Adult Rat Atria Research Articles

Do Cardiac Neurons Play a Role in the Intrinsic Control of Heart Rate in the Rat?

Three experiments were performed to see whether cardiac neurons contribute to the intrinsic control of heart rate in right atria of adult rats. The intrinsic heart rate response (IRR) was examined by raising right atrial pressure from 2 to 8 mmHg for 3 min. In isolated preparations of the right atrium, the IRR was not significantly altered by the addition of either 1 microM atropine (n =6; control +19+/- 3 min(-1) ; atropine+18+/-3 min(-1); (mean /+/-S.E.M.)) or 1 microM propranolol (n = 5; control +22+/- 4 min(-1); ; propranolol +21+/-3 min(-1); ). Tetrodotoxin (0.5 microm) had no effect on the IRR (n = 6; control +37+/-5 min(-1); tetrodotoxin 38+/-5 min(-1); ). In another experiment, 2-day-old rat pups were injected with capsaicin (50 mg kg(-1); treated) or with vehicle(control). There was no difference in the IRR of right atrial preparations taken from control and treated animals after they reached adulthood (control (n = 7) and treated (n = 8): +30+/- 4 and +32+/- 4 min(-1)). The influence of right atrial pressure on the efficacy of vagal stimulation was examined. The rate response to vagal stimulation was reduced similarly in control and treated preparations when pressure was elevated from 2 to 4 mmHg (control and treated: -34+/- 5% and -33+/- 3%). The effectiveness of the capsaicin treatment was confirmed by the depletion of substance P-immunoreactive nerve fibres in cardiac tissues. Together, these results strongly suggest that cardiac neurons are not involved in intrinsic heart rate control.

The Regeneration of Peripheral Noradrenergic Nerves after Chemical Sympathectomy in Diabetic Rats: Effects of Nerve Growth Factor

The study investigated the role of nerve growth factor (NGF) in the regeneration of noradrenergic nerves of the right atria from control and 8-week diabetic rats, after lesion caused by a single injection of 6-hydroxydopamine (6-OHDA, 100 mg/kg ip). This treatment caused a profound depletion of tissue noradrenaline (NA) of the right atria from both control and diabetic groups, followed by a progressive repletion that was not complete at 49 days. Immunoreactivity for the NGF receptorstrkA and p75NTRwas decreased and increased, respectively, between days 3 and 28 in right atria from diabetic rats and returned to pretreatment levels at day 49. Receptor levels were not significantly altered in controls. In contrast to tissue NA, at day 14 functional responses to electrical nerve stimulation of the right atria had completely returned to the pretreatment state in diabetic rats and were very close to normal in nondiabetic rats. NGF treatment (1 mg/kg, three times/week, for 2 weeks) increased tissue NA only in control rats; the pattern was similar after 6-OHDA. These findings are consistent with the hypothesis that NGF normally plays a role in the regulation of autonomic sympathetic nerves in the adult rat atrium and that mature and uninjured sympathetic neurons remain responsive to NGF. In injured noradrenergic neurons, NGF promotes regeneration in nondiabetic rats. The ability of NGF to promote regeneration of noradrenergic nerves is lost in diabetes and this may relate to the loss oftrkA receptor on prejunctional nerve terminals after denervation.

Differential cholinoceptor subtype-dependent activation of signal transduction pathways in neonatal versus adult rat atria

In this study, we investigated the expression and distribution of muscarinic cholinergic receptors (mAChRs) and the different signaling pathways associated with mAChR activation in atria isolated from adult and neonatal rats. Carbachol stimulation of mAChRs in both neonatal and adult rat atria led to a negative inotropic response with activation of phosphoinositide hydrolysis, an increase in cyclic GMP levels, and a decrease in cyclic AMP production. However, compared with adult atria, neonatal atria showed hypersensitivity in the contractile effect induced by carbachol. Pharmacological analysis with mAChR antagonists indicated that M 1 and M 2 mAChR subtypes are important mediators of the response to carbachol in neonatal atria. In contrast, in adult atria the effect of the agonist was coupled only to the M 2 mAChR subtype. Moreover, an increased number of total mAChRs was labeled in neonatal atrial membranes compared with those of adults. Although a predominant M 2 mAChR population is expressed in atria at both stages of development studied, competition binding parameters calculated for carbachol indicated the presence of high-affinity binding sites, with higher affinity in neonates than in adults. These results suggest that the differences observed between neonatal and adult atria in their response to a cholinergic agonist may be related to differential expression of mAChR subtypes and/or changes in functional coupling of mAChR subtypes during development.

