Mitochondrial dysfunction in kidney cortex and medulla of subtotally nephrectomized rats.

Five-sixths nephrectomy is a widely used experimental model of chronic kidney disease (CKD) that is associated with severe mitochondrial dysfunction of the remnant tissue. In this study, we assessed the effect of CKD on mitochondrial respiration separately in the rat kidney cortex and medulla 10 weeks after induction of CKD by subtotal 5/6 nephrectomy (SNX). Mitochondrial oxygen consumption was evaluated on mechanically permeabilized samples of kidney cortex and medulla using high-resolution respirometry and expressed per mg of tissue wet weight or IU citrate synthase (CS) activity. Mitochondrial respiration in the renal cortex of SNX rats was significantly reduced in all measured respiratory states if expressed per unit wet weight and remained lower if recalculated per IU citrate synthase activity, i.e. per mitochondrial mass. In contrast, the profound decrease in the activity of CS in SNX medulla resulted in significantly elevated respiratory states expressing the OXPHOS capacity when Complexes I and II or II only are provided with electrons, LEAK respiration after oligomycin injection, and Complex IV-linked oxygen consumption per unit CS activity suggesting compensatory hypermetabolic state in remaining functional mitochondria that is not sufficient to fully compensate for respiratory deficit expressed per tissue mass. The results document that CKD induced by 5/6 nephrectomy in the rat is likely to cause not only mitochondrial respiratory dysfunction (in the kidney cortex), but also adaptive changes in the medulla that tend to at least partially compensate for mitochondria loss.

Norepinephrine turnover in the left ventricle of subtotally nephrectomized rats.

Increased activity of the sympathetic nervous system (SNS) has been proposed as a risk factor for increased cardiovascular mortality in patients with chronic kidney disease (CKD). Information on the activity of cardiac sympathetic innervation is non-homogeneous and incomplete. The aim of our study was to evaluate the tonic effect of SNS on heart rate, norepinephrine turnover and direct and indirect effects of norepinephrine in left ventricles of subtotally nephrectomized rats (SNX) in comparison with sham-operated animals (SHAM). Renal failure was verified by measuring serum creatinine and urea levels. SNX rats developed increased heart rates and blood pressure (BP). The increase in heart rate was not caused by sympathetic overactivity as the negative chronotropic effect of metipranolol did not differ between the SNX and SHAM animals. The positive inotropic effects of norepinephrine and tyramine on papillary muscle were not significantly different. Norepinephrine turnover was measured after the administration of tyrosine hydroxylase inhibitor, pargyline, tyramine, desipramine, and KCl induced depolarization. The absolute amount of released norepinephrine was comparable in both groups despite a significantly decreased norepinephrine concentration in the cardiac tissue of the SNX rats. We conclude that CKD associated with renal denervation in rats led to adaptive changes characterized by an increased reuptake and intracellular norepinephrine turnover which maintained normal reactivity of the heart to sympathetic stimulation.

Beneficial effects of mesenchymal stem cells on adult porcine cardiomyocytes in non-contact co-culture.

Mesenchymal stem cells (MSCs) have been reported to improve survival of cardiomyocytes (CMCs) and overall regeneration of cardiac tissue. Despite promising preclinical results, interactions of MSCs and CMCs, both direct and indirect, remain unclear. In this study, porcine bone marrow MSCs and freshly isolated porcine primary adult CMCs were used for non-contact co-culture experiments. Morphology, viability and functional parameters of CMCs were measured over time and compared between CMCs cultured alone and CMCs co-cultured with MSCs. In non-contact co-culture, MSCs improved survival of CMCs. CMCs co-cultured with MSCs maintained CMCs morphology and viability in significantly higher percentage than CMCs cultured alone. In viable CMCs, mitochondrial respiration was preserved in both CMCs cultured alone and in CMCs co-cultured with MSCs. Comparison of cellular contractility and calcium handling, measured in single CMCs, revealed no significant differences between viable CMCs from co-culture and CMCs cultured alone. In conclusion, non-contact co-culture of porcine MSCs and CMCs improved survival of CMCs with a sufficient preservation of functional and mitochondrial parameters.

Propofol-induced mitochondrial and contractile dysfunction of the rat ventricular myocardium.

