Bimodal delta-opioid receptors regulate vagal bradycardia in canine sinoatrial node

Methionine-enkephalin-arginine-phenylalanine (MEAP) introduced into the interstitium of the canine sinoatrial (SA) node by microdialysis interrupts vagal bradycardia. In contrast, raising endogenous MEAP by occluding the SA node artery improves vagal bradycardia. Both are blocked by the same delta-selective antagonist, naltrindole. We tested the hypothesis that vagal responses to intranodal enkephalin are bimodal and that the polarity of the response is both dose- and opioid receptor subtype dependent. Ultralow doses of MEAP were introduced into the canine SA node by microdialysis. Heart rate frequency responses were constructed by stimulating the right vagus nerve at 1, 2, and 3 Hz. Ultralow MEAP infusions produced a 50-100% increase in bradycardia during vagal stimulation. Maximal improvement was observed at a dose rate of 500 fmol/min with an ED50 near 50 fmol/min. Vagal improvement was returned to control when MEAP was combined with the delta-antagonist naltrindole. The dose of naltrindole (500 fmol/min) was previously determined as ineffective vs. the vagolytic effect of higher dose MEAP. When MEAP was later reintroduced in the same animals at nanomoles per minute, a clear vagolytic response was observed. The delta1-selective antagonist 7-benzylidenenaltrexone (BNTX) reversed the vagal improvement with an ED50 near 1 x 10-21 mol/min, whereas the delta2-antagonist naltriben had no effect through 10-9 mol/min. Finally, the improved vagal bradycardia previously associated with nodal artery occlusion and endogenous MEAP was blocked by the selective delta1-antagonist BNTX. These data support the hypothesis that opioid effects within the SA node are bimodal in character, that low doses are vagotonic, acting on delta1-receptors, and that higher doses are vagolytic, acting on delta2-receptors.     

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.     

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.     

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.     

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.     

Cholinergic location of delta-opioid receptors in canine atria and SA node

Delta-opioid receptors (DORs) are associated with ischemic preconditioning and vagal transmission in the sinoatrial (SA) node and atria. Although functional studies suggested that DORs are prejunctional on parasympathetic nerve terminals, their precise location remains unconfirmed. DORs were colocalized in tissue slices and synaptosomes from the canine right atrium and SA node along with cholinergic and adrenergic markers, vesicular acetylcholine transporter (VAChT), and tyrosine hydroxylase (TH). Synapsin I immunofluorescence verified the neural character of tissue structures and isolated synaptosomes. Acetylcholine and norepinephrine measurements suggested the presence of both cholinergic and adrenergic synaptosomes. Fluorescent analysis of VAChT and TH signals indicated that >80% of the synapsin-positive synaptosomes were of cholinergic origin and <8% were adrenergic. DORs colocalized 75-85% with synapsin in tissue slices from both atria and SA node. The colocalization was equally strong (85%) for nodal synaptosomes but less so for atrial synaptosomes (57%). Colocalization between DOR and VAChT was 75-85% regardless of the source. Overlap between DOR and TH was uniformly low, ranging from 8% to 17%. Western blots with synaptosomal extracts confirmed two DOR-positive bands at molecular masses corresponding to those reported for DOR monomers and dimers. The abundance of DOR was greater in nodal synaptosomes than in atrial synaptosomes, largely attributable to a greater abundance of monomers in the SA node. The abundant nodal and atrial DORs predominantly associated with cholinergic nerve terminals support the hypothesis that prejunctional DORs regulate vagal transmission locally within the heart.     

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.     

Vagal blockade does not prevent adrenocorticotropin, cortisol, and cardiopulmonary responses to thromboxane A2.

