Subgroup comparison according to clinical phenotype and serostatus in autoimmune encephalitis: a multicenter retrospective study.

BACKGROUND AND PURPOSE: Autoimmune encephalitides (AE) include a spectrum of neurological disorders whose diagnosis revolves around the detection of neuronal antibodies (Abs). Consensus-based diagnostic criteria (AE-DC) allow clinic-serological subgrouping of AE, with unclear prognostic implications. The impact of AE-DC on patients' management was studied, focusing on the subgroup of Ab-negative-AE. METHODS: This was a retrospective multicenter study on patients fulfilling AE-DC. All patients underwent Ab testing with commercial cell-based assays (CBAs) and, when available, in-house assays (immunohistochemistry, live/fixed CBAs, neuronal cultures) that contributed to defining final categories. Patients were classified as Ab-positive-AE [N-methyl-d-aspartate-receptor encephalitis (NMDAR-E), Ab-positive limbic encephalitis (LE), definite-AE] or Ab-negative-AE (Ab-negative-LE, probable-AE, possible-AE). RESULTS: Commercial CBAs detected neuronal Abs in 70/118 (59.3%) patients. Testing 37/48 Ab-negative cases, in-house assays identified Abs in 11 patients (29.7%). A hundred and eighteen patients fulfilled the AE-DC, 81 (68.6%) with Ab-positive-AE (Ab-positive-LE, 40; NMDAR-E, 32; definite-AE, nine) and 37 (31.4%) with Ab-negative-AE (Ab-negative-LE, 17; probable/possible-AE, 20). Clinical phenotypes were similar in Ab-positive-LE versus Ab-negative-LE. Twenty-four/118 (20.3%) patients had tumors, and 19/118 (16.1%) relapsed, regardless of being Ab-positive or Ab-negative. Ab-positive-AE patients were treated earlier than Ab-negative-AE patients (P = 0.045), responded more frequently to treatments (92.3% vs. 65.6%, P < 0.001) and received second-line therapies more often (33.3% vs. 10.8%, P = 0.01). Delays in first-line therapy initiation were associated with poor response (P = 0.022; odds ratio 1.02; confidence interval 1.00-1.04). CONCLUSIONS: In-house diagnostics improved Ab detection allowing better patient management but was available in a patient subgroup only, implying possible Ab-positive-AE underestimation. Notwithstanding this limitation, our findings suggest that Ab-negative-AE and Ab-positive-AE patients share similar oncological profiles, warranting appropriate tumor screening. Ab-negative-AE patients risk worse responses due to delayed and less aggressive treatments.

Cholesterylphosphoserine as inhibitor of cell adhesion and actin polymerization in human T cells.

To further investigate the immunosuppressive activity of cholesterylphosphoserine (CPHS), we examined a variety of human T cell responses including proliferation, adhesion and cytoskeletal organization. The CPHS-induced inhibition of T cell response is greater in the integrin-dependent mixed lymphocyte reaction than in the integrin-independent proliferation elicited by anti-TCR-CD3 or anti-CD28 antibodies in the presence of tetradecanoylphorbol acetate. Consistently, CPHS inhibits the homotypic T cell adhesion involving the integrin alphaLbeta2 (LFA-1) and the cell adhesion to fibronectin and rVCAM-1 involving the integrins of the beta1 family. Since CPHS does not change integrin expression but inhibits post-receptor events such as cell spreading and pseudopodal projections, it seems likely that the site of CPHS influence is distal to the adhesion receptors. In agreement, the steroid prevents the reorganization of actin cytoskeleton occurring when T cells are allowed to spread on immobilized anti-CD3 in the absence of integrin activation. We suggest that CPHS acts on the metabolic pathway in which signals from integrin and growth factor receptors converge to induce the reorganization of the actin cytoskeleton. Selectivity in the action of CPHS is indicated by its ineffectiveness in the integrin-mediated adhesion of the monocytic cell line U-937 to fibronectin.

Cell cycle-independent death of prostate adenocarcinoma is induced by the trk tyrosine kinase inhibitor CEP-751 (KT6587).

