Tonic Component Research Articles

Out of the cleft: the source and target of extra‐synaptic glutamate in the CA1 region of the hippocampus

Out of the cleft: the source and target of extra‐synaptic glutamate in the CA1 region of the hippocampus

Biomechanical and electromyogram characterization of neuroleptic-induced rigidity in the rat

Rodent models of Parkinson’s disease (PD) are usually assessed using measures of akinesia, but other important parkinsonian symptoms such as rigidity are only rarely quantified. This is in part due to technical difficulties in obtaining such measures in small animals. In the present study we developed quantitative methods to provide time-course assessment of the alternations of muscle tone of parkinsonian rats. A portable and miniature biomechanical stretching device was established to manually stretch the hindlimb of awake rats with muscle rigidity induced by dopamine D2-receptor antagonist raclopride (5 mg/kg, i.p.). From the measured angular displacement angle and reactive torque of sinusoidal stretches at five varied frequencies, viscoelastic components of the muscle tone can be derived. In addition, non-invasive multielectrode was applied to record the tonic and phasic components of the gastrocnemius muscle electromyogram (EMG). Our biomechanical measurements showed not only increase in stiffness (P<0.05) but also increase in viscous components (P<0.05) that matched the time course of increased amplitude of EMG activity (P<0.05). There was a significant positive correlation between all of these measures and akinesia, as measured by the conventional bar-test for catalepsy (with a correlation coefficient of 0.87 at stiffness, 0.92 at viscosity and 0.96 at amplitude of EMG). Phasic contraction counts (PCC) of voluntary EMG exhibited a significantly negative correlation with the bar test scores (correlation coefficient=−0.78). These results confirm that akinesia induced by D2-receptor blockade also induces a rigidity that shares many features with human PD. These novel techniques for quantifying biomechanical and electromyographic parameters provide objective assessment methods for investigating the time-course changes of abnormal muscle tone in rat models of PD that will be useful for evaluating novel treatments.

Unity and diversity of tonic and phasic executive control components in episodic and working memory

The present study aimed to delineate the extent to which unitary executive functions might be shared across the separate domains of episodic and working memory. A mixed blocked/event-related functional magnetic resonance imaging (fMRI) design was employed to assess sustained (tonic control) and transient (phasic control) brain responses arising from incrementing executive demand (source versus item episodic memory – vis-à-vis – two-back versus one-back working memory) using load-dependent activation overlaps as indices of common components. Although an extensive portion of the regional load effects constituted differential control modulations in both sustained and transient responses, commonalities were also found implicating a subset of executive core mechanisms consistent with unitary or domain general control. ‘Unitary’ control modulations were temporally dissociated into (1) shared tonic components involving medial and lateral prefrontal cortex, striatum, cerebellum and superior parietal cortex, assumed to govern enhanced top-down context processing, monitoring and sustained attention throughout task periods and (2) stimulus-synchronous phasic components encompassing posterior intraparietal sulcus, hypothesized to support dynamic shifting of the ‘focus of attention’ among internal representations. Taken together, these results converge with theoretical models advocating both unity and diversity among executive control processes.

Sexual Function in Chronic Kidney Disease

Endocrine abnormalities are common in patients with chronic kidney disease (CKD) and lead to sexual dysfunction, anemia, hyperparathyroidism, and altered mineral metabolism. Common clinical problems include disturbances in menstruation in women, erectile dysfunction in men, and decreased libido and infertility in both sexes. Organic factors tend to be prominent and are related to uremia and other comorbid illnesses. Psychological factors and depression may exacerbate the primary problem. Alterations in the hypothalamic-pituitary axis are seen early in CKD and tend to worsen after patients start dialysis. Hypogonadism plays a dominant role in male sexual function, whereas changes in hypothalamic-pituitary function predominate in female sexual dysfunction. In patients on dialysis, treatment strategies include optimizing dose of dialysis, correction of anemia with erythropoietin, and correction of hyperparathyroidism. Successful kidney transplantation may restore normal sexual function, especially in younger patients.