Propofol is a short-acting hypnotic agent used in human medicine for sedation and general anesthesia. Its administration can be associated with serious cardiovascular side-effects that include decrease in arterial blood pressure and cardiac output. The aim of the present study was to evaluate propofol effects on mitochondrial respiration, myocardial contractility and electrophysiology in the same samples isolated from the heart ventricles of adult rats. Mitochondrial oxygen consumption was measured in permeabilized samples dissected from free walls of both ventricles using high-resolution respirometry. State LEAK was determined with malate and glutamate. Active respiration was induced by ADP (state PI) and further by succinate, a Complex II substrate (PI+II). Rotenone was injected to measure state PII. Antimycin A, a Complex III inhibitor was used to determine residual oxygen consumption (ROX). N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride and ascorbate were injected simultaneously for respirometric assay of cytochrome c oxidase activity (CIV). Isometric contractions and membrane potentials were determined on multicellular preparations isolated from right and left ventricles. Propofol concentrations used ranged from 0.005 to 0.5 mmol/l. All respiratory parameters were significantly higher in the left control ventricles compared to the right ones. Propofol significantly decreased Complex I activity at concentration 0.025 mmol/l and papillary muscle contraction force at 0.1 mmol/l. Propofol did not affect action potential duration at any concentration studied. Our study suggests that mechanisms contributing to the impaired myocardial contraction during propofol anesthesia might include also mitochondrial dysfunction manifested by compromised activity of the respiratory Complex I.

[Contractile functions of myocardium and their regulation]. / Kontraktilní funkce myokardu a jejich regulace.

Myocardial contractility is the ability of the cardiac muscle to contract, thereby generating force. Contractile functions of the myocardium are influenced by a number of intrinsic and extrinsic factors. The intrinsic factors, including the initial length of the muscle fibers, stimulation frequency and cardiac rhythm are modulated by neurohumoral mechanisms and extrinsic factors (ions and energy balance, temperature, pH, drugs, etc.). The mechanism of the cardiac contraction, intrinsic and neurohumonal regulation of the cardiac activity and changes in contractile functions of the myocardium and their regulation under pathological conditions are described in this article.

Tissue concentrations of vasoactive intestinal peptide are affected by peritonitis-induced sepsis and hemofiltration in pigs.

Vasoactive intestinal peptide (VIP) is a neuropeptide released from the autonomic nerves exerting multiple antiinflammatory effects. The aim of the present study was to investigate the impact of severe sepsis and hemofiltration in two settings on plasma and tissue concentrations of VIP in a porcine model of sepsis. Thirty-two pigs were divided into 5 groups: 1) control group; 2) control group with conventional hemofiltration; 3) septic group; 4) septic group with conventional hemofiltration; 5) septic group with high-volume hemofiltration. Sepsis induced by faecal peritonitis continued for 22 hours. Hemofiltration was applied for the last 10 hours. Hemodynamic, inflammatory and oxidative stress parameters (heart rate, mean arterial pressure, cardiac output, systemic vascular resistance, plasma concentrations of tumor necrosis factor-alpha, interleukin-6, thiobarbituric acid reactive species, nitrate + nitrite, asymmetric dimethylarginine) and the systemic VIP concentrations were measured before faeces inoculation and at 12 and 22 hours of peritonitis. VIP tissue levels were determined in the left ventricle, mesenteric and coronary arteries. Sepsis induced significant increases in VIP concentrations in the plasma and mesenteric artery, but it decreased peptide levels in the coronary artery. Hemofiltration in both settings reduced concentrations of VIP in the mesenteric artery. In severe sepsis, VIP seems to be rapidly depleted from the coronary artery and, on the other hand, upregulated in the mesenteric artery. Hemofiltration in both settings has a tendency to drain away these upregulated tissue stores which could result in the limited secretory capacity of the peptide.

Experimental models of spontaneous ventricular arrhythmias and of sudden cardiac death.

Sudden cardiac death defined as natural death from cardiac causes occurring within one hour of the onset of acute symptoms is one of the most significant non-injury causes of death in the adult population of industrialised countries. The understanding of the mechanisms leading to sudden cardiac death is so far limited. Recently, a number of experimental animal models with high incidence of sudden cardiac death were developed and are intensively studied to get new insights into the sudden cardiac death mechanisms. In this review the animal models of sudden cardiac death are summarized and their principal properties shortly described.

