Animal Models in Diabetic Research-History, Presence, and Future Perspectives.

Diabetes mellitus (DM) is a very serious disease, the incidence of which has been increasing worldwide. The beginning of diabetic research can be traced back to the 17th century. Since then, animals have been experimented on for diabetic research. However, the greatest development of diabetes research occurred in the second half of the last century, along with the development of laboratory techniques. Information obtained by monitoring patients and animal models led to the finding that there are several types of DM that differ significantly from each other in the causes of the onset and course of the disease. Through different types of animal models, researchers have studied the pathophysiology of all types of diabetic conditions and discovered suitable methods for therapy. Interestingly, despite the unquestionable success in understanding DM through animal models, we did not fully succeed in transferring the data obtained from animal models to human clinical research. On the contrary, we have observed that the chances of drug failure in human clinical trials are very high. In this review, we will summarize the history and presence of animal models in the research of DM over the last hundred years. Furthermore, we have summarized the new methodological approaches, such as "organ-on-chip," that have the potential to screen the newly discovered drugs for human clinical trials and advance the level of knowledge about diabetes, as well as its therapy, towards a personalized approach.

Regulation of Prepro-NeuropeptideW/B and Its Receptor in the Heart of ZDF Rats: An Animal Model of Type II DM.

Neuropeptide B (NPB) and neuropeptide W (NPW) are neuropeptides, which constitute NPB/W signaling systems together with G-protein coupled receptors NPBWR1. The location and function of NPB/W signaling systems have been predominantly detected and mapped within the CNS, including their role in the modulation of inflammatory pain, neuroendocrine functions, and autonomic nervous systems. The aim of the study is to investigate the impact of diabetes on the neuropeptide B/W signaling system in different heart compartments and neurons which innervates it. In the RT-qPCR analysis, we observed the upregulation of mRNA for preproNPB in RV, for preproNPW in LA, and for NPBWR1 in DRG in diabetic rats. On the contrary, the expression of mRNA for NPBWR1 was downregulated in LV in diabetic rats. In the WB analysis, significant downregulation of NPBWR1 in LV (0.54-fold, p = 0.046) in diabetic rats was observed at the proteomic level. The presence of NPBWR1 was also confirmed in a dissected LCM section of cardiomyocytes and coronary arteries. The positive inotropic effect of NPW described on the diabetic cardiomyocytes in vitro could point to a possible therapeutic target for compensation of the contractile dysfunction in the diabetic heart. In conclusion, the NPB/W signaling system is involved in the regulation of heart functions and long-term diabetes leads to changes in the expression of individual members of this signaling system differently in each cardiac compartment, which is related to the different morphology and function of these cardiac chambers.

iPSCs in Neurodegenerative Disorders: A Unique Platform for Clinical Research and Personalized Medicine.

In the past, several animal disease models were developed to study the molecular mechanism of neurological diseases and discover new therapies, but the lack of equivalent animal models has minimized the success rate. A number of critical issues remain unresolved, such as high costs for developing animal models, ethical issues, and lack of resemblance with human disease. Due to poor initial screening and assessment of the molecules, more than 90% of drugs fail during the final step of the human clinical trial. To overcome these limitations, a new approach has been developed based on induced pluripotent stem cells (iPSCs). The discovery of iPSCs has provided a new roadmap for clinical translation research and regeneration therapy. In this article, we discuss the potential role of patient-derived iPSCs in neurological diseases and their contribution to scientific and clinical research for developing disease models and for developing a roadmap for future medicine. The contribution of humaniPSCs in the most common neurodegenerative diseases (e.g., Parkinson's disease and Alzheimer's disease, diabetic neuropathy, stroke, and spinal cord injury) were examined and ranked as per their published literature on PUBMED. We have observed that Parkinson's disease scored highest, followed by Alzheimer's disease. Furthermore, we also explored recent advancements in the field of personalized medicine, such as the patient-on-a-chip concept, where iPSCs can be grown on 3D matrices inside microfluidic devices to create an in vitro disease model for personalized medicine.

Role of Peptides in Diagnostics.