17 β-estradiol modulates GABAergic synaptic transmission and tonic currents during development in vitro

Estrogens exert a variety of modulatory effects on the structure and function of the nervous system. In particular, 17 β-estradiol was found to affect GABAergic inhibition in adult animals but its action on GABAergic currents during development has not been elucidated. In the present study, we investigated the effect of 17 β-estradiol on hippocampal neurons developing in vitro. In this model, mIPSC kinetics showed acceleration with age along with increased α1 subunit expression, similarly as in vivo. Long-term treatment with 17 β-estradiol increased mIPSC amplitudes in neurons cultured for 6–8 and 9–11 DIV and prolonged the mIPSC decaying phase only in the 9–11 DIV group. The time needed for the onset of 17 β-estradiol effect on mIPSC amplitude was approximately 48 h. In the period of 9–11 DIV, treatment with 17 β-estradiol strongly reduced the tonic conductance activated by low GABA concentrations. The effects of 17 β-estradiol on mIPSCs and tonic conductance were not correlated with any change in expression of considered GABA AR subunits (α1–3, α5–6, γ2) while α4 and δ subunits were at the detection limit. In conclusion, we provide evidence that 17 β-estradiol differentially affects the phasic and tonic components of GABAergic currents in neurons developing in vitro.

In vitro Characterization of Parasympathetic and Sympathetic Responses in Cyclophosphamide‐Induced Cystitis in the Rat

In cyclophosphamide-induced cystitis in the rat, detrusor function is impaired and the expression and effects of muscarinic receptors altered. Whether or not the neuronal transmission may be affected by cystitis was presently investigated. Responses of urinary strip preparations from control and cyclophosphamide-pretreated rats to electrical field stimulation and to agonists were assessed in the absence and presence of muscarinic, adrenergic and purinergic receptor antagonists. Generally, atropine reduced contractions, but in contrast to controls, it also reduced responses to low electrical field stimulation intensity (1-5 Hz) in inflamed preparations. In both types, purinoceptor desensitization with alpha,beta-methylene adenosine-5'-triphosphate (alpha,beta-meATP) caused further reductions at low frequencies (<10 Hz). The muscarinic receptor antagonists atropine, 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) ('M(1)/M(3)/M(5)-selective'), methoctramine ('M(2)-selective') and pirenzepine ('M(1)-selective') antagonized the tonic component of the electrical field stimulation-evoked contractile response more potently than the phasic component. 4-DAMP inhibited the tonic contractions in controls more potently than methoctramine and pirenzepine. In inflamed preparations, the muscarinic receptor antagonism on the phasic component of the electrical field stimulation-evoked contraction was decreased and the pirenzepine and 4-DAMP antagonism on the tonic component was much less efficient than in controls. In contrast to controls, methoctramine increased -- instead of decreased -- the tonic responses at high frequencies. While contractions to carbachol and ATP were the same in inflamed and in control strips when related to a reference potassium response, isoprenaline-induced relaxations were smaller in inflamed strips. Thus, in cystitis substantial changes of the efferent functional responses occur. While postjunctional beta-adrenoceptor-mediated relaxations are reduced, effects by prejunctional inhibitory muscarinic receptors may be increased.