Pharmacological evidence for the existance of different subtypes of muscarinic acetylcholine receptors for phosphoinositide hydrolysis in neonatal versus adult rat atria

The negative inotropic effect of carbachol, as well as phosphoinositide hydrolysis, was measured in atria from neonatal and adult rats. Carbachol increased phosphoinositide hydrolysis and decreased dF dt of both neonatal and adult atria; however, the neonatal atria showed hyperactivity to carbachol as compared with adult atria. Inhibition of phospholipase C reduced the supersensitivity to carbachol upon contractility in neonatal atria producing values similar to those of the adult atria, suggesting that muscarinic acetylcholine receptor (mAchR) stimulation is secondary to receptor-mediated hydrolysis of phosphoinositides. Pharmacological analysis with mAchR antagonists tends to support the idea that ml and m2 subtypes are the most important mediators of the response to carbachol in neonatal atria. In adult atria, the effect of carbachol is coupled only to mAchR m2 subtypes.

Selective changes in natriuretic peptide and early response gene expression in isolated rat atria following stimulation by stretch or endothelin-1

Important physiological and pathophysiological conditions are associated with changes in secretion and synthesis of atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP). The aim of this study was to examine the effects of mechanical stretch and endothelin-1 on ANF and BNP secretion and gene expression in isolated adult rat atria, as paradigms for mechanical and endocrine stimulation of atrial tissue. The expression of the early response genes c-fos, c-jun, Egr-1, and c-myc was also studied, since their protein products may be involved in controlling natriuretic peptide gene expression. Isolated rat atria were stimulated by stretch (5 g) or endothelin-1 (10(-7) M) for 30 min, 2 h, or 4 h. ANF and BNP secretion was measured by radioimmunoassay, and relative mRNA levels were determined by northern blotting. Atrial stretch resulted in an immediate 1.8-fold increase in ANF release, which returned to basal levels after 160 min. Endothelin-1 caused a gradual increase in ANF release, up to 2.3 times basal levels, and thereafter returned towards basal levels. BNP secretion was increased threefold by endothelin-1, and remained significantly raised for 90 min. BNP mRNA levels were transiently increased by 33% after 2 h of endothelin-1 stimulation. Stretch increased c-fos mRNA levels (+55%) and Egr-1 mRNA levels (+70%) after 2 h, and increased c-myc mRNA levels (+69%) after 4 h. Endothelin-1 increased Egr-1 mRNA levels up to +767% after 4 h. Endothelin-1 stimulates BNP secretion from rat atria; this is followed by an increase in BNP mRNA levels. Conversely, acute secretion of ANF by stretch or endothelin-1 is not accompanied by changes in ANF mRNA levels. Atrial stretch results in changes in the expression of the early response genes c-fos, Egr-1, and c-myc, while endothelin-1 stimulates Egr-1 expression. The specific changes in natriuretic peptide and early response gene expression reveal distinct mechanisms of modulation of atrial gene expression by mechanical and endocrine stimuli.

