Organ culture of rat heart: maintained high sensitivity of fetal atria before innervation to norepinephrine

Changes in sensitivity to norepinephrine (NE) of fetal and neonatal rat right atria placed in organ culture were examined. The high sensitivity to NE of the 17-day fetal atria was maintained during organ culture for 5 days. The pD2 value for NE at the 17th day of gestation was 8.66 +/- 0.09, and that after organ culture for 5 days was 8.62 +/- 0.09. The sensitivity of 1-day-old neonatal artia was significantly lower than that of fetal atria; but when they were cultured for 24 h, there was a 10-fold increase in sensitivity. The pD2 value before culture was 7.59 +/- 0.05, and that after culture was 8.54 +/- 0.04. NE added to the culture medium prevented this increase in sensitivity. Similar changes were observed in the sensitivity to isoproterenol, but not in the sensitivity to forskolin, indicating that these sensitivity changes were of a postjunctional nature and most likely due to some changes in the beta-receptor and (or) its coupling to adenylate cyclase. Therefore, the decrease in myocardial sensitivity to NE observed during the late fetal period is most likely to be caused by factor(s) related to sympathetic innervation.     

Neural influences and norepinephrine sensitivity in the rat portal vein

Experimental evidence suggests that innervation can exert a long-term control and modulation on effector cell homeostasis. These trophic influences are known to occur between the motor innervation and skeletal muscles, tissues in which these phenomena have been well studied. A similar picture is also emerging to indicate that smooth muscles may be subjected to a neurotrophic influence. The present paper reviews and presents data obtained after chemical sympathectomy of rat portal vein with 6-hydroxydopamine. Basically three experimental protocols were utilized: 1) studies before and after complete in vitro denervation, using the vessel as its own control; 2) in vivo sympathectomy followed by in vitro studies at various times thereafter; and 3) experiments in which the veins were denervated in vitro, placed in organ culture, and treated with either norepinephrine (NE) or the vehicle. The results indicate that in rat portal vein the sympathetic innervation normally exerts a trophic influence mediated, at least in part, by NE. This evidence is discussed in relation to other smooth muscles insofar as supersensitivity mechanisms and other possible effects triggered by transmitter withdrawal. The discussion is extended to encompass ways by which NE could exert its effects, and the possibility of other trophic factors.     

Autonomic degeneration and altered blood pressure control in humans

The study of patients with partial or total defects of their sympathetic nerves can help clarify the role of the sympathetic nervous system in blood pressure control. Denervation supersensitivity of both beta and alpha receptors develops in humans and is proportional to the degree of sympathetic withdrawal. Although alterations in beta-receptor sensitivity are familiar responses to beta agonists or antagonists, patients with decreased norepinephrine (NE) levels appear to develop alpha-receptor supersensitivity of greater hemodynamic importance. When beta supersensitivity is marked, there may be a pressor response to beta blockade even when circulating levels of NE and epinephrine are very low. Indomethacin blocks prostaglandin synthesis and causes an increase in blood pressure in patients with partial autonomic neuropathies. The drug increases blood pressure by enhancing alpha- and diminishing beta-receptor sensitivity to NE, so it is effective only in patients who have some residual NE release.     

Cardiac norepinephrine transporter protein expression is inversely correlated to chamber norepinephrine content

The cardiac neuronal norepinephrine (NE) transporter (NET) in sympathetic neurons is responsible for uptake of released NE from the neuroeffector junction. The purpose of this study was to assess the chamber distribution of cardiac NET protein measured using [(3)H]nisoxetine binding in rat heart membranes and to correlate NE content to NET amount. In whole mounts of atria, NET was colocalized in nerve fibers with tyrosine hydroxylase (TH) immunoreactivity. NE content expressed as micrograms NE per gram tissue was lowest in the ventricles; however, NET binding was significantly higher in the left ventricle than the right ventricle and atria (P < 0.05), resulting in a significant negative correlation (r(2) = 0.922; P < 0.05) of NET to NE content. The neurotoxin 6-hydroxydopamine, an NET substrate, reduced NE content more in the ventricles than the atria, demonstrating functional significance of high ventricular NET binding. In summary, there is a ventricular predominance of NET binding that corresponds to a high NE reuptake capacity in the ventricles, yet negatively correlates to tissue NE content.     