Unique pharmacological profile of aripiprazole as the phasic component buster

Aripiprazole is a recently introduced antipsychotic with a unique pharmacological profile, a dopamine partial agonist. Dopaminergic neural transmission has two different components, tonic and phasic, which have different physiological functions, but the effects of aripiprazole on tonic and phasic components are not reported. Studies on antipsychotics including aripiprazole and tonic/phasic dopamine transmission are summarized. Antipsychotics exert efficacy without extrapyramidal side effects (EPS's) when their occupation of dopamine D2 receptors reaches 65-80%. When a "tightly binding" antipsychotic binds 70% of D2 receptors, the remaining 30% are available for endogenous dopamine to bind. These tight antipsychotics suppress dopamine transmission in both tonic/phasic components equally so that similar proportions are kept. Aripiprazole is effective when >90% of D2 receptors are occupied. In this condition, less than 10% of D2 receptors are available for endogenous dopamine to bind; however, EPS's do not occur because aripiprazole exerts partial dopaminergic agonistic activity. Because the concentration of aripiprazole in the brain is relatively constant and it binds to D2 receptors tightly, the added dopaminergic agonism may show a tonic nature. Thus, aripiprazole suppresses the phasic component relatively more than the tonic component. In contrast, under treatment with "loosely binding" antipsychotics, phasic dopaminergic transmission is relatively preserved. Tight antipsychotics suppress both tonic and phasic components equally. Aripiprazole suppresses the phasic component relatively more than the tonic; that is, aripiprazole is a phasic [corrected] component buster. By contrast, suppression of the phasic component by loosely binding antipsychotics may be relatively weak.

Effect of Cholecystokinin-Octapeptide and Cerulein on Phasic and Tonic Components in Ovine Duodenum with Special Reference to the 'Minute Rhythm'

Cholecystokinin (CCK) can affect phasic contractions and the minute rhythm (MR) in ovine duodenum but its effect on the tonic component remains unclear. Thus, the aim of this study was to assess whether the hormone exerts significant changes on phasic and tonic components of the duodenal motor activity and on phasic and tonic components of the duodenal MR. Mechanical and electrical activities of the duodenum were recorded in four sheep before and after slow intravenous cholecystokinin octapeptide (CCK-OP, doses 20, 200 and 2000 ng/kg b.w.) and cerulein (doses 1, 10 and 100 ng/kg b.w.) administration in the course of phase 2b of the migrating motor complex. During 5-20-minute periods the area under contraction curve for phasic, tonic and total motor activity was measured for the whole curve and separately for the MR- related activity. It was found that both CCK peptides stimulate phasic and tonic components of the duodenal motor activity as well as both these components of the duodenal MR. The effect of CCK peptides on the tonic component was stronger than on the phasic component. These effects were similar in non-fasted and fasted animals. CCK-OP evoked slightly greater effect than cerulein. The effects of these CCK peptides on phasic and tonic components of the MR were similar. It is concluded that CCK-OP and cerulein stimulate both phasic and tonic components of the duodenal motor activity and phasic and tonic components related to the MR in sheep.

Delta sleep-inducing peptide and its analogues alleviate the severity of metaphit-induced audiogenic seizures in rats

We investigated the potential of delta sleep-inducing peptide (DSIP) and its analogue DSIP-12 (a nonapeptide with alanine in position 2 of DISP molecule substituted by beta-alanine) and tetrapeptide analogue DSIP1-4, to antagonize metaphit(1-1 (3-isothiocyanatophenl)-cvyclohexyl piperidine) induced generalized reflex audiogenic seizures in adult male Wistar albino rats. Five groups of adult male Wistar rats were intraperitoneally treated with: (1) saline; (2) metaphit: (3) metaphit + DSIP, (4) metaphit + DSIPI-4 and (5) metaphit + DSIP-12. To examine the blocking effects of DSIP and its analogues on fully developed metaphit seizures, in the last three groups they were administered 8h after metaphit injection. The rats were stimulated using an electric bell (1003 dB, 5-8 kHz, 60 s) one hour after metaphit injection and afterwards at hourly intervals during the experiment. For EEG recordings and power spectra three gold-plated screws were implanted into the skull. In metaphit-treated animals EEGs appeared as polyspikes and spike-wave complexes, while power spectra were increasing. The incidence and severity of netaphit-induced audiogenic seizures reached a peak value at 7-12 h after injection. Both DSIP and DSIP analogues significantly increased power spectra of delta waves and decreased the incidence of seizures, as well as mean seizure grade and tonic component of metaphit-induced convulsions. Taken together, these results suggest that DSIP and its analogues should be considered as potential antiepileptic agents.