Previously, we reported that thromboxane A2 (TxA2) mediates heart rate, adrenocorticotropin (ACTH), cortisol, and blood gas responses, although the specific site of action was not identified. In the present study, we interrupted vagal nervous transmission in chronically instrumented conscious sheep and infused the TxA2 mimetic U46619 or saline into the carotid artery or U46619 into the vena cava to determine whether TxA2 acts at vagal afferent nerves. Heart rate increased in all three groups during vagal blockade, and responses were not different between groups. Carotid artery and intravenous infusions of U46619 resulted in an increase in blood pressure, but responses were not different between groups. PaO2 decreased in response to vagal blockade in all groups, and responses were not different among groups. Arterial pH increased and PaCO2 decreased during vagal blockade in response to carotid artery U46619 infusions but not in response to vagal blockade alone or combined with carotid artery saline or intravenous U46619. ACTH, cortisol, and hematocrit increased significantly in response to carotid artery infusions of U46619 during vagal blockade but not in response to carotid artery saline or intravenous U46619 infusions. In summary, carotid artery infusions of TxA2 mimetic result in ACTH, cortisol, PaCO2, pHa, and hematocrit responses that are not prevented by vagal blockade. We conclude that these responses are mediated at a site perfused by the carotid vasculature and not at a site innervated by the vagal nerves, findings consistent with the hypothesis that TxA2 acts on the brain to mediate cardiopulmonary and pituitary-adrenal responses.     

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.     

The influence of monovalent cations on trimeric G protein G(i)1α activity in HEK293 cells stably expressing DOR-G(i)1α (Cys(351)-Ile(351)) fusion protein

The effect of monovalent cations on trimeric G protein G(i)1α was measured at equimolar concentration of chloride anion in pertussis-toxin (PTX)-treated HEK293 cells stably expressing PTX-insensitive DOR- G(i)1α (Cys(351)-Ile(351)) fusion protein by high-affinity [(35)S]GTPgammaS binding assay. The high basal level of binding was detected in absence of DOR agonist and monovalent ions and this high level was inhibited with the order of: Na(+) > K(+) > Li(+). The first significant inhibition was detected at 1 mM NaCl. The inhibition by monovalent ions was reversed by increasing concentrations of DOR agonist DADLE. The maximum DADLE response was also highest for sodium and decreased in the order of: Na(+) > K(+) ~ Li(+). Our data indicate i) an inherently high activity of trimeric G protein G(i)1α when expressed within DOR- G(i)1α fusion protein and determined in the absence of monovalent cations, ii) preferential sensitivity of DOR- G(i)1alpha to sodium as far as maximum of agonist response is involved.     

Opioid receptor-induced GTPgamma35S binding during mouse development

Although a large superfamily of G-protein-coupled receptors serves multiple functions, little is known about their functional activation during ontogeny. To examine the functional activation of the mu-opioid receptor (MOR) and the delta-opioid receptor (DOR) during development, sections of mouse embryos and fetuses from e11.5 until birth were treated with DAMGO and DPDPE, respectively, and the ability of these drugs to induce G-protein coupling was assessed by using GTPgamma(35)S binding autoradiography. MOR activation was first detected in the caudate-putamen (CPU) at e12.5, and by e15.5, activity had not only increased in this region but also expanded to include the midbrain, medial habenula, hypothalamus, pons, and medulla. DOR activity first appeared at e17.5 in the hypothalamus, pons, medial habenula, and medulla and at p1 in the CPU at levels noticeably less than those of the MOR. In general, MOR and DOR activation lagged only slightly behind the appearance of MOR-1 and DOR-1 mRNA but delayed activation was particularly pronounced in the trigeminal ganglia, where MOR-1 gene expression was first detected at e13.5, but MOR activity was not observed even at birth. Thus, the data demonstrate temporal and often region-specific differences in the appearance and magnitude of functional activity in cell groups expressing either the MOR-1 or DOR-1 genes, suggesting that interaction between the opioid receptors, G-proteins, and other signaling cofactors is developmentally regulated.     

Cardiac sympathetic nerve stimulation does not attenuate dynamic vagal control of heart rate via alpha-adrenergic mechanism