The specificity of a diagnostic assay depends upon the purity of the biomolecules used as a probe. To get specific and accurate information of a disease, the use of synthetic peptides in diagnostics have increased in the last few decades, because of their high purity profile and ability to get modified chemically. The discovered peptide probes are used either in imaging diagnostics or in non-imaging diagnostics. In non-imaging diagnostics, techniques such as Enzyme-Linked Immunosorbent Assay (ELISA), lateral flow devices (i.e., point-of-care testing), or microarray or LC-MS/MS are used for direct analysis of biofluids. Among all, peptide-based ELISA is considered to be the most preferred technology platform. Similarly, peptides can also be used as probes for imaging techniques, such as single-photon emission computed tomography (SPECT) and positron emission tomography (PET). The role of radiolabeled peptides, such as somatostatin receptors, interleukin 2 receptor, prostate specific membrane antigen, αß3 integrin receptor, gastrin-releasing peptide, chemokine receptor 4, and urokinase-type plasminogen receptor, are well established tools for targeted molecular imaging ortumor receptor imaging. Low molecular weight peptides allow a rapid clearance from the blood and result in favorable target-to-non-target ratios. It also displays a good tissue penetration and non-immunogenicity. The only drawback of using peptides is their potential low metabolic stability. In this review article, we have discussed and evaluated the role of peptides in imaging and non-imaging diagnostics. The most popular non-imaging and imaging diagnostic platforms are discussed, categorized, and ranked, as per their scientific contribution on PUBMED. Moreover, the applicability of peptide-based diagnostics in deadly diseases, mainly COVID-19 and cancer, is also discussed in detail.

Laser Capture Microdissection Coupled Capillary Immunoassay to Study the Expression of PCK-2 on Spatially-Resolved Islets of Rat Langerhans.

The platform for precise proteomic profiling of targeted cell populations from heterogeneous tissue sections is developed. We demonstrate a seamless and systematic integration of LCM with an automated cap-IA for the handling of a very small-sized dissected tissues section from the kidney, liver and pancreatic Langerhans islet of rats. Our analysis reveals that the lowest LCM section area ≥ 0.125 mm2 with 10 µm thickness can be optimized for the detection of proteins through LCM-cap-IA integration. We detect signals ranging from a highly-abundant protein, ß-actin, to a low-abundance protein, LC-3AB, using 0.125 mm2 LCM section from rat kidney, but, so far, a relatively large section is required for good quality of results. This integration is applicable for a highly-sensitive and accurate assessment of microdissected tissue sections to decipher hidden proteomic information of pure targeted cells. To validate this integration, PCK2 protein expression is studied within Langerhans islets of normal and diabetic rats. Our results show significant overexpression of PCK2 in Langerhans islets of rats with long-term diabetes.

Identification of NPB, NPW and Their Receptor in the Rat Heart.

Members of neuropeptide B/W signaling system have been predominantly detected and mapped within the CNS. In the rat, this system includes neuropeptide B (NPB), neuropeptide W (NPW) and their specific receptor NPBWR1. This signaling system has a wide spectrum of functions including a role in modulation of inflammatory pain and neuroendocrine functions. Expression of NPB, NPW and NPBWR1 in separate heart compartments, dorsal root ganglia (DRG) and stellate ganglia was proven by RT-qPCR, Western blot (WB) and immunofluorescence. Presence of mRNA for all tested genes was detected within all heart compartments and ganglia. The presence of proteins preproNPB, preproNPW and NPBWR1 was confirmed in all the chambers of heart by WB. Expression of preproNPW and preproNPB was proven in cardiac ganglionic cells obtained by laser capture microdissection. In immunofluorescence analysis, NPB immunoreactivity was detected in nerve fibers, some nerve cell bodies and smooth muscle within heart and both ganglia. NPW immunoreactivity was present in the nerve cell bodies and nerve fibers of heart ganglia. Weak nonhomogenous staining of cardiomyocytes was present within heart ventricles. NPBWR1 immunoreactivity was detected on cardiomyocytes and some nerve fibers. We confirmed the presence of NPB/W signaling system in heart, DRG and stellate ganglia by proteomic and genomic analyses.

Novel insights into the mixed germ cell-sex cord stromal tumor of the testis: detection of chromosomal aneuploidy and further morphological evidence supporting the neoplastic nature of the germ cell component.