Atrial supersensitivity to noradrenaline in stressed female rats

Stress can change the responses to catecholamines in many tissues. The aim of this study was to investigate the influence of the estrous cycle on the sensitivity of right atria to noradrenaline in female rats subjected to acute swimming stress. Female Wistar rats in proestrus, estrus, metestrus or diestrus were submitted to a 50 min-swimming session. Immediately after the exercise, the rats were killed and their right atria were mounted for isometric recording of the spontaneous beating rate. Concentration-effect curves to noradrenaline were obtained before and after the inhibition of neuronal uptake with phenoxybenzamine (10 microM) and of extraneuronal uptake with estradiol (5 microM). Acute swimming stress did not change the right atrial sensitivity to noradrenaline in rats in estrus, metestrus and diestrus. However, swimming stress produced supersensitivity to noradrenaline in proestrus (pD(2) control: 7.14 +/- 0.03 vs. pD(2) swimming: 7.55 +/- 0.04; p<0.05). This supersensitivity was still observed after uptake inhibition. When catecholamine uptake was inhibited, the concentration-effect curve to noradrenaline was shifted to the left 2.5-fold in the proestrus control group and 1.7-fold in the proestrus stress group (p<0.05). In conclusion, the estrous cycle influenced the acute stress-induced atrial supersensitivity to noradrenaline.     

Norepinephrine release in the heart atria of diabetic rats

The content, release and uptake of norepinephrine (NE) in the sympathetic nerves of the rat heart atria were studied in the course of diabetes and in age-matched controls. Diabetes was induced by streptozotocin (STZ) and rats were subjected to further experiments 1, 4 or 7 months later (STZ1, STZ4, STZ7). Isolated atria were superfused with oxygenated Krebs-Henseleit (KH) solution. After equilibration, four 10-min fractions were collected: B1, basal release of NE; S1, potassium-evoked release (KER), where NE outflow was stimulated by depolarisation with 50 mmol/l KCl; B2, basal release of NE under the influence of the neuronal uptake blocker desipramine (DES); S2, KER under the influence of DES. The content of NE was measured by radioimmunoassay. In STZ4 and STZ7 rats, NE concentrations were significantly lower in both atria compared to controls. B1 and S1 were significantly higher in STZ4 than in control atria. DES increased KER of NE in controls only. In contrast, DES caused a significant decrease in B2 and S2 in STZ4 atria, suggesting that a substantial portion of NE release was due to a calcium-independent carrier-mediated process. In experiments with calcium-free KH solution in fractions B2 and S2, KER ill controls was nearly abolished. However, in STZ4 and STZ7 atria, S2 was still significantly higher than B2. In conclusion, the NE-releasing mechanism may be different in the chronically diabetic animals than in healthy subjects and may contribute to the decreased NE concentration in the STZ atria.     

Assay systems for screening food components that have anti-proliferative and anti-invasive activity to rat ascites hepatoma cells: In vitro and ex vivo effects of green tea extract

We have developed in vitro and ex vivo assay systems for screening food components and natural substances that suppress the proliferation and/or invasion of a rat ascites hepatoma cell line of AH109A and have used them to study the effect of green tea extract. AH109A cells were found to penetrate underneath the monolayer of primary cultured mesothelial cells isolated from Donryu rat mesentery in the presence of 10% newborn bovine serum. Green tea extract inhibited this AH109A penetration in a dose dependent manner and also inhibited AH109A proliferation in vitro dose-dependently. Green tea tannin, the major polyphenolic substances in green tea extract, also inhibited the proliferation and invasion of AH109A in vitro in a dose dependent manner. When rat serum obtained 0.5 h after oral intubation of green tea extract was added to the culture media instead of newborn bovine serum at a concentration of 10%, the invasion of AH109A was significantly inhibited as compared to control rat serum (before green tea extract intubation); the inhibitory effect lasted for 1 h and disappeared 3 h after oral intubation of green tea extract, but those rat sera showed no inhibition of AH109A proliferation. These results suggest that green tea extract has an inhibitory effect on the invasion of AH109A both in vitro and ex vivo, but on the proliferation of AH109A only in vitro, and that these assay systems are effective for the screening of food components which inhibit tumor cell proliferation and invasion.     