Post-translational processing of atrial natriuretic factor by adult rat atrial cardiocytes in culture

Post-translational processing of the cardiac polypeptide hormone atrial natriuretic factor (ANF) was studied using primary cultures of cardiocytes derived from adult rat atria. Atrial cardiocytes attached to microcarrier beads were maintained for up to 15 days under continuous superfusion in minichromatographic columns. The cultures were characterized for their ability to store, process, and release ANF and by immunofluorescence microscopy for ANF, desmin, and myosin. Nuclear staining using the fluorescent DNA stain Hoechst 33258 was carried out to determine the total number of cells in culture. Column eluates were assayed for ANF by radioimmunoassay and analyzed by reverse phase high-performance liquid chromatography. For comparison purposes, superfusion experiments using freshly isolated cardiocytes supported in Bio-Gel P2 were carried out. Freshly isolated atrial cardiocytes stored high molecular weight ANF (5.2 +/- 1.9 pmol/micrograms DNA) and released mostly (83.3 +/- 6.7%) low molecular weight ANF, at an average rate of 97 +/- 18 fmol.min-1 x micrograms-1 DNA. The cell content and the rate of release of ANF after 15 days in culture were 1.3 +/- 0.4 pmol/micrograms DNA and 1.7 +/- 0.4 fmol.min-1 x micrograms-1 DNA, respectively, and 62.7 +/- 6.3% of the released peptide was of a low molecular weight. There was no correlation between changes in cell population and the extent of processing. Cultures of noncardiocytes, superfused with exogenous proANF, did not significantly process proANF to a lower molecular weight peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

A Clathrin-Coated Vesicle-Mediated Pathway in Atrial Natriuretic Peptide (ANP) Secretion

Atrial Natriuretic Peptide (ANP), produced by atrial cardiomyocytes, is an endogenous hypotensive agent that brings about vasodilation and diuresis. Similar to other polypeptide hormones, ANP is synthesized as a precursor, preproANP. The preprohormone is processed intracellularly and is stored in secretory granules as the prohormone. During the events of exocytosis, the prohormone is converted to its active form, ANP. In this study, a double-label immunocytochemistry experiment was performed using ANP and clathrin antibodies to determine if the transport of this hormone is mediated by clathrin-coated vesicles. Additionally, we have isolated clathrin-coated vesicle (CVs) from adult rat atria using immunoadsorption, and have characterized the fraction by using SDS PAGE, TEM, and Western blot analysis. The data demonstrate that: (1) ANP and clathrin co-localize in myocardial tissue, (2) clathrin-coated vesicles can be isolated from adult rat atria, and (3) clathrin-coated vesicles isolated from adult atrial myocardium contain predominantly proANP. The presence of proANP in clathrin-coated vesicles suggest that this polypeptide hormone is transported intracellularly via a clathrin-mediated pathway and during transit the prohormone is not significantly converted to its active form.

Isolation of adult cardiomyocytes initiates a return of inositol trisphosphate phosphorylating activity.

1. We have previously reported that the addition of noradrenaline to [3H]-inositol-labelled adult rat atria or isolated perfused hearts caused the release of inositol-1,4,5-trisphosphate, which was metabolized by dephosphorylation to inositol-4-monophosphate. Inositol-1,3,4,5-tetrakisphosphate and its dephosphorylation products were not detected. 2. In the current study, the addition of noradrenaline to [3H]-inositol-labelled adult rat cardiomyocytes caused the release of inositol-1,4,5-trisphosphate, which was metabolized in part by phosphorylation to inositol-1,3,4,5-tetrakisphosphate. 3. These results demonstrate that the isolation and culture of rat adult cardiomyocytes initiates enhanced generation of inositol-1,3,4,5-tetrakisphosphate. This change would be expected to enhance the calcium response of the cells to stimulation of alpha 1-adrenoceptors.

Characteristics of binding of endothelin-1 and endothelin-3 to rat hearts. Developmental changes in mechanical responses and receptor subtypes.