Reduced rather than enhanced cholinergic airway constriction in mice with ablation of the large conductance Ca2+‐activated K+channel

The unique voltage- and Ca2+-dependent K+ (BK) channel, prominently expressed in airway smooth muscle cells, has been suggested as an important effector in controlling airway contractility. Its deletion in mice depolarized resting membrane potential of tracheal cells, suggesting an increased open-probability of voltage-gated Ca2+ channels. While carbachol concentration-dependently increased the tonic tension of wild-type (WT) trachea, mutant trachea showed a different response with rapid tension development followed by phasic contractions superimposed on a tonic component. Tonic contractions were substantially more dependent on L-type Ca2+ current in mutant than in WT trachea, even though L-type Ca2+ channels were not up-regulated. In the absence of L-type Ca2+ current, half-maximal contraction of trachea was shifted from 0.51 to 1.7 microM. In agreement, cholinergic bronchoconstriction was reduced in mutant lung slices, isolated-perfused lungs and, most impressively, in mutant mice analyzed by body plethysmography. Furthermore, isoprenaline-mediated airway relaxation was enhanced in mutants. In-depth analysis of cAMP and cGMP signaling revealed up-regulation of the cGMP pathway in mutant tracheal muscle. Inhibition of cGMP kinase reestablished normal sensitivity toward carbachol, indicating that up-regulation of cGMP signaling counterbalances for BK channel ablation, pointing to a predominant role of BK channel in regulation of airway tone.

Abnormal Neurotransmitter Release Underlying Behavioral and Cognitive Disorders: Toward Concepts of Dynamic and Function-Specific Dysregulation

Abnormalities in the regulation of neurotransmitter release and/or abnormal levels of extracellular neurotransmitter concentrations have remained core components of hypotheses on the neuronal foundations of behavioral and cognitive disorders and the symptoms of neuropsychiatric and neurodegenerative disorders. Furthermore, therapeutic drugs for the treatment of these disorders have been developed and categorized largely on the basis of their effects on neurotransmitter release and resulting receptor stimulation. This perspective stresses the theoretical and practical implications of hypotheses that address the dynamic nature of neurotransmitter dysregulation, including the multiple feedback mechanisms regulating synaptic processes, phasic and tonic components of neurotransmission, compartmentalized release, differentiation between dysregulation of basal vs activated release, and abnormal release from neuronal systems recruited by behavioral and cognitive activity. Several examples illustrate that the nature of the neurotransmitter dysregulation in animal models, including the direction of drug effects on neurotransmitter release, depends fundamentally on the state of activity of the neurotransmitter system of interest and on the behavioral and cognitive functions recruiting these systems. Evidence from evolving techniques for the measurement of neurotransmitter release at high spatial and temporal resolution is likely to advance hypotheses describing the pivotal role of neurotransmitter dysfunction in the development of essential symptoms of major neuropsychiatric disorders, and also to refine neuropharmacological mechanisms to serve as targets for new treatment approaches. The significance and usefulness of hypotheses concerning the abnormal regulation of the release of extracellular concentrations of primary messengers depend on the effective integration of emerging concepts describing the dynamic, compartmentalized, and activity-dependent characteristics of dysregulated neurotransmitter systems.

PKC δ-isoform translocation and enhancement of tonic contractions of gastrointestinal smooth muscle