Advanced prostate cancer remains largely incurable, primarily because the very low growth fraction present in these tumors makes them generally resistant to treatment with standard chemotherapeutic agents that target cell division. Effective therapies should therefore induce death of prostate cancer cells, independent of their growth rate. trkA, the high-affinity tyrosine kinase-linked receptor for nerve growth factor, has been implicated in prostatic cancer growth and may represent a molecular target for therapeutic agents. At low mg/kg doses, the trk tyrosine kinase inhibitor CEP-751 (KT6587) inhibits prostatic cancer growth in nine different animal models independent of the tumor growth rate, androgen sensitivity, metastatic ability, or state of tumor differentiation. CEP-751 is selective for cancerous versus normal prostate cells and affects the growth of only a limited number of nonprostate tumors. Importantly, CEP-751 induces cell death of prostate cancer cells in a cell cycle-independent fashion and, therefore, represents a novel therapeutic approach to the management of both hormone-dependent and hormone-independent prostate cancer.

Measurement of intracellular Ca2+ in cultured arterial smooth muscle cells using Fura-2 and digital imaging microscopy.

A rise in cytosolic free Ca2+ is the immediate trigger for contraction in vascular smooth muscle (VSM). We employed the fluorescent Ca2(+)-indicator, Fura-2, and digital imaging microscopy to study the spatial distribution of intracellular Ca2+ in cultured A7r5 cells and the changes evoked by activation with 5-HT. Several methodological considerations that affect the temporal and spatial resolution of Ca2+ images have been addressed. These include: cytoplasmic distribution of Fura-2, wavelength selection for ratio imaging, signal:noise ratio measurement and the effect of [Ca2+] on the limits of detectability under conditions in which [Ca2+] is changing. The distribution of apparent free Ca2+, [Ca2+]App, in A7r5 cells was heterogeneous. This reflects, in part, different pools of intracellular Ca2+. [Ca2+]App was lowest in the nucleus (113 +/- 14 nM; n = 20 cells) and highest in the organelle-rich perinuclear region (228 +/- 12; n = 20), while the surrounding cytoplasmic area (containing relatively few organelles) had intermediate [Ca2+]app levels (150 +/- 13; n = 20). 5-HT (1 microM) evoked transient increases in [Ca2+]App that began within 11 s as relatively modest elevations of [Ca2+]App in the periphery, near the sarcolemma, and subsequently spread to the entire cell, reaching a peak within 18-24 s. At the peak of the Ca2+ transients, [Ca2+]App was highest in the perinuclear region where it sometimes exceeded the maximal detectable levels of the system (1.9 microM). The average peak Ca2+ transient amplitude in the non-nuclear cytoplasm was 1083 +/- 208 nM (1 microM 5-HT; n = 20 cells). Despite the continued presence of 5-HT following the Ca2+ transients, [Ca2+]App then returned to pre-stimulation levels within 5 min. These observations indicate that digital imaging microscopy enables the study of subcellular regulation of intracellular Ca2+ in VSM. The results provide new insights into the role of localized changes in Ca2+ in the regulation of VSM contractility.

Autocrine-paracrine role of endothelin-1 in the regulation of aldosterone synthase expression and intracellular Ca2+ in human adrenocortical carcinoma NCI-H295 cells.

The role played by endothelin (ET-1) and its receptor subtypes A and B (ET(A) and ET(B)) in the functional regulation of human NCI-H295 adrenocortical carcinoma cells has been investigated. Reverse transcription-PCR with primers specific for prepro-ET-1, human ET-1 converting enzyme-1, ET(A), and ET(B) complementary DNAs consistently demonstrated the expression of all genes in NCI-H295 cells. The presence of mature ET-1 and both its receptor subtypes was confirmed by immunocytochemistry and autoradiography, respectively. Aldosterone synthase (AS) messenger RNA was also detected in NCI-H295 cells, and AS gene expression was enhanced by both ET-1 and the specific ET(B) agonist IRL-1620; this effect was not inhibited by either the ET(A) antagonist BQ-123 or the ET(B) antagonist BQ-788. A clear-cut increase in the intracellular Ca2+ concentration in NCI-H295 cells in response to ET(B), but not ET(A), activation was observed. In light of these findings, the following conclusions can be drawn: 1) NCI-H295 cells possess an active ET-1 biosynthetic pathway and are provided with ET(A) and ET(B) receptors; 2) ET-1 regulates in an autocrine/paracrine fashion the secretion of aldosterone by NCI-H295 cells by enhancing both AS transcription and raising the intracellular Ca2+ concentration; and 3) the former effect of ET-1 probably involves the activation of both receptor subtypes, whereas calcium response is exclusively mediated by the ET(B) receptor.

