Jana Slavikova

Catecholaminergic Neurons in the Rat Intrinsic Cardiac Nervous System

Immunoreactivities (IR) for catecholamine-synthesizing enzymes tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DβH), phenylethanolamine N-methyl transferase (PNMT), serotonin-synthesizing enzyme tryptophan hydroxylase, and neuropeptide Y were investigated in the intrinsic cardiac nervous system of 27–40–day-old rats using fluorescent immunohistochemistry. Individual neurons were identified by the general neuronal marker protein gene product 9.5. The presence of DβH and PNMT in the atrial specimens was verified using reverse transcriptase-polymerase chain reaction. Two types of catecholamine-handling intrinsic ganglion neurons were observed: small intensely fluorescent (SIF) cells and large-diameter neurons. SIF cells exhibited TH- and tryptophan hydroxylase-IR, but they were not positive for DβH. In contrast, large-diameter intrinsic TH-positive neurons, showing in majority also NPY-IR, displayed also DβH- and PNMT-IR, thus indicating the capacity for the synthesis of norepinephrine and epinephrine, respectively. In conclusion, the SIF cells are most probably dopaminergic and serotonergic neurons, whereas large-diameter intrinsic cells seem to represent a subpopulation of norepinephrine- and/or epinephrine-secreting neurons.

Developmental changes in the expression of nicotinic acetylcholine receptor α-subunits in the rat heart

Neuronal nicotinic acetylcholine receptors (nAChR) are ligand-gated ion channels that consist of various subunits. During ontogeny, muscular and neuronal nAChR undergo changes in the distribution and subunit composition in skeletal muscle and brain, respectively. Here, we have investigated the occurrence of the ligand-binding α-subunits of neuronal nAChR by means of reverse transcription/polymerase chain reaction and immunohistochemistry in the rat heart during prenatal and postnatal development and after capsaicin-induced sensory denervation. mRNAs coding for the α4, α5, α7 and α10 subunits were detected throughout all developmental stages. Messenger coding for the α2 subunit was first detectable at developmental stage E20; α3 subunit mRNA was expressed throughout all prenatal developmental stages, whereas it was restricted postnatally to the atria. mRNA for α6 was observed at E14-P8 but was absent thereafter. At no developmental stage could an unequivocal signal for α9 nAChR subunit mRNA be obtained. The expression pattern was unchanged by capsaicin treatment. Immunohistochemistry demonstrated α7 subunits on cardiac neurons, fibroblasts and cardiomyocytes and α2/4 subunits on cardiomyocytes with a postnatal redistribution to intercalated discs, as shown by cryo-immunoelectron microscopy. Our results indicate an additional non-neuronal expression of nAChR subunits in the rat heart that, as in skeletal muscle, precedes functional innervation and then undergoes changes in its distribution on the surface of cells.

Expression of neuropeptide Y and its receptors Y1 and Y2 in the rat heart and its supplying autonomic and spinal sensory ganglia in experimentally induced diabetes

Diabetic cardiomyopathy, involving both cardiomyocytes and the sensory and autonomic cardiac innervation, is a major life-threatening complication in diabetes mellitus. Here, we induced long-term (26-53 weeks) diabetes in rats by streptozotocin injection and analyzed the major cardiac neuropeptide signaling system, neuropeptide Y (NPY) and its receptors Y1R and Y2R. Heart compartments and ganglia supplying sympathetic (stellate ganglion) and spinal sensory fibers (upper thoracic dorsal root ganglia=DRG) were analyzed separately by real-time reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Ventricular, but not atrial innervation density by NPY-immunoreactive fibers was diminished, and preproNPY expression was transiently (26 weeks) reduced in left atria, but remained unchanged in sympathetic neurons and was not induced in DRG neurons. In all ganglia and heart compartments, Y1R expression dominated over Y2R, and Y1R-immunoreactivity was observed on cardiomyocytes and neuronal perikarya. Atrial, but not ventricular Y1R expression was up-regulated after 1 year of diabetes. Collectively, these data show that a disturbance of the cardiac NPY-Y1R/Y2R signaling system develops slowly in the course of experimentally induced diabetes and differentially affects atria and ventricles. This is in parallel with the clinically observed imbalances of the cardiac autonomic innervation in diabetic cardiac autonomic neuropathy.

