down regulation of the vagolytic DOR-2 phenotype in favor of the vagotonic DOR-1 phenotype. DOR-1 blockade may accelerate the process by sequestering newly emerging receptors.     

Vagotonic effects of enkephalin are not mediated by sympatholytic mechanisms

This study examined the hypothesis that vagotonic and sympatholytic effects of cardiac enkephalins are independently mediated by different receptors. A dose-response was constructed by administering the delta-receptor opioid methionine-enkephalin-arginine-phenylalanine (MEAP) by microdialysis into the interstitium of the canine sinoatrial node during vagal and sympathetic stimulation. The right cardiac sympathetic nerves were stimulated as they exited the stellate ganglion at frequencies selected to increase heart rate approximately 35 bpm. The right cervical vagus was stimulated at frequencies selected to produce a two-step decline in heart rate of 25 and 50 bpm. A six-step dose-response was constructed by recording heart rates during nerve stimulation as the dose of MEAP was increased between 0.05 pmol/min and 1.5 nmol/min. Vagal transmission improved during MEAP at 0.5 pmol/min. However, sympathetically mediated tachycardia was unaltered with any dose of MEAP. In Study 2, a similar dose-response was constructed with the kappa-opioid receptor agonist trans(+/-)-3-4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide-HCl (U-50488H) to illustrate an independent sympatholytic effect and to verify its kappa-receptor character. U-50488H gradually suppressed the sympathetic tachycardia, with a significant effect obtained only at the highest dose (1.5 nmol/min). U-50488H had no effect on vagally mediated bradycardia. Surprisingly, the sympatholytic effect was not reversed by withdrawing U-50488H or by the subsequent addition of the kappa-antagonist 17,17'-(dichloropropylmethyl)-6,6',7,7'-6,6'-imino-7,7'-binorphinan-3,4',14,14'-tetroldi-hydrochloride (norBNI). Study 3 was conducted to determine whether the sympatholytic effect of U-50488H could be prevented by norBNI. NorBNI blocked the sympatholytic effect of the U50488H for 90 mins. When norBNI was discontinued afterward and U-50488H was continued alone, a sympatholytic effect emerged within 30 mins. Collectively these observations support the hypothesis that the vagotonic influence of MEAP is not dependent on a sympatholytic influence. Furthermore, the sympatholytic effect is mediated independently by kappa-receptors. The sympatholytic effect of sustained kappa-receptor stimulation appears to evolve gradually into a functional state not easily reversed.     

Characterization of [3H]naltrindole binding to delta opioid receptors in rat brain.

[3H]Naltrindole binding characteristics were determined using homogenized rat brain tissue. Saturation binding studies at 25 degrees C measured an equilibrium dissociation constant (Kd) value of 37.0 +/- 3.0 pM and a receptor density (Bmax) value of 63.4 +/- 2.0 fmol/mg protein. Association binding studies showed that equilibrium was reached within 90 min at a radioligand concentration of 30 pM. Naltrindole, as well as the ligands selective for delta (delta) opioid receptors, such as pCI-DPDPE and Deltorphin II inhibited [3H]naltrindole binding with nanomolar IC50 values. Ligands selective for mu (mu) and kappa (kappa) opioid receptors were only effective in inhibiting [3H]naltrindole binding at micromolar concentrations. From these data, we conclude that [3H]naltrindole is a high affinity, selective radioligand for delta opioid receptors.     

The possibility of peripheral cholinolytic action of psychostimulants

The cholinolytic effect of sydnophen discovered in earlier anesthetized cats was confirmed on unanesthetized fish and frogs: the vagal bradycardia induced by electric stimulation of peripheral vagal end was decreased or even abolished by intravenous injection of sydnophen (0.2-20 mg/kg). The amphetamine (0.2-30 mg/kg) also blocked the vagal bradycardia in anesthetized cats and unanesthetized frogs. The maximum vagolytic action of amphetamine appeared later (in 4-8 min after injection) in compared with sydnophen (1-3 min). The small dose of amphetamine (0.2-0.3 mg/kg) in contrast to sydnophen didn't decrease the vagal bradycardia but even increased it. It was suggested that the cholinolytic effect of sydnophen and amphetamine is due to different mechanisms.     