Desipramine attenuates loss of cardiac sympathetic neurotransmitters produced by congestive heart failure and NE infusion

We reported recently that inhibition of neuronal reuptake of norepinephrine (NE) by desipramine prevented the reduction of sympathetic neurotransmitters in the failing right ventricle of right heart failure animals. In this study, we studied whether desipramine also reduced the sympathetic neurotransmitter loss in animals with left heart failure induced by rapid ventricular pacing (225 beats/min) or after chronic NE infusion (0.5 microg. kg(-1). min(-1)). Desipramine was given to the animals for 8 wk beginning with rapid ventricular pacing or NE infusion. Animals receiving no desipramine were studied as controls. We measured myocardial NE content, NE uptake activity, and sympathetic NE, tyrosine hydroxylase, and neuropeptide Y profiles by histofluorescence and immunocytochemical techniques. Effects of desipramine on NE uptake inhibition were evidenced by potentiation of the pressor response to exogenous NE and reduction of myocardial NE uptake activity. Desipramine treatment had no effect in sham or saline control animals but attenuated the reduction of sympathetic neurotransmitter profiles in the left ventricles of animals with rapid cardiac pacing and NE infusion. In contrast, the panneuronal marker protein gene product 9.5 profile was not affected by either rapid pacing or NE infusion, nor was it changed by desipramine treatment in the heart failure animals. The study confirms that excess NE contributes to the reduction of cardiac sympathetic neurotransmitters in heart failure. In addition, it shows that the anatomic integrity of the sympathetic nerves is relatively intact and that the neuronal damaging effect of NE involves the uptake of NE or its metabolites into the sympathetic nerves.     

Norepinephrine protects against apoptosis of mesenchymal stem cells induced by high glucose

In diabetes, the number of bone mesenchymal stem cells (MSCs) decreases and their differentiation is impaired. However, the exact mechanism is unclear. Patients with diabetes often experience sympathetic nerve injury. Norepinephrine (NE), a major mediator of the sympathetic nervous system, influences rat MSC migration in culture and in vivo. The present study aimed to investigate the effect of NE on MSCs under high glucose conditions; therefore MSCs were treated with high glucose and NE. High glucose-induced MSCs apoptosis, which was reversed by NE. To verify the effect of NE, mice underwent sympathectomy and were used to establish a diabetic model. Diabetic mice with sympathectomy had a higher apoptosis rate and higher levels of reactive oxygen species in their bone marrow-derived cells than diabetic mice without sympathectomy. High glucose inhibited p-AKT production and B-Cell CLL/Lymphoma 2 expression, and promoted BAX and caspase-3 expression. NE reversed these effects of high glucose. An AKT inhibitor enhanced the effects of high glucose. Thus, NE had a protective effect on MSC apoptosis induced by high glucose, possibly via the AKT/BCL-2 pathway.     

The topological characteristics of rat myocardial reactivity to noradrenaline, acetylcholine and a change in the electrostimulation frequency]

Dose-dependent effects of noradrenaline (10-7-10-6M), acetylcholine (10-8-3x10-6M) and stimulation rate (0.2-2.0 Hz) were obtained in experiments on myocardium preparations of the right and left atria and ventricles in rat. Three types of topological differences of the rat myocardium reactivity were observed: between the atria and ventricles (A/V), between the right and left atria and ventricles (R/L), between the right atrium (RA) and other cardiac chambers. A/V differences were most pronounced in the reactivity to acetylcholine (the atria were more reactive), the highest R/L differences were observed in the reactivity to noradrenaline (the myocardium of the right chambers was more reactive). RA reactivity greatly exceeded reactivity of other myocardial preparations to all three test influences. Topological peculiarities of chrono-inotropism permit supposing, that inotropic effects of rate changes in vivo are able to compensate, to some extent, the regional nonuniformity of cholin- and adrenergic regulatory inotropic effects.     