Endothelin-1 (ET-1) and endothelin-3 (ET-3) produced positive inotropic effects on electrically stimulated left atria and increased the frequency of spontaneously beating right atria of adult rats. The potency of the inotropic effect of ET-1 was greater than that of ET-3, but the potencies of the chronotropic effects of ET-1 and ET-3 were not significantly different. In the neonatal atria, ET-1 and ET-3 also induced positive inotropic and chronotropic responses. ET-1 and ET-3 showed weak or no cardiotonic effects on the adult ventricles, whereas they caused marked positive inotropy in the neonatal ventricles. The characteristics of binding sites for ET-1 and ET-3 were very similar between the atria and the ventricles of the rat neonate. Saturation and competition binding experiments have shown that neonatal cardiac membranes from both atria and ventricle have two distinct binding sites for endothelin, that is, a low-affinity and a high-affinity site. ET-1 was found to bind to the low-affinity sites with a significantly lower Kd than ET-3, whereas the estimated Kd values for ET-1 and ET-3 at the high affinity sites were similar. In contrast, the binding sites in adult atria were different from those of the ventricles: only a single binding site for both ET-1 and ET-3 was detected. Adult atrial membranes, on the other hand, had two distinct binding sites similar to those of neonatal membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning and expression of a rat cardiac delayed rectifier potassium channel.

We have cloned a cDNA (designated RAK) coding for a delayed-rectifier K current (IRAK) from adult rat heart atrium and expressed it in Xenopus oocytes. RAK differs from the cloned rat brain K current, BK2 [McKinnon, D. (1989) J. Biol. Chem. 264, 8230-8236], by one amino acid at residue 411. RAK expressed in oocytes compares closely to the intrinsic adult rat atrial delayed-rectifier current measured by using whole-cell recording of single isolated cells. Northern blot analysis confirmed the presence of the channel in adult rat atrium, and to a lesser extent, in rat ventricle. IRAK activates with time constants ranging from 58 ms at -20 mV to 6 ms at +60 mV and does not show significant inactivation over 800 ms. It is blocked by 4-aminopyridine greater than barium much greater than tetraethylammonium chloride, which is similar to the relative potencies of these blockers on the native delayed rectifier current. We conclude that the main delayed rectifier K current in adult rat atria is virtually identical to a neuronal delayed rectifier, BK2.

The membrane-bound bifunctional peptidylglycine alpha-amidating monooxygenase protein. Exploration of its domain structure through limited proteolysis.

The biosynthesis of alpha-amidated peptides from their glycine-extended precursors is catalyzed by the sequential action of peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL). The two enzymes are part of a bifunctional, integral membrane protein precursor, peptidylglycine alpha-amidating monooxygenase (PAM). The major forms of PAM mRNA in the adult rat atrium differ by the presence or absence of optional exon A, a 315-nucleotide segment separating the PHM and PAL domains. Using antipeptide antibodies specific to the PHM, exon A, PAL, and cytoplasmic domains of rat PAM, carbonate-washed atrial membranes were found to contain proteins corresponding to rPAM-1 and rPAM-2. Digestion of atrial membranes with a variety of endoproteinases released PHM and PAL catalytic activities. Dose-response curves indicated that both catalytic activities were extremely resistant to inactivation by trypsin. Endoproteolytic digestion of atrial membranes with trypsin, chymotrypsin, elastase, thermolysin, or endoproteinase Lys-C generated a 35-kDa PHM fragment. Digestion with trypsin, elastase, thermolysin, or endoproteinase Lys-C generated a 42-kDa PAL fragment. In contrast to the stability exhibited by the PHM and PAL domains, the cytoplasmic domain of PAM was destroyed by most of the enzymes; only digestion with endoproteinase Lys-C generated a stable fragment. Digestion with endoproteinase Arg-C removed the carboxyl-terminal tail from PAM but failed to release the PHM or PAL domains from the membranes. The PHM fragments generated by some of the endoproteinases showed a tendency to adhere to the membranes. Thus the bifunctional PAM protein consists of independent catalytic domains separated from each other and from the putative transmembrane domain by flexible regions accessible to attack by a wide variety of endoproteinases.