Cardiomyopathy in streptozotocin-induced diabetes involves intra-axonal accumulation of calcitonin gene-related peptide and altered expression of its receptor in rats

Calcitonin gene-related peptide (CGRP) is a vasorelaxant and positive inotropic and chronotropic peptide that binds to the calcitonin receptor-like receptor. In the heart, upon stimulation CGRP is released from sensory nerve terminals and improves cardiac perfusion and function. In the present study, we investigated alterations in the components of the CGRP signaling system during development of diabetic cardiomyopathy. Rats received a single injection of streptozotocin. Four, 8, and 16 weeks thereafter cardiac CGRP content (radioimmunoassay), calcitonin receptor-like receptor expression (by real-time RT-PCR), and CGRP and calcitonin receptor-like receptor tissue distribution (immunohistochemistry) were assessed. CGRP content of atria and ventricles progressively increased during the 4 months following streptozotocin-treatment, while the distribution of CGRP-immunoreactive fibers was not visibly altered. Conversely, cardiac expression of calcitonin receptor-like receptor initially (4 weeks after treatment) increased but then gradually declined to 47% of control levels in both atria after 16 weeks. These quantitative changes were not associated with altered cellular distribution patterns (primarily in venous and capillary endothelium). Since sensory neurons have been reported to decrease expression of the CGRP precursor in the course of diabetes, the intra-axonal accumulation of CGRP observed here reflects impaired release, which, coupled with the down-regulation of its cognate receptor, calcitonin receptor-like receptor, may contribute to the well-documented impairment of cardioprotective functions in diabetes.

Choline acetyltransferase in the heart of adult rats

The distribution of choline acetyltransferase (ChAT, EC 2.3.1.6) in the heart of adult rats has been reinvestigated in view of recent discoveries that acetylcholine (ACh) can be synthesized not only by ChAT, but also by carnitine acetyltransferase (CarAT, EC 2.3.1.7) and that it is possible to distinguish between the ACh-synthesizing activity of ChAT in intramuscular nerves and the CarAT-mediated extraneural synthesis of ACh by means of bromoacetylcholine (BrACh), a specific inhibitor of ChAT. BrACh (0.002 mmol/l) has been found to inhibit the synthesis of ACh in the atria by 66–85% and in the ventricles by only 19–29%. Bromoacetylcarnitine (BrACar, 0.02 mmol/l), an inhibitor of CarAT, inhibited the synthesis of ACh in the atria by 34% and in the ventricles by 74–80%. These findings indicate that ChAT is responsible for most of the synthesis of ACh observed in the homogenates of the atria; in the ventricles, it catalyses only a minor portion of the total ACh synthesis observed. In the investigation of the regional distribution of ChAT in the heart, the BrACh-sensitive part of ACh synthesis was taken as the measure of ChAT activity. The highest activity of ChAT (nmol ACh synthesized ·g−1·h−1) was found in the region of the sinoatrial aode (1775); it decreased in the order: interatrial septum (781) > rest of the right atrium (712) > left atrium (416) > basal part of the right ventricle (366) > apical part of the right ventricle (250) > inter-ventricular septum (239) > basal and apical part of the left ventricle (208 and 205). The results indicate that earlier investigations of the distribution of ChAT in the heart provided a basically correct picture although the contribution of CarAT to the synthesis of ACh measured had not been excluded, and confirm that ChAT is present throughout the heart, including the apical parts of the ventricles. However, the sino-atrio-ventricular gradient of ChAT distribution is steeper when the contribution of CarAT to the synthesis of ACh is excluded.