PKC is involved in mediating the tonic component of gastrointestinal smooth muscle contraction in response to stimulation by agonists for G protein-coupled receptors. Here, we present pharmacological and immunohistochemical evidence indicating that a member of the novel PKC isoforms, PKC-delta, is involved in maintaining muscarinic receptor-coupled tonic contractions of the guinea pig ileum. The tonic component of carbachol-evoked contractions was enhanced by an activator of conventional and novel PKCs, phorbol 12,13-dibutyrate (PDBu; 200 nM or 1 microM), and by an activator of novel PKCs, ingenol 3,20-dibenzoate (IDB; 100 or 500 nM). Enhancement was unaffected by concentrations of bisindolylmaleimide I (BIM-I; 22 nM) that block conventional PKCs or by a PKC-epsilon-specific inhibitor peptide but was attenuated by higher doses of BIM-I (2.2 microM). Relevant proteins were localized at a cellular and subcellular level using confocal analysis. Immunohistochemical staining of the ileum showed that PKC-delta was exclusively expressed in smooth muscles distributed throughout the layers of the gut wall. PKC-epsilon immunoreactivity was prominent in enteric neurons but was largely absent from smooth muscle of the muscularis externa. Treatment with PDBu, IDB, or carbachol resulted in a time- and concentration-dependent translocation of PKC-delta from the cytoplasm to filamentous structures within smooth muscle cells. These were parallel to, but distinct from, actin filaments. The translocation of PKC-delta in response to carbachol was significantly reduced by scopolamine or calphostin C. The present study indicates that the tonic carbachol-induced contraction of the guinea pig ileum is mediated through a novel PKC, probably PKC-delta.

The ratio between the phasic and tonic components of the frontal midline Θ rhythm in the attention test

The frontal midline Θ rhythm in the GO/NOGO paradigm was studied in a group of apparently healthy children at ages of 7–13 years. Calculated event-related synchronization in response to stimulus presentation in tests was used as an index of the phasic component, and the relative change in the EEG power in the Θ band (compared to the activity in the state of quiet wakefulness) in response to test performance was used as an index of the tonic component. Subjects were divided into two groups according to the characteristics of the baseline Θ activity. A statistically significant correlation between the phasic and tonic components of the Θ rhythm was found in the group of children characterized by the absence of the frontal midline Θ rhythm in the baseline EEG. No such correlation was found in the group of children characterized by a pronounced baseline Θ rhythm. The results testify to the functional heterogeneity of the phasic and tonic components of the human midline Θ rhythm.

Monday July 3, 2006 15:00-17:00 Hall 5B Discussion Group Session From functional brain anatomy to clinical seizure semiology

Abstract 1 C. Baumgartner ( 1 Department of Neurology, Medical University of Vienna, Austria ) Motor manifestations during epileptic seizures include focal clonic motor seizures, seizures with asymmetrical tonic posturing and seizures with hypermotor automatisms. Focal clonic motor seizures are generated by epileptic activity in the primary motor cortex and consist of unilateral, jerky, usually rhythmic movements, frequently also tonic components like tonic postural signs and tonic face contractions can be observed. Most frequently they start in the face or hand due to their large representation in primary motor cortex and due to a lower seizure threshold of these cortical regions. Asymmetric tonic seizures or supplementary motor area (SMA) seizures are mediated by epileptic activity in the supplementary sensorimotor area or the premotor cortex, but can also be generated by epileptic activity in other frontal lobe regions. Because frequently somatosensory auras like feelings of pulling, pulsing, heaviness, numbness or tingling which can occur ipsi-, contra- or bilaterally occur the term “supplementary sensorimotor seizures” has been introduced. Asymmetric tonic seizures are characterized by bilateral, asymmetric, tonic or dystonic posturing of the extremities and facial grimacing resulting in tonic posturing. Vocalizations frequently occurring during asymmetrical tonic seizures can be a reactive phenomenon, can be caused by a tonic contraction of the diaphragm and laryngeal muscles and finally can be a consequence of an activation of Broca's area or SMA. While hyperkinetic or ‘hypermotor’ automatisms are not associated with a seizure onset in any specific region within the frontal lobes. They are characterized by prominent and complex, coordinated, purposeless or semipurposefull movements (trembling, waving, rotating, stepping, cycling, rocking, hopping etc.) with an agitated or frenetic appearance. Vocalizations often are explosive and pronounced which seems to be unique for frontal lobe seizures with hypermotor automatisms. Seizures with hypermotor automatisms usually are associated with a loss of consciousness and therefore also are referred to as frontal lobe complex partial seizures.