The existence of a true mixed germ cell-sex cord stromal tumor (MGSCT) of the testis remains controversial. Based on our experience with rare testicular tumors in this spectrum, we sought to perform a detailed clinicopathologic and molecular study of MGCSCT. Eight cases of testicular MGSCT were morphologically reviewed, screened for chromosomal aberrations (using array comparative genomic hybridization (aCGH) and low pass genomic sequencing), and analyzed by next generation sequencing (The Illumina TruSight Tumor 170). Immunohistochemistry for OCT3/4, Nanog, SALL4, DMRT1, and inhibin was performed on the cohort. Clinical data and follow-up were assessed by medical record review. All patients were karyotypically normal men aged 27-74 years (median 41). All tumors had a similar biphasic morphology characterized by various proportions of the sex cord component resembling granulosa cell tumor of adult type and the germ cell component cytomorphologically akin to spermatocytic tumor. Germ cells were haphazardly scattered throughout the tumor or arranged in larger groups, without tubular formation. In 4 cases, atypical mitoses were found within the germ cells. Additionally, in 2 cases there was invasion into the spermatic cord, adjacent hilar soft tissue and into the tumor capsule, which contained both tumor components. Immunohistochemically, focal nuclear expression of DMRT1 was found in the germ cell component in 7/7 analyzable tumors, while SALL4 was positive in 6 cases and negative in one case. All tumors were negative with OCT3/4 and Nanog. The sex cord stromal component had immunoreactivity for inhibin in 7/7 analyzable cases. Four of 8 cases were cytogenetically analyzable: 4/8 by low pass genomic sequencing and 2/8 by aCGH. The results of both methods correlated well, revealing mostly multiple chromosomal losses and gains. One case revealed loss of chromosome 21; 1 case had loss of chromosomes 21 and 22 and partial gain of 22; 1 case had loss of chromosomes 22 and Y, partial loss of X, and gain of chromosomes 20, 5, 8, 9, 12, and 13; and the remaining one gain of chromosomes 20, 3, 6, 8, 2x(9), 11, 2x(12), 13, 14, 18, and 19. Three cases were analyzable by NGS; clinically significant activating mutations of either FGFR3 or HRAS were not detected in any case. Follow-up was available for 4 patients (12, 24, 84, and 288 months) and was uneventful in all 4 cases. The identification of extratesticular invasion of both the germ cell and sex cord stromal components, the DMRT1 expression, and the presence of atypical mitoses in germ cells argue for the neoplastic nature of the germ cell component. The molecular genetic study revealing multiple chromosomal losses and gains in a subset of the cases provides the first evidence that molecular abnormalities occur in testicular MGSCT. Multiple chromosomal aneuploidies, namely, recurrent losses of chromosomes 21 and 22 and gains of 8, 9, 12, 13, and 20, indicate that the germ cell component might be related to the morphologically similar spermatocytic tumor, which is characterized by extensive aneuploidies including recurrent gains of chromosomes 9 and 20 and loss of chromosome 7. In summary, our data support that rare examples of true MGSCT of the testis do exist and they represent a distinct tumor entity with admixed adult-type granulosa cell tumor and spermatocytic tumor components.

Distribution and Function of Neuropeptides W/B Signaling System.

Neuropeptide W (NPW) and neuropeptide B (NPB) are two structurally and functionally related regulatory peptides, which are highly expressed in several brain regions and, additionally, in some peripheral tissues. Nevertheless, their distributions in the tissues are not similar. They act on target tissues via two subtypes of G protein-coupled receptors which are designated as NPBWR1 (GPR7) and NPBWR2 (GPR8), respectively, and possess different binding affinities. NPB activates NPBWR1, whereas NPW stimulates both the receptors with similar potency. Both of these peptides takes a part in the central regulation of neuroendocrine axes, feeding behavior, energy homeostasis, cardiovascular functions, circadian rhythm, pain sensation, modulation of inflammatory pain, and emotions. Over the past few years, studies have shown that NPB is also involved in sleep regulation. On the contrary, NPW participates in regulation of vascular myogenic tone, inhibits gastric tension sensitive vagal afferents and insulin secretion. Also, expression of NPW in the stomach is regulated by feeding. Abovementioned findings clearly demonstrate the functional diversity among NPW versus NPB signaling systems. In this review, signal transduction pathways of NPW/NPB are critically evaluated and observed together with mapping of expression of their signaling systems.

Substance P Receptor in the Rat Heart and Regulation of Its Expression in Long-Term Diabetes.