Effect of local and systemic desipramine administration on extracellular noradrenaline in the myocardium of rats

Evaluation of a local activity of the sympathetic system on the basis of norepinephrine (NE) level in the blood of myocardial vessels depends on at least three processes: NE release by sympathetic neurons, its reuptake and spillover of NE into the blood. The relations between these processes are different in various organs. Direct investigations of changes in the myocardial NE level under the effect of reuptake blockade by desmethylimipramine (DMI) were performed after appearance of the microdialysis technology. In this work the effects of local (through microdialysis membrane) and systemic administration of DMI on the NE release in a rat myocardium, were compared. Local DMI delivery increased myocardial NE level to 153 +/- 13% of the control level (0.17 +/- 0.026 ng/ml dialysate). NE concentration increased to 582 +/- 84% of control as a result of i.m. administration of 5 mg/kg DMI. No changes of the NE level in the venous blood were registered after systemic DMI. It is suggested that a relatively weak effect of local DMI is determined by saturation of DMI receptors adjacent to the probe and/or progressive diminution of the DMI effect with an increasing of the distance from the probe. Effect of systemic DMI administration depends on uniform blockade of all myocardial DMI receptors as well as the DMI influence on higher levels of the sympathetic system.     

Sympathetic control of heart rate in nNOS knockout mice

Inhibition of neuronal nitric oxide synthase (nNOS) in cardiac postganglionic sympathetic neurons leads to enhanced cardiac sympathetic responsiveness in normal animals, as well as in animal models of cardiovascular diseases. We used isolated atria from mice with selective genetic disruption of nNOS (nNOS(-/-)) and their wild-type littermates (WT) to investigate whether sympathetic heart rate (HR) responses were dependent on nNOS. Immunohistochemistry was initially used to determine the presence of nNOS in sympathetic [tyrosine hydroxylase (TH) immunoreactive] nerve terminals in the mouse sinoatrial node (SAN). After this, the effects of postganglionic sympathetic nerve stimulation (1-10 Hz) and bath-applied norepinephrine (NE; 10(-8)-10(-4) mol/l) on HR were examined in atria from nNOS(-/-) and WT mice. In the SAN region of WT mice, TH and nNOS immunoreactivity was virtually never colocalized in nerve fibers. nNOS(-/-) atria showed significantly reduced HR responses to sympathetic nerve activation and NE (P < 0.05). Similarly, the positive chronotropic response to the adenylate cyclase activator forskolin (10(-7)-10(-5) mol/l) was attenuated in nNOS(-/-) atria (P < 0.05). Constitutive NOS inhibition with L-nitroarginine (0.1 mmol/l) did not affect the sympathetic HR responses in nNOS(-/-) and WT atria. The paucity of nNOS in the sympathetic innervation of the mouse SAN, in addition to the attenuated HR responses to neuronal and applied NE, indicates that presynaptic sympathetic neuronal NO does not modulate neuronal NE release and SAN pacemaking in this species. It appears that genetic deletion of nNOS results in the inhibition of adrenergic-adenylate cyclase signaling within SAN myocytes.     

Acetylcholine supersensitivity in the rat heart produced by neonatal sympathectomy

Effects of neonatal sympathectomy with antiserum to nerve growth factor or 6-hydroxydopamine on the acetylcholine sensitivity of the rat left atria were investigated. Sensitivities to acetylcholine of atria from immunologically and chemically sympathectomized rats were much higher than that of control at 4 weeks of age. These results suggest possible involvement of the sympathetic nervous system in regulation of cardiac cholinergic sensitivity.     

Cardioprotection of electroacupuncture against myocardial ischemia-reperfusion injury by modulation of cardiac norepinephrine release