Cardiovascular parameters in rat model of chronic renal failure induced by subtotal nephrectomy.

Chronic renal failure (CRF) is associated with high incidence of cardiovascular complications. To clarify pathogenesis of CRF numerous animal models have been developed. The aim of our work was to describe methodology of subtotal surgical renal ablation in rat and to characterize some biochemical and cardiovascular parameters of this animal model. Male rats underwent 5/6 surgical nephrectomy or sham operations in two steps. The following parameters were measured on day 10 and in week 10 after the surgery: plasma concentrations of creatinine and urea, blood pressure, resting heart rate, chronotropic response to atropine and metipranol, heart ventricles weight, contraction parameters and action potential duration in the left ventricle. Increased serum concentrations of creatinine and urea, decreased creatinine clearance, polyuria and alteration of the remnant kidney tissue were found in CRF rats. Changes in cardiovascular parameters identified after subtotal nephrectomy resembled alterations of cardiovascular system in uremic patients and included hypertension, elevated resting heart rate, diminished parasympathetic cardiac tone, hypertrophy of the left ventricle associated with weakened force of contraction, prolonged contraction and relaxation and shortening of action potential duration. These data suggest that the present model can be a useful tool in the study of CRF and its cardiovascular complications.

Chronic atropine administration diminishes the contribution of vasoactive intestinal polypeptide to heart rate regulation.

Vasoactive intestinal polypeptide (VIP) is implicated in the modulation of vagal effects on the heart rate. In this study, the impact of acute and chronic atropine administration on VIP levels in rat heart atria was investigated in relation to heart rate in the course of vagus nerves stimulation. Anaesthetised control and atropinised (10 mg/kg/day for 10 days) rats pretreated with metipranolol and phentolamine that were either given or not a single dose of atropine were subjected to bilateral vagus nerve stimulation (30 min: 0.7 mA, 20 Hz, 0.2 ms). VIP concentrations in the atria were determined after each stimulation protocol. In control rats with or without single atropine administration, the heart rate upon vagal stimulation was higher than in atropinised animals with or without single atropine dose, respectively. VIP concentrations in the control atria were significantly decreased after the stimulation; the decrease was comparable both in the absence and presence of a single dose of atropine. Compared to controls, VIP levels were significantly decreased after chronic atropine treatment and they were not further reduced by vagal stimulation and single atropine administration. Administration of VIP antagonist completely abolished the differences in the heart rate upon vagal stimulation between control and atropinised groups. In conclusion, the data indicate that chronic atropine administration affects VIP synthesis in rat heart atria and consequently it modifies the heart rate regulation.

Depressed cardiac contractility and its postnatal development in rats after chemical sympathectomy.

The contribution of the sympathetic innervation to the postnatal development of cardiac contractility remains unclear. In this study, the postnatal maturation of cardiac contractility was compared in control rats and rats after chemical sympathectomy. The chemical sympathectomy was induced by administration of 6-hydroxydopamine to newborn rats. At days 20, 40 and 60 of postnatal life, the contractile parameters and concentrations of sympathetic neurotransmitters were measured in both right and left ventricles. In rats with chemical sympathectomy, concentrations of norepinephrine were reduced almost completely in both ventricles at all time points. The contractility of the left ventricle papillary muscles was substantially decreased at all time points. In contrast, the contractility of the right ventricle papillary muscles was decreased only transiently, showing a recovery at day 60 regardless of the permanently decreased concentration of norepinephrine. The concentration of neuropeptide Y, another neurotransmitter present in sympathetic nerves, showed the same developmental trend as contractility: permanent reduction in the left ventricle, transient reduction with a recovery at day 60 in the right ventricle. The data indicate that the sympathetic nervous system plays an important role in the postnatal development of cardiac contractility and neuropeptide Y may contribute to this effect.

Negative inotropic effect of insulin in papillary muscles from control and diabetic rats.