Substance P (SP) is a neuropeptide engaged in the signal transmission of neural C fibers afferents in the myocardium. The actions of SP in the heart are extensive and they are mediated by the neurokinin 1 receptor (NK1R), a member of the tachykinin subfamily of G-protein coupled receptors. The receptors have been found in the heart, but to our knowledge, their exact localization in the heart has not been described yet. Here, we investigated the presence of NK1R protein in separate rat heart compartments by means of western blot and its tissue distribution by means of immunofluorescence. Specificity of NK1R immunolabeling was controlled by preabsorption of the antiserum with its corresponding peptide. Additionally, we investigated abundance of gene for NK1R in separated heart chambers by means of quantitative real-time PCR (RT-PCR). Relative abundance of NK1R mRNA was expressed as a ratio of target gene Cq value to Cq value of control gene - beta-actin. Finally, we studied abundance of NK1R mRNA in different cell types of heart isolated by laser capture microdissection. Immunofluorescence showed NK1R immunoreactivity on the surface of some intracardiac neurons and smooth muscle cells of coronary vessels. The results of quantitative RT-PCR indicate abundance of mRNA for NK1R in all heart chambers with highest level in the left atrium. The presence of NK1R mRNA was detected in some samples of dissected intracardiac neurons, but not in cardiomyocytes or smooth muscle cells of coronary vessels. In the course of long-term diabetes, a significant downregulation of the NK1R mRNA was seen in the right atrium and upregulation in the right ventricle 53 weeks after the induction of diabetes. Our results indicate localization of NK1R in some intracardiac neurons and smooth muscle cells. Impaired transcription of the NK1R gene in the diabetic heart may be induced by unidentified genes or factors involved in the development of diabetic cardiomyopathy.

Expression of classical mediators in hearts of rats with hepatic dysfunction.

Liver cirrhosis is associated with impairment of cardiovascular function including alterations of the heart innervation, humoral and nervous dysregulation, changes in systemic circulation and electrophysiological abnormalities. Choline acetyltransferase (ChAT), enzyme forming acetylcholine, tyrosine hydroxylase (TH), and dopamine-ß-hydroxylase (DBH), enzymes participating in noradrenaline synthesis, are responsible for the production of classical neurotransmitters, and atrial natriuretic peptide (ANP) is produced by cardiomyocytes. The aim of this study was to evaluate the influence of experimentally induced hepatic dysfunction on the expression of proANP, ChAT, TH, and DBH in the heart. Hepatic dysfunction was induced by application of thioacetamide (TAA) or by ligation of bile duct. Biochemical parameters of hepatic injury and levels of peroxidation in the liver and heart were measured. Liver enzymes measured in the plasma were significantly elevated. Cardiac level of peroxidation was increased in operated but not TAA group animals. In the left atrium of operated rats, the expression of TH and DBH was lower, while expression of ChAT remained unchanged. In TAA group, no significant differences in the expression of the genes compared to controls were observed. Liver injury induced by ligation leads to an imbalance in the intracardiac innervation, which might impair nervous control of the heart.

Basal Cell Carcinoma With Matrical Differentiation: Clinicopathologic, Immunohistochemical, and Molecular Biological Study of 22 Cases.

Basal cell carcinoma (BCC) with matrical differentiation is a fairly rare neoplasm, with about 30 cases documented mainly as isolated case reports. We studied a series of this neoplasm, including cases with an atypical matrical component, a hitherto unreported feature. Lesions coded as BCC with matrical differentiation were reviewed; 22 cases were included. Immunohistochemical studies were performed using antibodies against BerEp4, ß-catenin, and epithelial membrane antigen (EMA). Molecular genetic studies using Ion AmpliSeq Cancer Hotspot Panel v2 by massively parallel sequencing on Ion Torrent PGM were performed in 2 cases with an atypical matrical component (1 was previously subjected to microdissection to sample the matrical and BCC areas separately). There were 13 male and 9 female patients, ranging in age from 41 to 89 years. Microscopically, all lesions manifested at least 2 components, a BCC area (follicular germinative differentiation) and areas with matrical differentiation. A BCC component dominated in 14 cases, whereas a matrical component dominated in 4 cases. Matrical differentiation was recognized as matrical/supramatrical cells (n=21), shadow cells (n=21), bright red trichohyaline granules (n=18), and blue-gray corneocytes (n=18). In 2 cases, matrical areas manifested cytologic atypia, and a third case exhibited an infiltrative growth pattern, with the tumor metastasizing to a lymph node. BerEP4 labeled the follicular germinative cells, whereas it was markedly reduced or negative in matrical areas. The reverse pattern was seen with ß-catenin. EMA was negative in BCC areas but stained a proportion of matrical/supramatrical cells. Genetic studies revealed mutations of the following genes: CTNNB1, KIT, CDKN2A, TP53, SMAD4, ERBB4, and PTCH1, with some differences between the matrical and BCC components. It is concluded that matrical differentiation in BCC in most cases occurs as multiple foci. Rare neoplasms manifest atypia in the matrical areas. Immunohistochemical analysis for BerEP4, EMA, and ß-catenin can be helpful in limited biopsy specimens. From a molecular biological prospective, BCC and matrical components appear to share some of the gene mutations but have differences in others, but this observation must be validated in a large series.