Effects of an endogenous ouabainlike compound on heart and aorta.

An endogenous ouabainlike compound (OLC) has been purified from human plasma, and mass spectrometry has shown it to be indistinguishable from plant-derived ouabain. This human OLC was tested for its effects on evoked tension in guinea pig left atria and aortic rings. The tissues were incubated at 37 degrees C in bicarbonate-buffered physiological salt solution gassed with 95% O2-5% CO2. In atria stimulated electrically at 1 Hz, 85 and 170 nM human OLC increased peak active force to 177 +/- 15% and 313 +/- 32% of control, respectively (n = 3), with little effect on the duration of contraction. On washout of the OLC, peak systolic force returned to the control level with a half-time of 4.3 +/- 0.5 minutes. Similar results were obtained with 160 nM plant-derived ouabain: peak systolic force increased to 310 +/- 31% of control (n = 4) and returned to the control level with a half-time of 3.8 +/- 0.2 minutes during washout. In aortic rings, neither 170 nM human OLC nor 160 nM plant ouabain (30-minute treatments) affected resting (unstimulated) tension, but they increased the contractions evoked by histamine (0.2-1.0 microM) to 156 +/- 13% (n = 4) and 143 +/- 6% (n = 4) of control responses, respectively. The mean half-time for washout of the OLC and plant ouabain-induced augmentation of histamine-evoked tension exceeded 35 minutes. These data show that human OLC has cardiotonic and vasotonic actions qualitatively and quantitatively similar to those observed with plant ouabain.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuropsychological testing may predict early progression of asymptomatic adrenoleukodystrophy.

OBJECTIVES: To investigate the correlation between neuropsychological and MRI findings in children with the childhood cerebral (CCALD) and asymptomatic forms of X-linked adrenoleukodystrophy (ALD) and to identify early cognitive markers that may predict disease progression in asymptomatic children with ALD. BACKGROUND: The few published neuropsychological studies on CCALD suggest a correlation between the pattern of cognitive deficit and lesion site; however, neuropsychological performance in asymptomatic children with ALD has not been investigated. METHODS: The authors assessed cognitive function and cerebral MRI findings in seven CCALD and eight asymptomatic ALD children. RESULTS: The CCALD children's cognitive skills were severely compromised, especially Wechsler and executive functions. Visual perception, short-term memory, and language were generally preserved, except that naming was severely impaired. All had extensive posterior white matter deterioration. The asymptomatic children had relatively intact neuropsychological performance, but their verbal fluency was compromised and naming severely impaired. All except one had mild white matter alterations. For all the children, the majority of neuropsychological test performance correlated significantly with extent of white matter lesions. CONCLUSIONS: The pattern of cognitive deterioration in children with CCALD and the significant correlation of neuropsychological test performance with extent of white matter lesions indicate a white matter dementia similar to that observed in adults with demyelinating diseases. The deficits found in asymptomatic children, despite their normal intelligence, suggest that careful neuropsychological investigation can identify early signs of malfunction. These may be markers of disease progression useful for selecting children for bone marrow transplant, although this will require confirmation by prospective longitudinal studies.

Effects of amiloride in guinea-pig and rat left atrial contraction as affected by frequency of stimulation and [Ca2+]0-[Na+]0 ratio: role of Na+/Ca2+ exchange.