Characterization of novel mRNAs encoding enzymes involved in peptide alpha-amidation

The COOH-terminal alpha-amidation of bioactive peptides is a 2-step process catalyzed by two separable enzymatic activities both derived from the peptidylglycine alpha-amidating monooxygenase (PAM) precursor. Two forms of PAM mRNA (rPAM-1 and -2), differing by the presence or absence of optional Exon A, were previously characterized; both encode precursors predicted to have an NH2-terminal signal sequence, an intragranular domain containing both enzymatic activities, and a single transmembrane domain followed by a short, cytoplasmic COOH-terminal domain. In this report, two novel types of PAM mRNA were identified in adult rat atrium. A cDNA of each type was sequenced, and the results indicate that rPAM-3 and -4 could be related to each other and to the previously characterized rat PAM cDNAs by alternative mRNA splicing. Deletion of a 258-nucleotide segment (optional Exon B) encoding the transmembrane domain from rPAM-3 and the presence of a novel 3'-exon in rPAM-4 mean that both rPAM-3 and -4 mRNAs encode precursor proteins that have an NH2-terminal signal peptide but lack a transmembrane domain. The rPAM-4 precursor protein lacks the region of the PAM precursor catalyzing the second step in the alpha-amidation reaction. Low levels of rPAM-3 and -4 type mRNA were detected in atrium. Utilizing the polymerase chain reaction, two major patterns of distribution of forms of PAM mRNA were found. In the heart and central nervous system, PAM mRNAs both containing and lacking optional Exon A were prevalent and almost all of the PAM mRNAs detected contained optional Exon B. In the pituitary and submaxillary glands, PAM mRNAs lacking optimal Exon A were prevalent, as were PAM mRNAs lacking all or part of optional Exon B. Since the distribution of PAM activity between soluble and membrane fractions is tissue-specific and developmentally regulated and since rPAM-4 lacks an enzymatic portion of the PAM precursor, the tissue-specific expression of these forms of rat PAM mRNA is expected to be of functional significance.

Developmental regulation of peptidylglycine α-amidating monooxygenase (PAM) in rat heart atrium and ventricle: Tissue-specific changes in distribution of PAM activity, mRNA levels, and protein forms

The high levels of peptidylglycine alpha-amidating monooxygenase (PAM, EC 1.14.17.3) found in adult rat atrium led us to examine PAM expression in rat atrium and ventricle from embryonic day 14 through adulthood. Immunocytochemical studies using antisera to PAM identified cardiocytes as the major site of PAM expression in atrium and ventricle throughout development. Levels of PAM mRNA and PAM activity exhibited distinctly different developmental profiles in atrium and ventricle. Ventricular PAM mRNA and PAM activity were highest from embryonic days 14 through 18, declined at the time of birth, rose slightly during the first postnatal week, and declined toward adult levels. Atrial PAM mRNA and PAM activity were low at embryonic day 14, rose to a peak immediately before birth, declined at the time of birth, and then rose after birth. Levels of atrial PAM mRNA and PAM activity were not directly correlated at all developmental stages. Two major forms of PAM mRNA (4.2 +/- 0.1 and 3.8 +/- 0.1 kilobase(s] were identified in atrium and ventricle throughout development. The prevalence of the two forms varied with developmental stage, with atrium and ventricle containing similar forms at each stage. Western blots of atrial and ventricular membranes revealed the existence of a developmental stage-specific distribution of PAM protein among forms ranging in mass from 125 to 94 kDa. In both atrium and ventricle PAM activity was primarily soluble from embryonic days 14 through 16 and primarily particulate after birth. The role of PAM in the heart is not yet clear, but the presence of tissue-specific and developmentally regulated alterations in PAM mRNA, PAM protein, and PAM activity suggests that this peptide processing enzyme plays a key role in the heart.