In vivo labeling of delta opioid receptors in mouse brain by [3H]benzylidenenaltrexone,a ligand selective for the Delta1 subtype

(E)-7-Benzylidenenaltrexone (BNTX) is a selective ligand for the putative deltai (61) opioid receptor. To explore the feasibility of labeling δ₁ sites in vivo, we determined the cerebral distribution of radioactivity after systemic administration of [3H]BNTX to CDI mice. Uptake was, highest in striatum and lowest in cerebellum throughout the 4 hr time course. Specific radioligand binding, approximated as the difference in radioactivity concentrations between striatum and cerebellum, peaked at 0.32 percent injected dose/g at 30 min and comprised a modest 23% of total striatal radioactivity. For seven brain regions, radioactivity concentrations correlated with S site densities known from prior in vitro studies (rs = 0.79, p = 0.03), and also with the uptake of Nl'-([HC]methyl) naltrindole in vivo (rs = 0.78, p = 0.04) in mice. Specific binding in striatum, olfactory tubercles and cortical regions was saturable by BNTX, and was inhibited stereoselectively by the optical isomers of naloxone. Naltrindole and naltriben (NTB), δ antagonists, blocked 65 -99% of [3H]BNTX specific binding at a dosage of 5.0 μmol/kg. Similar doses of the μ antagonist cyprodime, or the k agonist U50.488H, did not inhibit binding. Adjusted for the four-fold greater brain penetration of NTB relative to BNTX, dose-response studies suggested that δ₁ selective BNTX (ED₅₀ = 1.51 μmolol/kg) was 50% more potent than 82 selective NTB (ED50 = 0.56 μmol/kg) in blocking specific [³H]BNTX binding in striatum. In CXBK mice, a strain with functional 81 but not 82 receptors in antinociceptive assays, radioligand uptake and distribution proved similar to that in CD1 mice. In sum, [³H]BNTX labels murine 5 opioid receptors in vivo with a low extent of specific binding. The data is consistent with, but not conclusive for, selective labeling of the δ₁ subtype.     

Inhibition of an opioid-evoked vagal reflex in rats by naloxone,SMS 201-995 and ICI 154,129

Intravenous injection of opioid agonists in rats evokes a vagal reflex resulting in a fall in heart rate and blood pressure. Three opioid antagonists, naloxone, SMS 201-995, and ICI 154,129 were used to assess the nature of the opioid receptors that mediate the vagal reflex. The agonists used were morphine, Tyr-Pro-NMePhe-d-Pro-NH₂ (PLO17), and d-Ala²-Leu⁵-enkephalin (DADL). At challenge doses of morphine, PLO17, and DADL at five times the ED50 for bradycardia, the naloxone ED50 for DADL was nine times greater than that for morphine and PLO17. The pA₂ value of naloxone against DADL was significantly less than that for morphine and PLO17. The antagonist properties of SMS 201-995 were similar to those of naloxone. ICI 154,129, a putative delta receptor antagonist, was not, however, selective in its antagonism of opioid bradycardia. Both SMS 201-995 and ICI 154,129, when injected alone, produced changes in heart rate and blood pressure. The cardiovascular actions of the peptide antagonists were not affected by naloxone hydrochloride at doses up to 4 mg/kg i.v.     

Peripheral opioid receptors influencing heart rate in rats: evidence for endogenous tolerance

Opioid peptides injected into the circulation of rats evoke a vagally mediated bradycardia. The intravenous ED50 of morphine for producing a greater than or equal to 10% fall in heart rate was determined in urethane-anesthetized rats. Hypophysectomy, or adrenalectomy plus treatment with dexamethasone (0.5 microgram/h, s.c., 1 day), procedures that remove endogenous sources of opioid peptides, increased the sensitivity of the animal to morphine bradycardia by 6-10-fold. Conversely, stressing the animals by exposure to cold (4-6 degrees C for two days) elevated the ED50 for morphine sulfate and for beta h-endorphin by about 5-fold. Dexamethasone infusions prevented the cold-induced desensitization to morphine. Intravenous administration of rat corticotropin-releasing factor (CRF) also desensitized the animals to morphine. CRF alone produced a fall in blood pressure and heart rate. The bradycardia was prevented by pretreatment with naloxone. These results indicate that the sensitivity of vagal opioid chemoreceptors is influenced by endogenous sources of opioid peptides. This phenomenon can be called 'endogenous tolerance'.     

Interactions with the cardiac cholinergic system: effects of disopyramide and its mono-N-dealkylated metabolite.

The cardiac vagolytic effects of disopyramide and its mono-N-dealkylated metabolite (MND), and their interactions with the cardiac cholinergic system, were assessed using in vivo and in vitro experiments. In chloralose anesthetized dogs, disopyramide phosphate (0.25 mg/kg/min) and MND at equimolar dose (0.173 mg/kg/min) reduced vagal bradycardia. As indicated by the ED80, MND exhibits a vagolytic activity 1.5-2 times less potent than disopyramide. Concomitantly, increases in heart rate and mean blood pressure were observed with disopyramide, whereas with MND only a rise in mean blood pressure occurred. In conscious dogs, where vagal tone is fully expressed, disopyramide and MND increased heart rate and, interestingly, prevented any atropine-induced additional tachycardia, though heart rate was relatively low. Binding studies on rat heart membranes yielded Ki values 2-2.5 times higher for MND than for disopyramide, and demonstrated that neither disopyramide nor MND binding modified the cardiac muscarinic receptor sites. Taken together, these results show that disopyramide exhibits a more potent cardiac vagolytic action than MND, very likely linked to a greater ability to bind to cardiac muscarinic receptors. They also show that disopyramide and MND are very potent in preventing atropine-induced "excess tachycardia", very likely by inhibiting the ionic pacemaker current(s) involved in its genesis.     