Role of substance P in the cardiovascular system.

This article provides an overview of the structure and function of substance P signalling system and its involvement in the cardiovascular regulation. Substance P is an undecapeptide originating from TAC1 gen and belonging to the tachykinin family. The biological actions of substance P are mainly mediated through neurokinin receptor 1 since substance P is the ligand with the highest affinity to neurokinin receptor 1. Substance P is widely distributed within the central and peripheral nervous systems as well as in the cardiovascular system. Substance P is involved in the regulation of heart frequency, blood pressure and in the stretching of vessels. Substance P plays an important role in ischemia and reperfusion and cardiovascular response to stress. Additionally, it has been also implicated in angiogenesis, pain transmission and inflammation. The substance P/neurokinin receptor 1 receptor system is involved in the molecular bases of many human pathological processes. Antagonists of neurokinin receptor 1 receptor could provide clinical solutions for a variety of diseases. Neurokinin receptor 1 antagonists are already used in the prevention of chemotherapy induced nausea and vomiting.

Adrenomedullin and the calcitonin receptor-like receptor system mRNA expressions in the rat heart and sensory ganglia in experimentally-induced long-term diabetes.

Both adrenomedullin and calcitonin gene-related peptide (CGRP) regulate vascular tone in the heart, being cardioprotective in hypoxia. Additionally, adrenomedullin exhibits antiproliferative and antiapoptotic functions in the myocardium, while CGRP exerts positive chronotropic effect. Their actions are mediated through the specific G protein-coupled receptor, CRLR, whose ligand affinity is determined by receptor activity modifying proteins RAMP1-3. CGRP binds to the complex formed by CRLR/RAMP1, whereas CRLR/RAMP2 and CRLR/RAMP3 serve as receptors for adrenomedullin. Here, we quantified expression of this signaling system in the rat heart and supplying sensory ganglia (dorsal root ganglia T1-T4 and vagal nodose ganglia) in streptozotocin-induced diabetes. In the course of diabetes, an increase of CRLR mRNA was noticed in the right ventricle 8 weeks and of RAMP3 mRNA in the left ventricle and right atrium 26 weeks after induction of diabetes. Relative expressions of other tested genes were not significantly altered. In the nodose vagal supplying specific cardiac afferents, but not in dorsal root ganglia which provide cardiac pain fibres, a small upregulation of CGRP expression was detected. In summary, the shifts observed in diabetes may favour a trend of a pronounced adrenomedullin signaling. These observations may provide a new possible therapeutic strategy for diabetic cardiomyopathy.

Effects of acute stressors on the expression of oxytocin receptor mRNA in hearts of rats with different activity of HPA axis.

OBJECTIVES: Cardiovascular system is regulated by a diverse array of hormones, neurotransmitters and neuropeptides. Oxytocin and its receptors (OTR) were also shown to regulate cardiovascular functions and this hormone was even called cardiovascular hormone. In recent publication, we demonstrated the expression of mRNA of OTR by real-time quantitative PCR (RT qPCR) in all rat heart compartments. The aim of this study was to investigate the effects of acute restraint stress on OTR mRNA expression in two rat strains with different activity of HPA axis. METHODS: Adult male Sprague-Dawley and Lewis rats, the latter strain reported to have lower HPA activity, were used in RT qPCR studies and Wistar rats in immunofluorescent ones. Both acute restraint (IS) and this stress combined with the immersion of rats in water (ICS) lasted 60 min. Gene expression of OTR mRNA was estimated in all heart compartments after 1 or 3 hours after stress termination (IS1, IS3, ICS1, ICS3). The relative expression was calculated using 2(-ΔΔC)T method. In immunofluorescent studies we used commercial specific OTR antibodies. RESULTS: In RT qPCR studies we found higher expression of OTR mRNA in atria than in ventricles and no statistical differences between Sprague-Dawley and Lewis rats under basal conditions. Relative expression of OTR mRNA after 60 min lasting stress exposure differed in dependence on the stress type and partly on the time interval after the stress termination. When compared to controls, in rat left atria both stressors caused inhibition of OTR mRNA expression in both rat strains. In rat ventricles, which have very low OTR mRNA expression, there was a significant difference in the effect of two stressors. In most groups ICS displayed the increase of OTR mRNA expression if compared to IS groups. Immunofluorescent studies revealed changes induced by acute restraint stress in all heart compartments. The immunofluorescent studies suggested that acute stress induces higher colocalization of OTR with the nuclei than it was observed in the controls. CONCLUSIONS: The expression of OTR mRNA in all heart compartments of controls as well as after stress exposure in Sprague-Dawley and Lewis rats support the notion that OTR plays a regulatory role in the cardiovascular system and is also involved in the regulations in the heart after stress. The immunofluorescent observation that OTRs coexpress in areas of cell nuclei in certain heart compartments and after acute stress, compared to controls, requires further studies.