The inotropic effects of insulin in the rat heart are still incompletely understood. In this study, the effects of insulin on cardiac contraction were studied in right ventricular papillary muscles from both control rats and rats with chronic diabetes (lasting 16 weeks). Diabetes was induced by the application of streptozotocin (STZ) and the development of diabetes was documented by increased levels of blood glucose, by reduction in body weight and by decreased plasma concentrations of insulin. The contraction was significantly smaller in diabetic rats. Insulin (80 IU/l) reduced the contraction force in both control and diabetic groups. The post-rest potentiation of contraction was not influenced by insulin in control rats, but insulin increased it in diabetic rats. The negative inotropic effect of insulin was preserved in the presence of cyclopiazonic acid (3 micromol/l), a blocker of sarcoplasmic reticulum (SR) Ca2+ pump, in both control and diabetic groups. In contrast, the negative inotropic effect of insulin was completely prevented in the presence of nifedipine (3 micromol/l), a blocker of L-type Ca2+ current. We conclude that insulin exerts a significant negative inotropic effect in rat myocardium, both control and diabetic. This effect is probably related to processes of SR Ca2+ release triggering, whereas SR Ca2+ loading is not involved.

Oscillations of the transepithelial potential of moth olfactory sensilla are influenced by octopamine and serotonin.

The biogenic amine octopamine is known to enhance the sensitivity of male moths to their species-specific pheromones in flight-tunnel experiments. This sensitization of pheromone-guided upwind flight is at least partly due to octopamine-dependent increases in the peak nerve impulse frequency of the pheromone response of olfactory receptor neurons. It is not known, however, whether octopamine exerts its effects directly on the electrical properties of the olfactory receptor neurons or indirectly, via modulation of the accessory cells of the sensillum. In extracellular tip recordings of pheromone-dependent trichoid sensilla on the antennae of male Manduca sexta moths, we investigated the effects of octopamine and serotonin on the transepithelial potential, which is generated by the activity of V-ATPases in sensillar accessory cells. In addition, the action potential activity of unstimulated olfactory receptor neurons was examined in the presence of biogenic amines. Under constant environmental conditions, the transepithelial potential oscillated regularly with periods of 2-8 min and with a 1-25mV peak-to-peak amplitude over periods of several hours. These oscillatory intervals were interrupted by periods of relatively stable transepithelial potential, correlated with flight activity by the moth. Octopamine reduced the amplitude of the transepithelial potential oscillation and decreased the resistance of the sensillum preparation in a dose-dependent manner. Serotonin altered the waveform of the transepithelial potential, but did not change the resistance of the preparation. Thus, both amines affect the accessory cells, but have different targets in the regulation of the transepithelial potential. Neither amine significantly influenced the spontaneous action potential activity of the olfactory receptor neurons.

Abrupt rate accelerations or premature beats cause life-threatening arrhythmias in mice with long-QT3 syndrome.

Deletion of amino-acid residues 1505-1507 (KPQ) in the cardiac SCN5A Na(+) channel causes autosomal dominant prolongation of the electrocardiographic QT interval (long-QT syndrome type 3 or LQT3). Excessive prolongation of the action potential at low heart rates predisposes individuals with LQT3 to fatal arrhythmias, typically at rest or during sleep. Here we report that mice heterozygous for a knock-in KPQ-deletion (SCN5A(Delta/+)) show the essential LQT3 features and spontaneously develop life-threatening polymorphous ventricular arrhythmias. Unexpectedly, sudden accelerations in heart rate or premature beats caused lengthening of the action potential with early afterdepolarization and triggered arrhythmias in Scn5a(Delta/+) mice. Adrenergic agonists normalized the response to rate acceleration in vitro and suppressed arrhythmias upon premature stimulation in vivo. These results show the possible risk of sudden heart-rate accelerations. The Scn5a(Delta/+) mouse with its predisposition for pacing-induced arrhythmia might be useful for the development of new treatments for the LQT3 syndrome.

Localization of cGMP immunoreactivity and of soluble guanylyl cyclase in antennal sensilla of the hawkmoth Manduca sexta.