1. The effect of amiloride (0.5 mM) on guinea-pig and rat left atria driven at various rates of stimulation and different [Ca2+]0-[Na+]0 ratios has been studied. 2. Amiloride elicited a positive inotropic response in guinea-pig left atria driven at 0.1 Hz, 0.5 Hz and 1 Hz when [Ca2+]0 was 3.6 mM, 1.8 mM and 0.9 mM respectively but not when [Ca2+]0 was 2.7 mM at 0.1 Hz, 0.9 mM at 0.5 Hz and 0.45 mM at 1 Hz. 3. A positive inotropic response was obtained in guinea-pig left atria driven at 0.1 Hz and 1 Hz when [Ca2+]0-[Na+]0(2) was increased respectively from 8 x 10(-5) to 16 x 10(-5) and from 2 x 10(-5) to 8 x 10(-5). The positive inotropic effect was evident only when the ratio was increased by increasing [Ca2+]0 and not by decreasing [Na+]0. 4. In the presence of amiloride, the force of contraction of guinea-pig left atria decreased instead of increasing, when the rate of stimulation was lowered from 1 Hz to 0.01 Hz. Amiloride inhibited the post-rest potentiation. 5. In rat left atria amiloride was devoid of any effect in all the above-mentioned experimental conditions. 6. It is suggested that the pattern of cardiac actions of amiloride can be explained by the inhibition of the Na+/Ca2+ exchange system.

Na/Ca exchange and tension development in vascular smooth muscle: effect of amiloride.

1. The potassium-sparing diuretic, amiloride, has been shown to inhibit the Na/Ca exchange system in various preparations. The effects of this drug have been investigated on the contractions of guinea-pig aortic strips elicited by reduction of external K, by addition of ouabain and by removal of external Na. 2. Amiloride (5 X 10(-6) M-5 X 10(-4) M) inhibited the mechanical responses when it was added before giving the stimulus for contractions, but was not effective in relaxing the contracted strips. The drug shifted to the right the dose-response curve for Ca in low K solution. 3. The calcium antagonist diltiazem had no effect on the ouabain-, low K- and Na-free-induced contractions. 4. Amiloride decreased the rate of relaxation of aortic strips induced by removal of the low K solution. 5. The pattern of amiloride action on ouabain-, low K- and Na-free-induced contractions suggests that the drug interferes with Ca influx. The effect of amiloride on the relaxation rate of low K-contracted aortic strips is consistent with an interference with Ca efflux. 6. It is suggested that amiloride prevents Ca fluxes through the Na/Ca exchange system of guinea-pig aortic strips.

Electrocardiographic interactions between pinacidil, a potassium channel opener and class I antiarrhythmic agents in guinea-pig isolated perfused heart.

1. Drugs that shorten action potential duration could decrease the Na-channel blocking effect of class I antiarrhythmic agents by reducing the availability of Na channel in the inactivated state. 2. This hypothesis was tested in guinea-pig perfused heart, measuring the surface ECG effects of three class I drugs endowed with different binding kinetics (15 microM mexiletine, 10 microM quinidine and 3 microM flecainide) in the presence of increasing concentrations of pinacidil (10 microM, 30 microM, 50 microM), a potassium channel opener that shortens action potential duration. 3. The ECG parameters measured were: the QRS interval, i.e. the intraventricular conduction time; the JT interval, which reflects the duration of ventricular repolarization; the ratio between JT peak (the time from the end of QRS and the peak of T wave) and JT interval, which quantifies changes in the morphology of the T wave. 4. At the concentrations tested all the antiarrhythmic drugs widened the QRS complex by 55-60%. Flecainide did not significantly change JT interval, but quinidine prolonged and mexiletine shortened it. Mexiletine also decreased the JT peak/JT ratio. Pinacidil by itself decreased the JT interval and the JT peak/JT ratio in a dose-dependent way, but did not affect QRS duration. 5. In the presence of fixed antiarrhythmic drug concentrations, however, pinacidil decreased the QRS prolongation induced by mexiletine (-17%) and quinidine (-8%), but not that induced by flecainide: this effect was already maximal at the lower concentration tested (10 microM) and there was no relationship between pinacidil-induced JT shortening and QRS changes. To explain this unexpected result it has been supposed that, at the driving frequency used (4 Hz), myocardial cells were partially depolarized and that pinacidil could repolarize them, thus decreasing the number of inactivated Na channels and the effects of drugs that (mainly or partly) block the channels in the inactivated state. In agreement with this hypothesis, an additional series of experiments carried out with 15 microM mexiletine at a lower stimulation rate (2 Hz) showed only a negligible loss of QRS effect (- 2.3%) at any pinacidil concentration.6. Flecainide, but not quinidine and mexiletine, antagonized the JT shortening induced by pinacidil;furthermore, no drug modified the JTp/JT decrease induced by pinacidil.7. These results indicate that: (a) an antagonism between class I antiarrhythmic drugs and pinacidil is possible; (b) mexiletine is the most involved among the drugs tested; (c) the interaction is not related to pinacidil-induced repolarization shortening, but probably to changes in membrane resting potential. The possible clinical implications need to be defined.