Alpha-2 adrenergic and opioid receptor-mediated gastroprotection.

Clonidine inhibited the development of gastric mucosal lesions induced by either acidified ethanol or indomethacin. The ED50 values were: 7.1 and 5.2 microg x kg(-1) orally, respectively. The gastroprotective effect was antagonised by the pre-synaptic alpha-2 antagonist yohimbine, the more selective alpha-2 antagonist Ch-38083 and the pre-synaptic alpha-2B antagonist prazosin. Moreover, the non-selective opioid receptor antagonist naloxone, the delta receptor selective naltrindole also reversed the clonidine-induced mucosal protective action. Clonidine was also effective following intracerebroventricular administration with the ED50 of 37 ng/rat against ethanol-induced mucosal damage. Our results suggest that: 1) the gastroprotective effect of clonidine is likely to be mediated by alpha-2B adrenoceptor subtype; 2) there is an interaction between pre-synaptic alpha-2 adrenoceptors and opioid system; and 3) clonidine can induce gastroprotection by central mechanism.     

Evidence for delta opioid receptor subtypes regulating adenylyl cyclase activity in rat brain

Opioid agonists selective for mu- or delta opioid receptors inhibit adenyl yl cyclase in membranes from rat caudate-putamen and nucleus accumbens. The presence of subtypes of delta opioid receptors has been suggested. In both brain regions we have found that the inhibition of adenylyl cyclase by DPDPE was more readily antagonized by 7-benzylidenenaltrexone (BNTX), than by naltriben. In contrast, the inhibitory effects of deltorphin-II and DSLET were more readily antagonized by naltriben, than by BNTX. neither naltriben nor BNTX significantly antagonized the effect of a mu selective agonist. These results suggest that inhibition of adenylyl cyclase in caudate-putamen and nucleus accumbens is regulated by two forms of delta-opioid receptor with ligand selectivities similar to those two forms proposed to mediate analgesic effect.     

Modulation of cardiopulmonary depressor reflex in nucleus ambiguus by electroacupuncture: roles of opioids and γ-aminobutyric acid

Stimulation of cardiopulmonary receptors with phenylbiguanide (PBG) elicits depressor cardiovascular reflex responses, including decreases in blood pressure and heart rate mediated in part by the brain stem parasympathetic cardiac neurons in the nucleus ambiguus (NAmb). The present study examined NAmb neurotransmitter mechanisms underlying the influence of electroacupuncture (EA) on the PBG-induced hypotension and bradycardia. We hypothesized that somatic stimulation during EA modulates PBG responses through opioid and γ-aminobutyric acid (GABA) modulation in the NAmb. Anesthetized and ventilated cats were studied during repeated stimulation with PBG or cardiac vagal afferents while low-frequency EA (2 Hz) was applied at P5-6 acupoints overlying the median nerve for 30 min and NAmb neuronal activity, heart rate, and blood pressure were recorded. Microinjection of kainic acid into the NAmb attenuated the PBG-induced bradycardia from -60 ± 11 to -36 ± 11 beats/min. Likewise, EA reduced the PBG-induced depressor and bradycardia reflex by 52 and 61%, respectively. Cardiac vagal afferent evoked preganglionic cellular activity in the NAmb was reduced by EA for about 60 min. Blockade of opioid or GABA(A) receptors using naloxone and gabazine reversed the EA-related modulation of the evoked cardiac vagal activity by 73 and 53%, respectively. Similarly, naloxone and gabazine reversed EA modulation of the negative chronotropic responses from -11 ± 5 to -23 ± 6 and -13 ± 4 to -24 ± 3 beats/min, respectively. Thus EA at P5-6 decreases PBG evoked hypotension and bradycardia as well as the NAmb PBG-sensitive preganglionic cardiac vagal outflow through opioid and GABA neurotransmitter systems.     