Augmentation of cardiac sympathetic tone during myocardial ischemia has been shown to increase myocardial O(2) demand and infarct size as well as induce arrhythmias. We have previously demonstrated that electroacupuncture (EA) inhibits the visceral sympathoexcitatory cardiovascular reflex. The purpose of this study was to determine the effects of EA on left ventricular (LV) function, O(2) demand, infarct size, arrhythmogenesis, and in vivo cardiac norepinephrine (NE) release in a myocardial ischemia-reperfusion model. Anesthetized rabbits (n = 36) underwent 30 min of left anterior descending coronary artery occlusion followed by 90 min of reperfusion. We evaluated myocardial O(2) demand, infarct size, ventricular arrhythmias, and myocardial NE release using microdialysis under the following experimental conditions: 1) untreated, 2) EA at P5-6 acupoints, 3) sham acupuncture, 4) EA with pretreatment with naloxone (a nonselective opioid receptor antagonist), 5) EA with pretreatment with chelerythrine (a nonselective PKC inhibitor), and 6) EA with pretreatment with both naloxone and chelerythrine. Compared with the untreated and sham acupuncture groups, EA resulted in decreased O(2) demand, myocardial NE concentration, and infarct size. Furthermore, the degree of ST segment elevation and severity of LV dysfunction and ventricular arrhythmias were all significantly decreased (P < 0.05). The cardioprotective effects of EA were partially blocked by pretreatment with naloxone or chelerythrine alone and completely blocked by pretreatment with both naloxone and chelerythrine. These results suggest that the cardioprotective effects of EA against myocardial ischemia-reperfusion are mediated through inhibition of the cardiac sympathetic nervous system as well as opioid and PKC-dependent pathways.     

Selegiline improves cardiac sympathetic terminal function and beta-adrenergic responsiveness in heart failure

Selegiline is a centrally acting sympatholytic agent with neuroprotective properties. It also has been shown to promote sympathetic reinnervation after sympathectomy. These actions of selegiline may be beneficial in heart failure that is characterized by increased sympathetic nervous activity and functional sympathetic denervation. Twenty-seven rabbits with rapid cardiac pacing (360 beats/min, 8 wk) and twenty-three rabbits without pacing were randomly assigned to receive selegiline (1 mg/day, 8 wk) or placebo. Rapid pacing increased plasma norepinephrine (NE) and decreased left ventricular fractional shortening, baroreflex sensitivity, cardiac sympathetic nerve terminal profiles, cardiac NE uptake activity, and myocardial beta-adrenoceptor density. Selegiline administration to animals with rapid ventricular pacing attenuated the increase in plasma NE and decreases in fractional shortening, baroreflex sensitivity, sympathetic nerve profiles, NE uptake activity and beta-adrenoceptor density. Thus selegiline appears to exert a sympatholytic and cardiac neuroprotective effect in pacing-induced cardiomyopathy. The effects are potentially beneficial because selegiline not only improves cardiac function but also increases baroreflex sensitivity in heart failure.     

Endogenous substrates for epidermal transglutaminase.

Potential in vivo substrates for epidermal transglutaminase have been isolated and partially characterized in human stratum corneum and new born rat epidermis. [14C]Putrescine and dansylcadaverine were incorporated into epidermal proteins in vitro. Two high molecular weight proteins incorporated the labels in both the rat ahd human homogenates. One of the proteins was too large to enter a 4% sodium dodecyl sulfate-polyacrylamide spacer gel; the other was seen at the interface between the spacer gel and a 10% sodium dodecyl sulphate-polyacrylamide running gel. These proteins were present in a buffer extract, sodium dodecyl sulphate-dithiothreitol extract and NaOH extract. The labels were also incorporated into protein in the insoluble pellet remaining after the afore-mentioned extractions. The incorporation of putrescine and dansylcadaverine was time dependent, and was inhibited by known inhibitors of epidermal transglutaminase. The two high molecular weight proteins had similar amino acid composition, characterized by high glycine, glutamic acid, serine and aspartic acid. The amino acid composition was similar to, although not identical with, the amino acid composition of alpha-keratin proteins. Epidermal homogenates incubated in the presence of transglutaminase showed progressive insolubilization of the protein. This cross-linking was inhibited by putrescine. [14C]Glycine, [14C]histidine and [4C]proline were incorporated into epidermal proteins in newborn rats in vivo. The glycine-labelled protein became progressively more insoluble when incubated in vitro in the presence of transglutaminase. In vitro incubation with transglutaminase had no effect on the histidine-and proline-labelled proteins.     

Cholinesterases and postnatal development of the negative chronotropic effects of acetylcholine in albino rats

The negative chronotropic effects of acetylcholine were studied in the isolated atria of the hearts of albino rats aged 2, 15, 29 and 47 days and adult. In untreated preparations, i.e. with full cholinesterase activity, the strongest effects were observed in newborn animals; with advancing age the reaction grew weaker. If cholinesterase activity was inhibited with physostigmine, the differences between the various age groups were obliterated. It is thus evident that the actual acetylcholine sensitivity of the sinoatrial node tissue does alter during postnatal life, but that growing cholinesterase activity reduces the amount of acetylcholine diffusing from the medium into the acetylcholine receptor zone. The change which takes place in cholinesterase activity in the myocardial tissue during postnatal life is so great that is must be taken into account when considering the development of cholinergic control of cardiac function.     