Effects of pinacidil on guinea-pig isolated perfused heart with particular reference to the proarrhythmic effect.

1. The effects of pinacidil (10, 30, 50 microM) on contractility (+dP/dtmax), coronary perfusion pressure (cP), and ECG intervals (PR, QRS, QT) have been studied on constant-flow perfused guinea-pig hearts, driven at four frequencies (2.5, 3, 3.5, 4 Hz). 2. Pinacidil decreased +dP/dtmax, cP and the QT interval in a dose-dependent manner, whereas the PR interval was increased. QRS duration was not modified. All these effects were independent of driving frequency. Pinacidil decreased the interval from Q-wave to T-wave peak (QTpeak) to a greater extent than the QT interval, thus decreasing the QTpeak/QT ratio. This effect, unlike that on QT interval, was more evident at the highest frequency of stimulation. 3. In 4 out of 20 hearts treated with pinacidil sustained ventricular fibrillation (VF) occurred following a short run of premature ventricular beats (R on T phenomenon). 4. In separate experiments, an attempt to induce VF electrically was made at drug concentrations ranging from 10 microM to 100 microM (8 experiments for each concentration). In control conditions and at the lowest concentrations of pinacidil tested (10 microM) VF could never be induced; in the presence of 30 microM pinacidil VF was induced in 5 out of 8 experiments. Drug concentrations higher than 50 microM permitted the induction of VF in every case. 5. Although the concentrations of pinacidil producing ventricular fibrillation are 30-40 times higher than those found in patients under long term treatment with this agent, it is suggested that caution should be used in prescribing this drug, at least in patients suffering from myocardial ischaemia.

Effect of subthreshold ouabain on the tone of guinea-pig aortic strips following repeated noradrenaline stimulation.

1. The effect of ouabain at a concentration (0.8 microM) that does not induce contractile response in guinea-pig aortic strips has been studied on endothelium-denuded strips repeatedly stimulated with 1 microM noradrenaline or 60 mM K+ applied for 5 min every 30 min. 2. The resting tone (i.e. the tone between one noradrenaline stimulation and the following) of the aortic strips exposed to ouabain increased progressively, whereas the control strips (no ouabain) completely relaxed on washout of the agonist. In the aortic strips stimulated by 60 mM K+, the resting tone did not increase. 3. The calcium antagonist, verapamil, did not affect the increase in tone, that was nevertheless strictly dependent on external calcium, since the contracted strips completely relaxed on calcium removal and promptly contracted again on calcium readdition. This finding indicates a mechanism independent of voltage-gated calcium channels. 4. Caffeine-induced contractions, taken as a measure of sarcoplasmic reticulum calcium content, were amplified by the presence of ouabain in aortic strips either stimulated by noradrenaline or unstimulated, with a larger increase in the former. 5. These results suggest that the repeated stimulation of guinea-pig aortic strips by noradrenaline in the presence of ouabain, by raising both intracellular Na+ and Ca2+, decreases the ouabain threshold concentration required for contraction, thus increasing the responsiveness of vascular smooth muscle to the glycoside.

Vasorelaxant properties of norbormide, a selective vasoconstrictor agent for the rat microvasculature.