Acetylcholinesterase molecular forms in rat heart

A comparison of the molecular forms of acetylcholinesterase (AChE) in adult rat atria and ventricles was undertaken. The major forms present in both the atria and ventricles were globular 4S (G1) and 10S (G4) and asymmetric 16S (A12) with minor contributions from 6S (G2) and 12S (A8). Although the total specific AChE activity was higher in atrial samples, no differences in the proportions of the major AChE forms between the atrial and ventricular samples were seen. For example, 16S AChE accounted for 8% to 10% of the total AChE activity in all examined regions of the heart. Cardiac 16S AChE was shown to be soluble in high ionic strength buffer. The addition of EDTA to the extraction buffer resulted in no further solubilization of 16S AChE, indicating that only Type I asymmetric AChE is present in the heart. Additionally, 16S AChE did not require Triton X-100 for extraction. In contrast, 35% of the globular AChE required non-ionic detergent for extraction, which indicates that a percentage of globular AChE in rat heart is membrane-associated.

The effects of catecholamines on tension reactivation in cardiac muscle.

The effects of adrenaline and the beta-agonist isoprenaline on the time course of tension reactivation were studied in several cardiac tissues. The aim of the study was to assess whether experimental evidence can be found for a role of the sarcoplasmic reticulum in the reactivation of tension. It was assumed that calcium recycles between different parts of the reticulum, and that this recycling may affect tension repriming. Isoprenaline was assumed to enhance such recycling by increasing the uptake of calcium, following its release during a preceding contraction. Isoprenaline (in the range of 40 nM to 4 microM) was found to enhance tension repriming in adult guinea pig atria. However, in adult rat atria, isoprenaline often gave a complex effect, with a smaller degree of repriming at short intervals, and enhanced repriming at longer intervals. This was thought to reflect the balance between the enhancing effect of the drug on calcium recycling and an augmented release from the sarcoplasmic reticulum (SR). In striking contrast, there was no effect of isoprenaline on tension repriming in neonatal guinea pig atria and a retardation in neonatal rat atria. This was interpreted as reflecting the lack of a sarcoplasmic network in the neonatal tissue. The effects of isoprenaline on tension repriming in the frog atrium (which also has a sparse sarcoplasmic reticulum network) were also found to be complex; low concentrations (40 nM) enhanced the process, and high concentrations (0.4 microM) retarded it. Intermediate levels often produced a 'crossover' effect: more reactivation at short intervals, and less at long intervals. The interpretation of these results was that there are two processes which interact to determine the amount of tension produced at short intervals after each contraction: the basal reactivation process and some augmenting mechanism superimposed on it. This mechanism is probably related to other behavioural features of cardiac muscle, such as rate-dependent increases in membrane calcium currents. It is relevant mainly in those cases where tension repriming depends on membrane calcium currents. Further experiments (in the frog atrium) with elevated calcium and with the alpha-adrenergic agonist phenylephrine (both of which slowed down the reactivation process) also support this idea. These agents elevate internal calcium levels, and presumably saturate the augmenting mechanism (by producing maximal tension responses).(ABSTRACT TRUNCATED AT 400 WORDS)

Atriopeptin distribution in the developing rat heart.

The embryonic distribution of atriopeptin (atrial natriuretic factor) in the Sprague-Dawley rat heart was mapped by immunoperoxidase staining of embryonic and neonatal hearts using rabbit antiserum to atriopeptigen purified from adult rat atrium. During the period of cardiac septation (days 14 and 16), immune serum reacted strongly with myocardial cytoplasmic granules in two sites: the inner cell layer along the cephalic curvature of the atria and the trabeculae of the incompletely divided ventricles. The youngest hearts studied (gestational day 11) displayed only nonspecific diffuse peroxidase reactivity within blood cells, indistinguishable from control sections incubated with normal rabbit serum. One week following birth, intense anti-atriopeptin reactivity was widely distributed through both atria. In addition, immunoreactive cytoplasmic granules were found at several sites in the ventricular myocardium. Along the fiber tracts of the concentric layers of the ventricular walls and interventricular septum, scattered granular foci were seen between nuclei of contiguous elongated myocytes. Positive staining was also seen within the papillary muscles and trabeculae carnae, regions shown by Alcian blue/periodic acid-Schiff base staining of sister sections to be relatively rich in glycogen. These patterns of antibody reactivity suggest the coupling of early atriopeptin secretory activity with developing cardiac function.