On the Phospholipid Metabolism of Glial Cell Primary Cultures: Cell Characterization and Their Utilization of 1-Alkyl-Glycerophosphoethanolamine

Abstract: Primary cultures prepared from newborn rat brain were grown for 16 or 17 days in culture. Addition of brain extract from newborn rats to the medium stimulated the maturation of astrocytes and the development of oligodendrocytes. Both cell types were characterized by morphology and by immunohistochemistry. The phospholipid composition of these cells was estimated. Incubations were performed with l-[³H]alkyl- sn -glycerophosphoethanolamine in varying concentrations for 3 h. About one-third of the substrate supplied was internalized by the cells. Several enzymic reactions were observed. The acylating enzyme system was the most active one—a K _m was determined with 5 nmol intracellular 1-alkyl- sn -glycerophosphoethanolamine/mg cell protein. Plasmalogen formation was rather low. 1-Alkyl- sn -glycerol, a hydrolysis product, was found in small amounts. Some radioactivity was also incorporated into the phosphatidylcholine fraction.     

Formation of a functional adrenergic input to intraocular cerebellar grafts: ingrowth of inhibitory sympathetic fibers.

The intraocular transplantation technique was used to study the ingrowth of peripheral sympathetic adrenergic nerves from the iris into transplants of fetal rat cerebellum, and the possible function of these nerves. The transplants, grown in oculo for one-half to eight months, were analyzed by fluorescence histochemistry and electrophysiological techniques. Peripheral sympathetic adrenergic fibers from the iris were able to grow into the cerebellar transplants and arborize in a pattern similar to that in situ, appearing in all three cortical layers and the noncortical areas of the transplants. The density of visible nerves without pretreatment and after preincubation in 10(-6) or 10(-5) M alpha-methylnorepinephrine was comparable to mature rat cerebellum. The spontaneous discharge of the Purkinje cells in oculo was inhibited by microiontophoresis of norepinephrine (NE) and amphetamine in sympathetically innervated, as well as sympathectomized transplants denervated by ganglionectomy. The NE response was blocked by the adrenergic beta-receptor blocker MJ-1999. GABA also inhibited the Purkinje cell activity while glutamate accelerated the discharge. Parenteral amphetamine inhibited Purkinje cell activity in sympathetically innervated transplants, but was ineffective in denervated transplants. The Purkinje cell spontaneous activity was inhibited by electrical stimulation of the NE fiber input through the cervical sympathetic trunk. This inhibition could be antagonized by parenteral reserpine or the beta-adrenergic antagonist propranolol. The responses of the Purkinje cells within the transplants to drugs and transmitters mimic those of the adult rat in situ. In view of the fluorescence histochemical evidence for an ingrowth of peripheral sympathetic adrenergic fibers into the cerebellar transplants, and the results of stimulating the sympathetic trunk, it is suggested that peripheral adrenergic fibers may be able to establish functional connections with the Purkinje cells similar to the cerebellar adrenergic synapses normally formed in situ by fibers from the locus coeruleus.     

Atrial natriuretic factor: atrial conversion of high to low molecular weight forms

The atrial natriuretic factor elutes by gel filtration in high and low molecular weight fractions. Extraction and elution of rat atria in 1.0 M acetic acid yielded a predominance of the high molecular weight form(s); whereas when these procedures were carried out in 0.1 M acetic acid, there was a predominance of the low molecular weight forms. When partially purified high molecular weight natriuretic activity was eluted in 0.1 M acetic acid, the high molecular weight form(s) remained intact. When partially purified high molecular weight natriuretic activity was mixed with crude atrial extract in 0.1 M acetic acid, there was an apparent conversion to the low molecular weight forms. Extraction of rat atria in boiling 0.1 M acetic acid blocked this conversion. It is concluded that rat atria contain a heat labile factor that converts high molecular weight natriuretic activity to the low molecular weight forms.