The intracellular messenger cGMP (cyclic guanosine monophosphate) has been suggested to play a role in olfactory transduction in both invertebrates and vertebrates, but its cellular location within the olfactory system has remained elusive. We used cGMP immunocytochemistry to determine which antennal cells of the hawkmoth Manduca sexta are cGMP immunoreactive in the absence of pheromone. We then tested which antennal cells increase cGMP levels in response to nitric oxide (NO) and to long pheromonal stimuli, which the male encounters close to a calling female moth. In addition, we used in situ hybridization to determine which antennal cells express NO-sensitive soluble guanylyl cyclase. In response to long pheromonal stimuli with NO donors present, cGMP concentrations change in at least a subpopulation of pheromone-sensitive olfactory receptor neurons. These changes in cGMP concentrations in pheromone-dependent olfactory receptor neurons cannot be mimicked by the addition of NO donors in the absence of pheromone. NO stimulates sensilla chaetica type I and II, but not pheromone-sensitive trichoid sensilla, to high levels of cGMP accumulation as detected by immunocytochemistry. In situ hybridizations show that sensilla chaetica, but not sensilla trichodea, express detectable levels of mRNA coding for soluble guanylyl cyclase. These results suggest that intracellular rises in cGMP concentrations play a role in information processing in a subpopulation of pheromone-sensitive sensilla in Manduca sexta antennae, mediated by an NO-sensitive mechanism, but not an NO-dependent soluble guanylyl cyclase.

Phase response curves of a molecular model oscillator: implications for mutual coupling of paired oscillators.

Increasing evidence indicates that the accessory medulla is the circadian pacemaker controlling locomotor activity rhythms in insects. A prominent group of neurons of this neuropil shows immunoreactivity to the peptide pigment-dispersing hormone (PDH). In Drosophila melanogaster, the PDH-immunoreactive (PDH-ir) lateral neurons, which also express the clock genes period and timeless, are assumed to be circadian pacemaker cells themselves. In other insects, such as Leucophaea maderae, a subset of apparently homologue PDH-ir cells is a candidate for the circadian coupling pathway of the bilaterally symmetric clocks. Although knowledge about molecular mechanisms of the circadian clockwork is increasing rapidly, very little is known about mechanisms of circadian coupling. The authors used a computer model, based on the molecular feedback loop of the clock genes in D. melanogaster, to test the hypothesis that release of PDH is involved in the coupling between bilaterally paired oscillators. They can show that a combination of all-delay- and all-advance-type interactions between two model oscillators matches best the experimental findings on mutual pacemaker coupling in L. maderae. The model predicts that PDH affects the phosphorylation rate of clock genes and that in addition to PDH, another neuroactive substance is involved in the coupling pathway, via an all-advance type of interaction. The model suggests that PDH and light pulses, represented by two distinct classes of phase response curves, have different targets in the oscillatory feedback loop and are, therefore, likely to act in separate input pathways to the clock.

[Na+/Ca+ exchange: structure, mechanism, regulation and function]. / Na+/Ca2+ výmena: struktura, mechanismus, regulace a funkce.

The Na+/Ca2+ exchange is a plasma membrane system for the countertransport of Na+ and Ca2+ ions. The system can transport Ca2+ both into and out of the cell and therefore it plays a crucial role in the calcium homeostasis. The system works in electrogenic way (3 Na+ are exchanged for 1 Ca2+) and thus it influences the electrogenic properties of excitable cells. Due to new techniques of electrophysiology and molecular biology a lot of information about transport mechanism, regulation and possible physiological and pathophysiological roles of the exchanger was accumulated. This review summarises new findings and shows progress in this attractive field of biological research.

Calcitonin gene-related peptide suppresses the transient outward current in rat ventricular myocytes.

The effects of calcitonin gene-related peptide (CGRP) on the transient outward current (Ito) and the L-type calcium current (ICa,L) were investigated in isolated rat ventricular cardiomyocytes using the whole-cell, patch-clamp technique. CGRP influenced neither the amplitude nor the time course of ICa,L. On the other hand, at all membrane potentials at which a significant Ito was elicited, CGRP decreased its amplitude. The effect on Ito was completely reversible and independent of membrane potential. The steady-state activation and inactivation curves of Ito were not influenced by CGRP. The time course of Ito inactivation was satisfactorily fitted by two exponentials. Both the fast and the slow time constants of inactivation were voltage independent and were not influenced by CGRP. The effect of CGRP on Ito was concentration dependent with half-maximum inhibition at 99 nM. Chelerythrine, a selective inhibitor of protein kinase C, prevented the effect of CGRP on Ito. The data indicate that CGRP suppresses Ito in a concentration-dependent, but membrane potential-independent manner and that the effect is probably mediated via a protein kinase C-dependent pathway.