Down-regulation of vasoactive intestinal peptide and altered expression of its receptors in rat diabetic cardiomyopathy

Vasoactive intestinal peptide (VIP) is a vasorelaxant peptide that addresses two receptor subtypes, VPAC1 and VPAC2. It stimulates insulin secretion and mediates anti-inflammatory effects and has been proposed for treatment of type 2 and autoimmune diabetes. In the heart, VIP is produced and released primarily by intrinsic neurons and improves cardiac perfusion and function. Here, we investigated the involvement of this system in the events underlying development of experimentally induced diabetic cardiomyopathy. Rats received a single streptozotocin injection, and cardiac VIP content [radioimmune assay (RIA)], expression of the VIP precursors VPAC1 and VPAC2 [real-time reverse transcription-polymerase chain reaction (RT-PCR)], and VPAC1 and VPAC2 tissue distribution (immunohistochemistry) were assessed 4, 8, and 16 weeks thereafter and compared with corresponding vehicle-treated controls. Cardiac neuropathy manifests progressively during the first 4 months of diabetes at the preproVIP mRNA and VIP peptide level and is accompanied by initial down-regulation of VPAC2 at one prime target of VIP-containing axons, i.e., smooth muscle cells of coronary arterioles. VPAC1 is expressed by macrophages. After initial changes that are specific for atria and ventricles, respectively, VPAC1 and VPAC2 expression return to control levels at 16 weeks despite ongoing loss of VIP. Given the cardioprotective role of the VIP signaling system, the persistence of receptors has therapeutic implications since it is the prerequisite for trials with VPAC2 agonists.

Heterogenous changes in neuropeptide Y, norepinephrine and epinephrine concentrations in the hearts of diabetic rats

The changes in concentrations of neuropeptide Y (NPY), norepinephrine and epinephrine were investigated in the rat hearts 1, 2, 4, 6, 9 and 12 months after administration of streptozotocin (STZ; 65 mg/kg i.v.). About 30% of diabetic animals displayed symptoms of partial spontaneous recovery, i.e. decreasing blood glucose levels and increasing insulin concentrations in the plasma and pancreas. NPY concentrations in the atria of diabetic rats did not differ from those in age-matched control rats 1, 2, 4, 6 months in the right atria and even 9 months after STZ in the left atria. However, uncompensated diabetes led to a significant decrease in NPY levels 9 and 12 months after STZ administration in the right and left atria, respectively. In the ventricles, NPY concentrations were significantly decreased 6 months after the onset of diabetes. Interestingly, partial spontaneous recovery of diabetes was associated with increased NPY levels in the atria. Myocardial norepinephrine concentrations increased 1 month after STZ and then declined reaching approximately 60% of the respective control values 12 months after the onset of the disease. Partial spontaneous recovery of diabetes had no effect on norepinephrine concentrations. Myocardial epinephrine concentrations did not differ from those found in controls till month 9 of the disease and they became significantly lower at month 12. Partial recovery of diabetes resulted in epinephrine concentrations not differing from the control values at month 12 of diabetes. Regarding to preferential localization of norepinephrine in the sympathetic postganglionic fibers and that of NPY also in intrinsic ganglion neurons, intrinsic neuronal circuits seem to be less susceptible to STZ-induced damage than extrinsic nerves and they might be able to recover after amelioration of diabetes.

Muscarinic acetylcholine receptors in the heart of rats before and after birth

Atropine-displaceable binding of (3H)quinuclidinyl benzilate (QNB) to homogenates was used to identify the muscarinic binding sites in rat heart atria and ventricles and to investigate developmental changes in their concentration and binding properties between the 15th day of prenatal life and 3 months after birth. On the 15th day of prenatal life, muscarinic binding sites were already present in the heart. Their concentration increased steeply between the 15th and 19th days of prenatal development; in the atria, it remained high until the 1st day after birth and thereafter it diminished throughout the postnatal life, while in the ventricles the decrease started before the first postnatal day. The concentration of the binding sites was 1.8–3.0 times higher in the atria than in the ventricles at all time points investigated. Their affinity for QNB (the antagonist) was the same in the atria and ventricles and did not change during postnatal development (KD of 17.8 pmol/l at an infinitely low concentration of the binding sites). The binding of carbamoylcholine (the agonist) to muscarinic bindig sites was analysed in experiments with the displacement of (3H)QNB binding, assuming the presence of high- and low-affinity binding sites for agonists. The proportion between the concentrations of the two classes of agonist binding sites is close to 1:1 both in the atria and the ventricles and does not change with age. No statistical significant differences were discovered between the affinities of the high- and low-affinity binding sites for carbamoylcholine between the atria and the ventricles and between new-born and adult rats. It is suggested that the steep increase in the number of muscarinic binding sites between the 15th and 19th days of prenatal development is triggered by the arrival of nerve cells and fibres into the heart (occuring on the 14th–16th day) and that it is one of the factors responsible for the onset of effective neuro-effector transmission in the heart (21st day of prenatal development). The commencement of tonic cardioinhibitory vagal control (18th day after birth) appears unrelated to developmental changes of cardiac muscarinic binding sites.