1. The effects of norbormide on the contractility of endothelium-deprived rat, guinea-pig, mouse, and human artery rings, and of freshly isolated smooth muscle cells of rat caudal artery were investigated. In addition, the effect of norbormide on intracellular calcium levels of A7r5 cells was evaluated. 2. In resting rat mesenteric, renal, and caudal arteries, norbormide (0.5-50 microM) induced a concentration-dependent contractile effect. In rat caudal artery, the contraction was very slowly reversible on washing, completely abolished in the absence of extracellular calcium, and antagonized by high concentrations (10-800 microM) of verapamil. The norbormide effect persisted upon removal of either extracellular Na+ or K+. The contractile effect of norbormide was observed also in single, freshly isolated smooth muscle cells from rat caudal artery. 3. In resting rat and guinea-pig aortae, guinea-pig mesenteric artery, mouse caudal artery, and human subcutaneous resistance arteries, norbormide did not induce contraction. When these vessels were contracted by 80 mM KCl, norbormide (10-100 microM) caused relaxation. Norbormide inhibited the response to Ca2+ of rat aorta incubated in 80 mM KCl/Ca2(+)-free medium. Norbormide (up to 100 microM) was ineffective in phenylephrine-contracted guinea-pig and rat aorta. 4. In A7r5 cells, a cell line from rat aorta, norbormide prevented high K(+)- but not 5-hydroxytryptamine-induced intracellular calcium transients. 5. These findings indicate that in vitro, norbormide induces a myogenic contraction, selective for the rat small vessels, by promoting calcium entry in smooth muscle cells, presumably through calcium channels. In rat aorta and arteries from other mammals, norbormide behaves like a calcium channel entry blocker.

Calcium-antagonist effects of norbormide on isolated perfused heart and cardiac myocytes of guinea-pig: a comparison with verapamil.

1. Cardiac effects on norbormide and verapamil were compared in single ventricular myocytes, right atria, and Langendorff perfused hearts isolated from guinea-pigs. 2. In ventricular myocytes, norbormide 50 microM inhibited the peak calcium current (ICa) by 49.6 +/- 3.9% without altering the shape of the current-voltage relationship; verapamil 1 microM inhibited ICa by 83.2 +/- 3.3%. Neither norbormide nor verapamil affected ICa at the first beat after a 3 min quiescence period; during repeated depolarizations, both drugs cumulatively blocked ICa (use-dependence), with time constants of 23.0 +/- 7.0 s for norbormide and 91.3 +/- 8.4 s for verapamil. 3. In constant-flow perfused hearts electrically driven at 2.5 Hz or 3.3 Hz, both norbormide and verapamil concentration-dependently decreased ventricular contractility (dP/dtmax), atrio-ventricular (AV) conduction velocity and coronary pressure. Intraventricular conduction velocity was slightly decreased by norbormide but not by verapamil. At an equivalent change in AV conduction, norbormide depressed heart contractility less than verapamil. The effects of norbormide on AV conduction, intraventricular conduction, and contractility were frequency-dependent. Furthermore, the curves correlating the mechanical and electrical effects of norbormide at the two frequencies used were apparently coincident, while those of verapamil were clearly separated. 4. In spontaneously beating right atria, norbormide and verapamil decreased the frequency of sinus node (SA) in a concentration-dependent way. At an equivalent effect on the AV conduction, norbormide exerted a greater effect on sinus frequency than verapamil. 5. These results indicate that in guinea-pig heart norbormide has the pharmacological profile of a Ca-antagonist with strong electrophysiological properties. In comparison with verapamil, norbormide is more selective on SA and AV node tissues and exerts a weaker negative inotropic effect on ventricles. In principle, this pattern of effects may be an advantage in treating supraventricular tachyarrhythmias in patients with heart failure. The effect of norbormide on intraventricular conduction may represent an additional antiarrhythmic mechanism.

Alpha1-adrenoceptor-mediated formation of glycerophosphoinositol 4-phosphate in rat heart: possible role in the positive inotropic response.