Effects of strontium ions on contraction and action potential in rabbit papillary muscles. A comparison with effects of tetraethylammonium ions.

The effects of Sr2+ on contraction and action potential were studied in rabbit papillary muscles and compared with effects of tetraethylammonium (TEA+). The membrane potential was measured with KCl-filled microelectrodes and the contraction was simultaneously recorded using a mechanoelectrical transducer. A partial (90%) substitution of extracellular Ca2+ (Ca2+e) by Sr2+ produced stimulation frequency-dependent prolongation of the action potential (AP) with a dominant phase "plateau" as well as prolongation of the contraction. At low frequencies where the AP prolongation was well pronounced, the contraction became biphasic. The effect of Sr2+ on both AP and contraction was blocked by nifedipine (10 mumol/l) or by increasing Ca2+e. Ryanodine suppressed the early contraction component only. AP was prolonged to a similar extent and in the same frequency-dependent manner by TEA+ (20 mmol/l). Despite similar AP configuration, no biphasic contraction developed in the presence of TEA+. High Ca2+e (10 mmol/l) or low Na+e (70 mmol/l) suppressed the TEA+ effect on AP. The data indicate that the two components of the biphasic contraction are of different origin; the early one is activated by activator cation released from the sarcoplasmic reticulum while the late one results from the Sr2+ entry across the sarcolemma via L-type Ca2+ channels.

Presumptive insect circadian pacemakers in vitro: immunocytochemical characterization of cultured pigment-dispersing hormone-immunoreactive neurons of Leucophaea maderae.

The accessory medulla with its associated pigment-dispersing hormone-immunoreactive neurons appears to be the pacemaker that controls the circadian locomotor activity rhythm of the cockroach Leucophaea maderae. To permit studies at the level of individual, identified, pacemaker neurons, we developed specific long-term primary cell cultures of fully differentiated adult neurons of the accessory medulla. As judged from soma diameter distribution, the cultures contain an unbiased representation of apparently all neuronal types of the accessory medulla. The cultured cells survive and grow processes for more than 2 months with or without additional hemocyte coculturing. However, a strong positive effect on initial outgrowth was observed with hemocyte coculturing. At least six different morphological cell types of the accessory medulla could be distinguished in vitro. Among these only one cell type, the monopolar type C cell, was recognized in vitro with an antiserum against the neuropeptide pigment-dispersing hormone. Thus, the identifiable monopolar type C cells are candidates for circadian pacemaker neurons and will be the focus of further physiological characterizations.

Organization of the circadian system in insects.

The circadian systems of different insect groups are summarized and compared. Emphasis is placed on the anatomical identification and characterization of circadian pacemakers, as well as on their entrainment, coupling, and output pathways. Cockroaches, crickets, beetles, and flies possess bilaterally organized pacemakers in the optic lobes that appear to be located in the accessory medulla, a small neuropil between the medulla and the lobula. Neurons that are immunoreactive for the peptide pigment-dispersing hormone (PDH) arborize in the accessory medulla and appear to be important components of the optic lobe pacemakers. The neuronal architecture of the accessory medulla with associated PDH-immunoreactive neurons is best characterized in cockroaches, while the molecular machinery of rhythm generation is best understood in fruit flies. One essential component of the circadian clock is the period protein (PER), which colocalizes with PDH in about half of the fruit fly's presumptive pacemaker neurons. PER is also found in the presumptive pacemaker neurons of beetles and moths, but appears to have different functions in these insects. In moths, the pacemakers are situated in the central brain and are closely associated with neuroendocrine functions. In the other insects, neurons associated with neuroendocrine functions also appear to be closely coupled to the optic lobe pacemakers. Some crickets and flies seem to possess central brain pacemakers in addition to their optic lobe pacemakers. With respect to neuronal organization, the circadian systems of insects show striking similarities to the vertebrate circadian system.