Expression of heart oxytocin receptor and its mRNA in two rat strains with different activity of HPA axis.

OBJECTIVES: Oxytocin (OT) is a neuropeptide acting both as a peripheral hormone and in the brain as neurotransmitter and neuromodulator. In addition to its well-known effects on milk-ejection and uterine contraction, OT was shown to exert neuroendocrine regulation of heart functions. The aim of this study was to investigate the expression of mRNA of OT receptors (OTR) in rat hearts by real-time quantitative PCR (qPCR). The study was performed in Sprague-Dawley (SD) and Lewis (LE) rat strains, the latter having lower activity of HPA axis. METHODS: We used adult male SD and LE rats. OTR mRNA expression was detected in all heart chambers by comparing their threshold cycle values (CT) to CT of reference gene ß-actin. The relative expression ratios were calculated using the 2-ΔΔCT method. The specificity of reaction of primary antibody with OTRs was tested by Western Blot and localization of OTR in the heart compartments was performed by immunofluorescence with commercial OTR specific antibodies. RESULTS: We found expression of OTR mRNA in all heart compartments. The expression of OTR mRNA in both atria (LA, RA) was much higher than in the ventricles (RV, LV). By using two-way ANOVA we found no statistical differences between corresponding compartments of SD and LE rats. Immunohistochemical studies showed that OTR staining is not related to neuronal tissue and findings from left atrium indicate that prevalent localization of OTR is on cell membranes of cardiomyocytes. CONCLUSIONS: The finding of expression of OTR mRNA by real-time qPCR and proof of OTR staining by immunohistochemistry in all heart compartments indicate that OT and its receptors may have function as a cardiovascular hormone. The differences in the HPA axis activity, as is exemplified in Sprague-Dawley and Lewis rat strain, do not project in the expression of OTR mRNA under basal condition. The effect of activity of HPA on OTR expression should be studied under stimulated conditions as it was performed in the behavioral studies.

Parasympathetic regulation of heart rate in rats after 5/6 nephrectomy is impaired despite functionally intact cardiac vagal innervation.

BACKGROUND: Chronic renal failure is frequently associated with a high risk of sudden cardiac death due to dysfunction of the autonomic nervous system. The pathogenic mechanisms underlying the parasympathetic cardiac dysautonomia are not fully elucidated yet. METHODS: Chronic renal failure was induced in rats by 5/6 nephrectomy. Blood pressure, resting heart rate and plasma levels of creatinine, urea and asymmetric dimethylarginine (ADMA) were measured. To characterize the parasympathetic innervation of the heart, chronotropic responses to atropine, metipranolol and to vagal stimulation in the absence or presence of ADMA were investigated in vivo. In vitro, chronotropic and inotropic effects of carbachol and ADMA and mRNA expression of muscarinic M2 receptors, high affinity choline transporter (CHT1), vesicular acetylcholine transporter (VAChT) and choline acetyltransferase (ChAT) were assessed in the isolated cardiac tissues. RESULTS: In 5/6 nephrectomy rats, the resting heart rate was significantly higher and the parasympathetic tone, measured as the effect of atropine after administration of metipranolol was significantly lower than in control animals. Plasma ADMA levels were significantly elevated in the uraemic rats and significantly inversely correlated with the effect of atropine on the heart rate. No differences were revealed in the plasma norepinephrine concentrations, negative chronotropic responses to stimulation of the vagus nerves, chronotropic and inotropic responses to carbachol and the relative expression of M2 receptors, CHT1, VAChT and ChAT. CONCLUSION: The data suggest that cardioacceleration in chronic renal failure is caused by a diminished cardiac parasympathetic tone in the presence of a functionally intact intrinsic cardiac cholinergic signalling system.