Ethanol blocks nicotine-induced seizures in mice: comparison with midazolam and baclofen

Low doses of ethanol may antagonize the pharmacological effects of nicotine. Recently, it has been shown that the effects of ethanol on nicotine discrimination are not correlated with blood ethanol levels. The aim of the present study was to evaluate whether ethanol (0.5–2 g/kg, i.p.) could block nicotine-induced seizures in C57BL/6J mice and to correlate ethanol's actions with blood ethanol concentrations. For comparison, the effects of a γ-aminobutyric acid A (GABA A)/benzodiazepine receptor positive modulator, midazolam (0.25–40 mg/kg, i.p.), and a γ-aminobutyric acid B receptor agonist, baclofen (2.5–20 mg/kg, i.p.), were assessed in the same procedure. Nicotine (3–9 mg/kg, s.c.) induced clonic–tonic seizures in a dose-dependent manner. Ethanol, administered 5 or 50 min before nicotine, dose dependently antagonized seizures elicited by 6 mg/kg nicotine. The anticonvulsant effects of ethanol correlated with blood ethanol levels and were comparable to those exerted by midazolam. Baclofen antagonized only the tonic component of nicotine-induced convulsions. The anticonvulsant doses of ethanol (0.5–2 g/kg), midazolam (0.5–1 mg/kg), and baclofen (5–10 mg/kg) did not affect spontaneous locomotor activity in a control experiment. The present results indicate that (i) ethanol may block nicotine-induced seizures in mice at doses that do not alter locomotor activity and (ii) the antiseizure effects of ethanol depend on blood ethanol levels and are comparable to those exerted by the GABA A positive modulator midazolam.

Endoscopic plication partially restores a missing tonic pressure component to the LES and the stiffness of relaxed esophageal muscle during tLESR in patients with GERD

Temple University Medical Center, United States Special Issue of the 8th World Congress of OESO Avignon, September 3–6, 2006 Oral, Video and Poster Sessions

γ-Aminobutyric Acid Type A Receptors and Alcoholism

Alcohol facilitates gamma-aminobutyric acid (GABA) function, and GABA type A (GABA(A)) receptor-facilitating agents suppress alcohol withdrawal symptoms. Advances in molecular neuroscience, genetics, and neuroimaging provide new insights into the role of brain GABA systems in short- and long-term alcohol effects. To review the role of brain GABA systems in alcohol response, alcohol dependence, alcoholism vulnerability, and alcoholism pharmacotherapy. Literature review. Alcohol increases GABA release, raises neurosteroid levels, and may potently enhance the function of a GABA(A) receptor subclass that shows high affinity for GABA and neurosteroids, relative insensitivity to benzodiazepines, low chloride conductance, and an extrasynaptic location. Variation in GABA(A) receptor subunit genes may contribute to the vulnerability to alcoholism, particularly in the context of environmental risk factors. Alcohol dependence is associated with time-dependent changes in brain GABA(A) receptor density and subunit gene expression levels that contribute to a withdrawal-related deficit in GABA(A) receptor function. However, cortical GABA levels are not reduced during acute withdrawal. Benzodiazepine-assisted detoxification enhances a phasic component of GABA function. However, novel treatments target the tonic component of GABA neurotransmission mediated by benzodiazepine-insensitive GABA(A) receptors. Smoking attenuates withdrawal-related disturbances in brain GABA function, perhaps contributing to comorbid nicotine and alcohol dependence. The GABA systems show recovery with long-term sobriety. Recent research deepens our understanding of the role of GABA systems in alcohol action, alcohol dependence, and the vulnerability to alcoholism. Also, GABA(A) receptor subtype-selective treatments merit exploration for reducing withdrawal symptoms and drinking in alcohol-dependent individuals.