Ligand binding profiles of delta-opioid receptor in human cerebral cortex membranes: evidence of delta-opioid receptor heterogeneity

In this study, receptor binding profiles of opioid ligands for subtypes of opioid delta-receptors were examined employing [3H]D-Pen2,D-Pen5-enkephalin ([3H]DPDPE) and [3H]Ile(5,6)-deltorphin II ([3H]Ile-Delt II) in human cerebral cortex membranes. [3H]DPDPE, a representative ligand for delta1 sites, labeled a single population of binding sites with apparent affinity constant (Kd) of 2.72 +/- 0.21 nM and maximal binding capacity (Bmax) value of 20.78 +/- 3.13 fmol/mg protein. Homologous competition curve of [3H]Ile-Delt II, a representative ligand for delta2 sites, was best fit by the one-site model (Kd = 0.82 +/- 0.07 nM). Bmax value (43.65 +/- 2.41 fmol/mg) for [3H]Ile-Delt II was significantly greater than that for [3H]DPDPE. DPDPE, [D-Ala2,D-Leu5]enkephalin (DADLE) and 7-benzylidenaltrexone (BNTX) were more potent in competing for the binding sites of [3H]DPDPE than for those of [3H]Ile-Delt II. On the other hand, deltorphin II (Delt II), [D-Ser2,Leu5,Thr6]enkephalin (DSLET), naltriben (NTB) and naltrindole (NTI) were found to be equipotent in competing for [3H]DPDPE and [3H]Ile-Delt II binding sites. These results indicate that both subtypes of opioid delta-receptors, delta1 and delta2, exist in human cerebral cortex with different ligand binding profiles.     

The effect of the leucopyrokinin analogue: [2-8]-leucopyrokinin on central opioid receptors in rats

The antinociceptive effect of intracerebroventricular injections of [2–8]-leucopyrokinin (LPK), a truncated leucopyrokinin analogue, was determined in rats, by means of a tail immersion test. We found a significant antinociceptive effect of three i.c.v. doses of [2–8]-LPK: 1, 5 and 10 nmol. Pre-treating animals with naloxone hydrochloride (1 mg/kg i.p.) completely blocked the effect of two high doses of [2–8]-LPK. To determine the sub-types of opioid receptors involved in [2–8]-leucopyrokinin-induced analgesia we injected specific blockers of μ-, δ- and κ-receptors namely, β-funaltrexamine hydrochloride, naltrindole hydrochloride and nor-binaltorphimine dihydrochloride, respectively, prior to [2–8]-leucopyrokinin at equimolar doses. We conclude that the antinociceptive effect of [2–8]-leucopyrokinin is mediated mainly by central μ- and δ-opioid receptors.     

Agonist and antagonist activities of ligands derived from naltrexone and oxymorphone.

The pharmacological profile of naltrindole (NTI) and three of its analogues, N-methyl-NTI (N-Me-NTI), oxymorphindole (OMI) and naltriben (NTB) were studied in antinociceptive assays. The compounds were found to have agonist activities that appear to be mediated mainly by kappa opioid receptors because norbinaltorphimine (nor-BNI), the selective kappa opioid receptor antagonist inhibited their effects significantly. All of the compounds, behaved as antagonists at doses that were lower than those that produced agonist effects and they possessed a profile that was very selective for inhibiting the antinociceptive activities of delta opioid receptor agonists. Differential antagonism by NTB of the activities of DSLET and DPDPE was demonstrated.     

Effect of systemic B-type natriuretic peptide on cardiac vagal motoneuron activity

Intravenous B-type natriuretic peptide (BNP) enhances the bradycardia of reflexes from the heart, including the von Bezold-Jarisch reflex, but its site of action is unknown. The peptide is unlikely to penetrate the blood-brain barrier but could act on afferent or efferent reflex pathways. To investigate the latter, two types of experiment were performed on urethane-anesthetized (1.4 g/kg iv) rats. First, the activity was recorded extracellularly from single cardiac vagal motoneurons (CVMs) in the nucleus ambiguus. CVMs were identified by antidromic activation from the cardiac vagal branch and by their barosensitivity. Phenyl biguanide (PBG), injected via the right atrium in bolus doses of 1-5 mug to evoke the von Bezold-Jarisch reflex, caused a dose-related increase in CVM activity and bradycardia. BNP infusion (25 pmol.kg(-1).min(-1) iv) significantly enhanced both the CVM response to PBG (n = 5 rats) and the reflex bradycardia, but the log-linear relation between those two responses over a range of PBG doses was unchanged by BNP. The reflex bradycardia was not enhanced in five matched time-control rats receiving only vehicle infusions. In five other rats the cervical vagi were cut and the peripheral right vagus was stimulated supramaximally at frequencies of 1-20 Hz. The bradycardic responses to these stimuli were unchanged before, during, and after BNP infusion. We conclude that systemic BNP in a moderate dose enhances the von Bezold-Jarisch reflex activation of CVM, in parallel with the enhanced reflex bradycardia. That enhancement is due entirely to an action before the vagal efferent arm of the reflex pathway.