In the present study, we investigated whether phospholipase A2 (PLA2)/lysophospholipase activity producing glycerophosphoinositols from phosphoinositides was operating in rat heart and could be stimulated by alpha1-adrenergic agonists. PLA2/lysophospholipase activity was found in homogenates from rat right ventricles. The stimulation of PLA2/lysophospholipase activity by noradrenaline (NA) was prevented either by the alpha1-adrenergic antagonist prazosin or arachidonyl trifluoromethyl ketone, a selective inhibitor of the 85-110 kDa, sn-2-arachidonyl-specific cytosolic PLA2. The selective alpha1-adrenergic agonist phenylephrine induced a concentration- and time-dependent increase in glycerophosphoinositol (GroPIns) and glycerophosphoinositol 4-phosphate (GroPIns4P) in rat right ventricle slices prelabelled with D-myo-[3H]inositol. In electrically driven strips of rat right ventricles, prelabelled with D-myo-[3H]inositol, the positive inotropic effect induced by 20 microM NA in the presence of propranolol was accompanied by the formation of GroPIns and GroPIns4P. The concentration of the formed GroPIns4P (1.33+/-0.12 microM, N = 6) was similar to that previously reported to inhibit the Na+/Ca2+ exchanger in cardiac sarcolemmal vesicles (Luciani S, Antolini M, Bova S, Cargnelli G, Cusinato F, Debetto P, Trevisi L and Varotto R, Biochem Biophys Res Commun 206: 674-680, 1995). These findings show that the stimulation of alpha1-adrenoceptors in rat heart is followed by an increase in the formation of GroPIns4P, which may contribute to the positive inotropic effect of alpha1-adrenergic agonists by inhibition of the Na+/Ca2+ exchanger.

Effects of SAS 1310, a new calcium antagonist, on isolated myocardial and smooth muscle preparations.

Some features of the effects of the diltiazem derivative SAS 1310 on in vitro myocardial and smooth muscle preparations were compared to those of the effects of diltiazem. Left atria, aortic strips and taenia coli of guinea-pigs were used. SAS 1310 induced a negative inotropic response of the left atria driven at 1 Hz similar to the response to diltiazem (IC50 values: SAS 1310 1.34 microM, diltiazem 0.8 microM). The inotropic effect of diltiazem (5 microM) was clearly rate-dependent whereas the reduction of left atria contractility induced by SAS 1310 (5 microM) was not modified by changes of the stimulation rate (the range of frequencies used was 0.5-2 Hz, with stepwise changes of 0.5 Hz). Diltiazem (0.1-0.5 microM) was more effective than SAS 1310 (0.1-5 microM) in inhibiting the contractile response to calcium of taenia coli depolarized by high K+ as well as in relaxing the aortic strips contracted by high K+ (IC50 SAS 1310 12.3 microM, diltiazem 0.41 microM). The response of aortic strips to norepinephrine (50 microM) in Ca2+-free medium was inhibited by SAS 1310 (50 microM) and was not affected by diltiazem (2 microM). The drug concentrations used were equiactive in inhibiting the high K+-induced contraction of the aortic strips. The different effects of diltiazem and its derivative on left atria contraction at different force-frequency ratios and on aortic strip contraction induced by norepinephrine in a Ca2+-free medium suggest that the actions of the two drugs differ qualitatively.

The influence of acidosis on the myocardial uptake and electrocardiographic effects of disopyramide.

The time course for the ECG effects and myocardial uptake of disopyramide was studied in isolated perfused guinea pig hearts under different pH conditions. At pH 7.46 the drug depressed the overall AV conduction time (PR) by 16.64%, the His-ventriculum conduction time (HV interval) by 30.46% and delayed the ventricular repolarization (QT interval) by 8.08%, on average. The maximum intraventricular pressure (Pmax) was also depressed by 35.6%. The maximum effect on the QT interval (constant rate: 0.609 min-1) was reached faster than the maximum effect on the PR and HV intervals (constant rates: 0.399 and 0.400 min-1, respectively), while the myocardium uptake process was complete before any ECG parameter reached a steady state (uptake constant: 1.58 min-1). Under conditions of extracellular acidosis (pH 6.92), the disopyramide disposition parameters (uptake rate constant and myocardial concentration) were not modified. However, the drug exerted significantly smaller effects on the HV and QT intervals and on myocardial contractility. These results are in contrast with those obtained previously with lidocaine and quinidine, and indicate that the influence of acidosis on class 1 antiarrhythmic agents may also depend on the characteristics of the individual drug.