Complex sympathovagal interactions govern heart rate (HR). Activation of the postjunctional beta-adrenergic receptors on the sinus nodal cells augments the HR response to vagal stimulation, whereas exogenous activation of the presynaptic alpha-adrenergic receptors on the vagal nerve terminals attenuates vagal control of HR. Whether the alpha-adrenergic mechanism associated with cardiac postganglionic sympathetic nerve activation plays a significant role in modulation of the dynamic vagal control of HR remains unknown. The right vagal nerve was stimulated in seven anesthetized rabbits that had undergone sinoaortic denervation and vagotomy according to a binary white-noise signal (0-10 Hz) for 10 min; subsequently, the transfer function from vagal stimulation to HR was estimated. The effects of beta-adrenergic blockade with propranolol (1 mg/kg i.v.) and the combined effects of beta-adrenergic blockade and tonic cardiac sympathetic nerve stimulation at 5 Hz were examined. The transfer function from vagal stimulation to HR approximated a first-order, low-pass filter with pure delay. beta-Adrenergic blockade decreased the dynamic gain from 6.0 +/- 0.4 to 3.7 +/- 0.6 beats x min(-1) x Hz(-1) (P < 0.01) with no alteration of the corner frequency or pure delay. Under beta-adrenergic blockade conditions, tonic sympathetic stimulation did not further change the dynamic gain (3.8 +/- 0.5 beats x min(-1) x Hz(-1)). In conclusion, cardiac postganglionic sympathetic nerve stimulation did not affect the dynamic HR response to vagal stimulation via the alpha-adrenergic mechanism.     

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.     

Influence of ethacizin (the diethyl analog of ethmozine) on phase-dependent parasympathetic effects on the heart

The influence of ethacizin (a diethylamine analog of ethmozine) (1.10(-7)-1.10(-6) g/ml) upon the phase-dependent chronotropic parasympathetic effects was studied on the perfused frog heart. The vagolytic influence of ethacizin (5.10(-7) and 1.10(-6) g/ml) was detected; the concentration of 1.10(-7) g/ml was found ineffective. The vagolytic effect consisted of a decreased maximum of phase-dependent effect, reduced latency and time required for the manifestation of the maximum increase. The period of inhibitory vagal stimulus effectiveness did not change significantly.     

AV blocking due to asynchronous vagal stimulation in rats

The parasympathetic nervous system innervates the heart through two cervical vagal branches. The right vagal branch mainly influences the heart rate by the modulation of the rhythmogenesis of the sinoatrial node. The left branch predominantly influences the conduction properties of the atrioventricular (AV) node. We investigated the effect of asynchronous stimulation by the vagal nerves on the occurrence of irregularities in heart rate. In rats, the vagal nerves were isolated and cut. Different vagal stimulation patterns (continuous, pulsed) were applied. The heart was beating spontaneously under continuous vagal stimulation. In case of pulsed vagal stimulation, the atria were paced at different rates. Asynchronicity was induced by delaying the right stimulus with respect to the left stimulus (early right) or the left stimulus with respect to the right stimulus (early left). The value of the fraction of deviated R-R or P-Q intervals in the distribution in the histogram was used to characterize irregularities during a stimulation protocol (duration in case of continuous stimulation: 20 s; pulsed stimulation: 120 s). Under both stimulation patterns (continuous or pulsed), we found that early left vagal stimulation introduced a much larger fraction of deviated intervals in the R-R or P-Q histogram (in R-R: 29.1 +/- 4.9%; in P-Q: 12.90 +/- 1.95%) than early right vagal stimulation (in R-R: 7.4 +/- 2.0%; in P-Q: 1. 05 +/- 0.50%) or synchronous stimulation (in R-R: 8.2 +/- 3.6%; in P-Q: 2.15 +/- 0.75%). We conclude that early stimulation by the left vagal nerve can introduce irregularities in heart rate, mainly due to different degrees of AV nodal blockade.     

Role of the medullary lateral tegmental field in reflex-mediated sympathoexcitation in cats