Distribution of peptide-containing neurons in the developing rat right atrium, studied using immunofluorescence and confocal laser scanning

The developmental pattern and distribution of peptide-containing neurons in the rat heart right atrium has been studied by indirect immunofluorescence. Antibodies against neuropeptide Y (NPY), substance P (SP), and vasoactive intestinal polypeptide (VIP) were applied to whole-mount stretch preparations of the right atria from hearts of newborn to 40-day-old animals. NPY-like immunoreactivity (LI) was compared with the synaptic vesicle marker SV2 in double immunoincubation studies. The distribution of immunofluorescence was studied by confocal laser scanning microscopy. NPY-LI and SP-LI were present throughout the atria already at birth, in contrast to VIP-LI that was observed at day 10. The postnatal changes of innervation were basically quantitative, with an increase in density of nerve fibres and number of varicosities, while the basic pattern of innervation was essentially established during the first 1–10 days. NPY- and SP-positive bundles of fibres appeared to enter the right atrium along the superior caval vein, having extrinsic origins. Nerve fibres with NPY-LI colocalized in most nerve terminals with SV2-LI, and showed a developmental pattern similar to that observed for adrenergic neurons earlier. These NPY/SV2 positive fibres probably represent the extrinsic NPY innervation. In addition, NPY-LI was identified in large intrinsic nerve cells bodies located near the atrioventricular (AV) region. Most of the VIP-LI was observed in short nerve fibres originating in intrinsic VIP-positive cell bodies, but a few apparently extrinsic VIP-positive fibres were found, probably representing preganglionic parasympathetic neurons. SP in the atria was probably of extrinsic (sensory) origin and no nerve cell bodies with SP-LI were detected. The results show that the peptidergic innervation in the developing rat right atrium involves both extrinsic and intrinsic peptidergic neurons which may participate in the regulation of neurotransmission in local neuronal circuits.

Distribution of vasoactive intestinal polypeptide in the rat heart: effect of guanethidine and capsaicin

Vasoactive intestinal polypeptide (VIP) is believed to coexist with acetylcholine in postganglionic parasympathetic neurones. However, the presence of VIP in extrinsic nerves and/or other types of intrinsic cardiac neurones has not been excluded. The aim of our study was to examine the distribution and origin of VIP-ergic innervation in the rat heart atria using immunocytochemistry and radioimmunoassay (RIA) combined with two types of denervation: sympathectomy, which was produced by guanethidine treatment and sensory denervation achieved by capsaicin administration. In whole-mount preparations of the intact atria, VIP-immunoreactive (IR) nerve fibres and ganglionic cells were found, the latter being much more numerous in the left atria (LA) than in the right ones. Some of VIP-IR nerve fibres forming bundles appeared to be extrinsic in origin. VIP-IR concentrations determined by RIA in the intact rats were significantly higher in the LA than in the right ones (p < 0.01). However, no changes in VIP-IR levels were found in either atrium after both guanethidine and capsaicin treatment protocols, thus indicating that VIP-immunoreactivity is not associated with either sympathetic or sensory innervation. In conclusion, the ganglionated plexus of the rat atria may comprise at least 3 different neuronal populations expressing VIP-positivity: 1. extrinsic preganglionic parasympathetic fibres, 2. intrinsic postganglionic parasympathetic neurones and 3. intrinsic local circuit neurones that do not express a cholinergic phenotype.