Effect of amiloride on inotropic and toxic actions of ouabain in guinea-pig left atria.

The effect of amiloride on the positive inotropic and toxic effects of ouabain in guinea-pig left atria has been studied. In atria driven at 1 Hz, amiloride (0.3 and 0.5 mM) decreased the EC50 but did not affect the maximal tension developed by ouabain. At 0.1 Hz, amiloride did not change either the EC50 or the maximal tension developed by ouabain. Ouabain toxicity (onset of arrhythmias) was not changed by amiloride at either frequency of stimulation. Therefore, amiloride did not antagonize either the positive inotropic or the toxic effect of ouabain. The positive inotropic effect of amiloride has been ascribed to the inhibition of the Na+/Ca2+ exchanger. Since amiloride inhibits also the Na+/H+ exchanger, 5-(N-ethyl-N-isopropyl)amiloride (EIPA), an amiloride derivative which selectively inhibits the Na+/H+ exchange, has been tested to evaluate the role of the Na+/H+ exchange in the amiloride-ouabain interaction. EIPA increased the EC50 values of ouabain and decreased the maximal developed tension by the glycoside in atria driven at 0.1 and 1 Hz, but did not antagonize the toxic response (arrhythmias) of atria to ouabain. It is suggested that the inhibition of Ca2+ exit through the Na+/Ca2+ exchange by amiloride and ouabain may explain the observation that the positive inotropic effects of amiloride and ouabain are additive.

Acquired epileptic frontal syndrome as long-term outcome in two children with CSWS.

Continuous spikes and waves during slow sleep (CSWS) are a well-known EEG pattern that can be associated with cognitive and behavioural deterioration. We present the long-term clinical, neuropsychological and EEG follow-up of two patients who developed CSWS during childhood. In both the CSWS onset was followed immediately by rapid cognitive and behavioural deterioration. Later the CSWS fragmented or fluctuated and the spike-wave discharges diminished and this was associated with progressive clinical improvement. At the same time bilateral frontal EEG abnormalities appeared awake and in sleep. After the initial period of rapid cognitive and linguistic improvement both patients stabilised. The latest neuropsychological assessment showed a frontal syndrome. The presence of frontal EEG abnormalities superimposed on CSWS, their persistence after CSWS resolution and, in addition, the finding of subtle frontal-type neuropsychological alterations early in recovery may indicate poor long-term outcome.

L-type Ca(2+) channel current characteristics are preserved in rat tail artery myocytes after one-day storage.

AIM: To develop a cheap and simple method of storing for 24-h vascular tissue and single myocytes while preserving therein the biophysical and pharmacological characteristics of L-type Ca(2+) channels and contractile activity. METHODS: Rings or vascular smooth muscle cells obtained from the rat tail main artery were used either freshly (R0h and VSMC0h) or stored for 24 h (R24h and VSMC24h) at 4 °C, to record whole-cell L-type Ca(2+) currents (IC a(L) ) or measure contractile responses. RESULTS: R0h/VSMC0h and R24h/VSMC24h comparably contracted when stimulated with phenylephrine, high KCl or ATP. In both VSMC0h and VSMC24h, IC a(L) was identified and characterized as a stable inward current for at least 35 min; IC a(L) was comparably inhibited by the Ca(2+) antagonists nifedipine, verapamil and diltiazem and increased by the Ca(2+) channel agonist (S)-(-)-Bay K 8644; current density and current-voltage relationships were similar; at more hyperpolarized holding potentials, IC a(L) intensity increased comparably; nifedipine shifted the steady-state inactivation curve towards more negative potentials, while verapamil blocked IC a(L) in a frequency-dependent manner and slowed down the rate of recovery from inactivation in a comparable way. CONCLUSION: Findings show that smooth muscle contractile activity and the biophysical and pharmacological features of L-type Ca(2+) channels are similar in VSMC24h and VSMC0h. The fact that reproducible results were obtained in vascular myocytes up to 24 h after dissociation may facilitate vascular smooth muscle cell investigation by increasing throughput and reducing the number of animals required.