We tested the hypothesis that blockade of N-methyl-D-aspartate (NMDA) and non-NMDA receptors on medullary lateral tegmental field (LTF) neurons would reduce the sympathoexcitatory responses elicited by electrical stimulation of vagal, trigeminal, and sciatic afferents, posterior hypothalamus, and midbrain periaqueductal gray as well as by activation of arterial chemoreceptors with intravenous NaCN. Bilateral microinjection of a non-NMDA receptor antagonist into LTF of urethane-anesthetized cats significantly decreased vagal afferent-evoked excitatory responses in inferior cardiac and vertebral nerves to 29 +/- 8 and 24 +/- 6% of control (n = 7), respectively. Likewise, blockade of non-NMDA receptors significantly reduced chemoreceptor reflex-induced increases in inferior cardiac (from 210 +/- 22 to 129 +/- 13% of control; n = 4) and vertebral nerves (from 253 +/- 41 to 154 +/- 20% of control; n = 7) and mean arterial pressure (from 39 +/- 7 to 21 +/- 5 mmHg; n = 8). Microinjection of muscimol, but not an NMDA receptor antagonist, caused similar attenuation of these excitatory responses. Sympathoexcitatory responses to the other stimuli were not attenuated by microinjection of a non-NMDA receptor antagonist or muscimol into LTF. In fact, excitatory responses elicited by stimulation of trigeminal, and in some cases sciatic, afferents were enhanced. These data reveal two new roles for the LTF in control of sympathetic nerve activity in cats. One, LTF neurons are involved in mediating sympathoexcitation elicited by activation of vagal afferents and arterial chemoreceptors, primarily via activation of non-NMDA receptors. Two, non-NMDA receptor-mediated activation of other LTF neurons tonically suppresses transmission in trigeminal-sympathetic and sciatic-sympathetic reflex pathways.     

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.     

Retention of supraspinal delta-like analgesia and loss of morphine tolerance in delta opioid receptor knockout mice

Gene targeting was used to delete exon 2 of mouse DOR-1, which encodes the delta opioid receptor. Essentially all 3H-[D-Pen2,D-Pen5]enkephalin (3H-DPDPE) and 3H-[D-Ala2,D-Glu4]deltorphin (3H-deltorphin-2) binding is absent from mutant mice, demonstrating that DOR-1 encodes both delta1 and delta2 receptor subtypes. Homozygous mutant mice display markedly reduced spinal delta analgesia, but peptide delta agonists retain supraspinal analgesic potency that is only partially antagonized by naltrindole. Retained DPDPE analgesia is also demonstrated upon formalin testing, while the nonpeptide delta agonist BW373U69 exhibits enhanced activity in DOR-1 mutant mice. Together, these findings suggest the existence of a second delta-like analgesic system. Finally, DOR-1 mutant mice do not develop analgesic tolerance to morphine, genetically demonstrating a central role for DOR-1 in this process.     

The role of different mechanisms of cholinergic regulation in controlled bradycardia evoked by vagal stimulation]

In experiments on anesthetised cats we investigated functional significance of different cholinergic mechanisms regulating the magnitude of vagal chronotropic effect components, inhibitory tonic and synchronizing. It was established that inhibitory tonic vagal component is determined by intensity of acetylcholine hydrolysis and total amount of excited cardiac M-cholinoreceptors. The magnitude of synchronizing vagal component depended on subtypes of cholinoreceptors selectively excited by acetylcholine released from vagal terminals. In particular, the blockade of M1- or M3-cholinoreceptors potentiated the synchronizing vagal component, whereas the blockade of M2-cholinoreceptors inhibited it.     

Effect of indomethacin-induced inhibition of arachidonic acid metabolism on the development of cardiogenic reflexes

In acute experiments on anesthetized cats, afferent spike activity from the parasympathetic (vagal) and sympathetic cardiac nerves, ECG, and cardiodynamic indices were recorded. The effects of indomethacin-induced blockade of cyclo-oxygenase pathway in metabolism of arachidonic acid on the development of cardiogenic reflex responses after intracoronary injections of veratrine, bradykinin, or prostacyclin were tested. It was found that after indomethacin injection depressor cardiogenic vagal reflexes, evoked by veratrine or bradykinin administrations, became significantly suppressed or practically disappeared. This was accompanied by a drop in the frequency of afferent vagal activity in the cardiac nerves. This effect could be observed throughout the entire period of influence of indomethacin (about 2 h after its injection). Veratrine or bradykinin, being injected simultaneously with prostacyclin, provided faster partial recovery of depressor responses (at 1 h) and promoted some activation of vagal cardiac nerves, despite the effect of indomethacin. Injection of indomethacin did not change the pattern of sympathetic afferent activity. It is suggested that the main derivative of cyclo-oxygenase pathway of arachidonic acid metabolism, prostacyclin, is able to modulate vagal nervous activity at the level of afferent structures in the heart. Prostacyclin may appear a humoral component of cardiogenic depressor reflexes of a vagal nature.Neirofiziologiya/Neurophysiology, Vol. 28, No. 1, pp. 53–61, January–February, 1996.