Muscarinic stimulation of the mouse isolated whole bladder: physiological responses in young and ageing mice

1 Peripheral autonomous bladder activity is an incompletely understood property that may be important both in normal bladder function and in functional problems of the lower urinary tract. We describe how a muscarinic agonist, arecaidine, influences intravesical pressure and intramural bladder contractions in the isolated mouse and how response varies in ageing mice. 2 A group of 12 mice aged 3-4 months was compared with an 'ageing' group of 12 mice age 28-34 months. Bladders were microsurgically removed and mounted in whole organ tissue baths. The effects of the muscarinic agonist arecaidine on intravesical pressure and intramural contractions were performed at different bladder volumes. 3 In normal mice, arecaidine elicited tonic and phasic contractions, the latter showing a more substantial increase in amplitude with bladder distension. Localized 'micromotion' contractions were seen in the bladder wall, with regional differences arising after exposure to arecaidine. A background release of acetylcholine was inferred from the pressure increase induced by the cholinesterase inhibitor physostigmine. 4 Both micromotion activity and the phasic component of the arecaidine response were substantially reduced in ageing mice; the tonic component was preserved in the same specimens. 5 We conclude that the enhanced pressure fluctuations seen at high bladder volumes may act as a peripheral determinant of bladder capacity, and that changes in such activity may contribute to altered functional capacity and lower urinary tract symptoms in ageing individuals.

Muscarinic stimulation of the rat isolated whole bladder: pathophysiological models of detrusor overactivity

1 Hypotheses as to the pathophysiological basis of bladder detrusor muscle overactivity (DO) have identified both central nervous and peripheral mechanisms as likely contributory factors. In this paper, we describe peripheral autonomous bladder activity in two animal models of DO and discuss how the differences observed between the two models support the likelihood that clinical DO has a multifactorial basis. 2 A total of 12 adult female Sprague-Dawley rats underwent obstruction or sham operation for 1 or 4 weeks. Six adult female spontaneously hypertensive rats (SHR) were compared with normal Wistar controls. Bladders were microsurgically removed and mounted in whole organ tissue baths. Recordings of intravesical pressure in response to the muscarinic receptor agonist arecaidine were performed under standardized conditions. 3 In the partially obstructed rat bladder, the amplitude of pressure fluctuations elicited by the muscarinic agonist arecaidine was significantly increased compared with sham-operated animals. The tonic component of the response was no different for the two groups. No difference from controls was apparent in the SHR. 4 We conclude that alterations in autonomous bladder activity in the obstructed rat model suggest that peripheral functional changes contribute to the pathophysiological abnormality. In contrast, the fundamental abnormality in the SHR appears to be at a more central level. The observations support the supposition that lesions at widely separate sites can give rise to apparently similar abnormalities of lower urinary tract function.

Dynamic changes in free Ca-calmodulin levels in adult cardiac myocytes

Despite its multifunctional role in cardiac myocyte function, little is known about dynamic changes in activation state of calmodulin (CaM). Thus, the purpose of this study was to develop a tool to measure Ca bound CaM (Ca–CaM) levels in intact cardiac myocytes. For dynamic measurements of Ca–CaM, we generated an adenoviral vector which expresses a cyan and a yellow fluorescent protein linked by a modified version of the Ca–CaM binding domain of avian smooth muscle myosin light chain kinase. Adult rabbit cardiac myocytes were infected with the Ca–CaM sensing probe or simultaneously infected with viruses containing CaM and the Ca–CaM sensing probe for 24–48 h. Myocytes were then field stimulated (1 Hz) and excited at 440 nm with emitted fluorescence measured at 485 and 535 nm. Changes in [Ca–CaM] are expressed as the ratio of 485 nm/535 nm. Small beat-to-beat changes of [Ca–CaM] were detected, but only when CaM was co-expressed with the sensor. However, upon β-adrenergic stimulation with isoproterenol, there was an increase in the amplitude of the signals during each beat (parallel to the shortening, which is an indirect measure of [Ca] i) and also a rise in the diastolic [Ca–CaM]. Total [CaM] measured by both competitive ELISA and semi-quantitative Western blots was 5–6 μM in isolated adult ventricular myocytes. Our results indicate that there are dynamic changes in free Ca–CaM levels (a phasic component tracking [Ca] i) as well as system memory that integrates the [Ca] i signals (a tonic component).