HHT: a rare disease with a broad spectrum of clinical aspects.

HHT is an autosomal dominant disease characterised by diffuse muco-cutaneous and visceral telangiectases in potentially all organs. Mutations in two different genes identify HHT type 1 and HHT type 2: endoglin located on chromosome 9q33-q34 and ALK-1 or ACVRL1 on chromosome 12q13, respectively. The existence of a third locus has also been hypothesised. HHT-1 is considered a more severe form of the disease with an earlier onset of epistaxis and telangiectases and a higher prevalence of pulmonary arteriovenous malformations than that found in HHT-2 subjects. Usually, a typical HHT patient has epistaxis, muco-cutaneous telangiectases and GI bleeding in later life, even though this clinical scenario represents only one of the possible HHT patterns. In fact, vascular malformations often remain silent until the onset of a severe complication, which frequently is the first clinical manifestation of HHT. The lung and brain are of particular concern because each may contain clinically silent lesions that can result in sudden morbidity and mortality. At present, awaiting the availability of genetic testing, only an expert in the clinical patterns and diagnostic imaging of HHT can permit a definite diagnosis in individuals at high risk for the disease.

Emergencies in hereditary haemorrhagic telangiectasia.

BACKGROUND: Hereditary haemorrhagic telangiectasia (HHT) is a systemic autosomal dominant vascular disease. Although the clinical picture is that of a chronic disabling disease, vascular malformations can suddenly lead to life-threatening conditions. AIM: To assess the frequency and type of emergency acute complications in HHT. DESIGN: Retrospective case-note review. METHODS: From August 2000 to December 2004, our specialized HHT centre saw 139 patients (74 males, 65 females, mean age 45.5 years, range 14-77) with a definite diagnosis of HHT. We reviewed their clinical files and recorded all visits for acute complications (massive nosebleeds, haematemesis, melaena, haematochezia, haemothorax, haemoptysis, TIA/ischaemic stroke, haemorrhagic stroke, brain abscess). RESULTS: Fifty patients (35.9%) had at least one acute complication. There were a total of 93 visits potentially involving the emergency department. Most commonly, patients sought urgent medical attention for nosebleeds and gastrointestinal bleeding (63.4%), but there were also disorders of the brain, lung, heart and liver. DISCUSSION: Acute complications of HHT are not uncommon and can be severe and wide-ranging. Physicians should be aware of HHT and its major complications, as a prompt diagnosis is essential to direct patients to the most appropriate therapies, and to suggest screening for visceral involvement in their relatives.

Changes in mitochondrial calcium concentration during the cardiac contraction cycle.

OBJECTIVE: The aim was to examine whether mitochondrial Ca2+ fluxes are high enough to change mitochondrial and cytosolic calcium concentration during the contraction cycle. METHODS: Isolated guinea pig ventricular myocytes were stimulated with paired voltage clamp pulses until contractions were maximal (2 mM [Ca2+]o, 36 degrees C). At defined times of diastole or systole, the cells were shock frozen. Electron-probe microanalysis measured the concentration of total calcium in mitochondria (sigma Ca(mito)) and surrounding cytosol (sigma Cac). Other experiments were performed to evaluate DNP sensitive mitochondrial Ca2+ uptake from depolarisation induced [Ca2+]c transients (K5indo-1 fluorescence). RESULTS: At end of diastole, sigma Ca(mito) was 446 mumol.litre-1. During systole, sigma Ca(mito) increased with a 20 ms delay. A peak sigma Ca(mito) of 1050 mumol.litre-1 was measured 40 ms after start of systole, while 95 ms after start of systole sigma Ca(mito) had fallen to 530 mumol.litre-1. From the changes in sigma Ca(mito) the rates of net mitochondrial Ca2+ flux were estimated at 100 nmol.s-1 x mg-1 protein for Ca2+ influx and 36 nmol.s-1 x mg-1 protein for Ca2+ egress. Decay of sigma Ca(mito) was coupled to a rise in sigma Na(mito). sigma Cl(mito) and sigma K(mito) rose and fell in parallel with sigma Ca(mito), suggesting Ca2+ activation of mitochondrial anion and cation channels. Activation of the non-specific permeability can be excluded. Block of mitochondrial Ca2+ uptake with DNP (100 microM) or FCCP (10 microM) increased the amplitude of the [Ca2+]c transients for 1-3 min by about 50%; evaluation of mitochondrial Ca2+ uptake from DNP sensitive difference signals, however, was hampered by sequestration of mitochondrial Ca2+ into the sarcoplasmic reticulum. CONCLUSIONS: Mitochondrial calcium content changes during each individual contraction cycle; a substantial amount of calcium is taken up during the systole and released during later systole and diastole.

Regulatory effects mediated by OKT8+ subsets on B cell response in the elderly.

The B cell responsiveness mediated by T cells has been evaluated in 50 aged donors by using a Pokeweed mitogen (PWM)-driven differentiation system. Peripheral blood mononuclear cells (PBMC) from aged donors exhibited a lower plaque-forming cell (PFC) generation in comparison with young donors. Similar results were obtained by using, on a per cell basis, purified T cells supplemented with Non-T lymphocytes. OKT4+ cell addition to Non-T cells enhanced PFC number, even if values were still lower when compared to young donors. Moreover, results similar to those seen with T-supplemented Non-T cell cultures were obtained by mixing Non-T cells with OKT4+ and OKT8+ lymphocytes. Interestingly, when OKT8+ cells were divided into OKT8+M1+ and OKT8+M1-subsets and used in the assay, both cell fractions suppressed B cell response in aged donors, while this effect was mediated by OKT8+M1+ cells only in the young counterpart. Soluble suppressive factors were responsible for the observed activity, since indomethacin supplementation to cultures or overnight incubation of cells before PWM stimulation abrogated the OKT8+M1--mediated suppression. Taken together, these findings suggest an additional alteration of T-B cell relationship in the elderly.

Impairment of T immunoregulatory activities in the induction of antibody specific response in aged humans.

T helper (Th) and T suppressor (Ts) functions on the induction of specific antibody response have been studied in 80 aged individuals by means of a plaque-forming cell assay. Of the subjects 45.2% exhibited a reduction of Ts activity on Ig production by adding Concanavalin A (Con A) to cultures on day 0, while 35.7% of aged donors showed a decrease of Th functions by supplementation of Con A on day 2. A small number of individuals displayed a combined deficit (Th + Ts). Furthermore, these defects seem to be related to soluble suppressive factors which might adhere to cell surface. In fact, preincubation of peripheral blood mononuclear cells (PBMC) before their addition to cultures and resuspension in fresh medium normalized the immunoregulatory defects. On the other hand, overnight supernatants from old PBMC transferred to young PBMC cultures induced the same deficit observed in the aged cell suspensions. Finally, Zinc chloride supplementation to cultures was able to correct the deficient Th activity only. These data suggest an additional defect of immunoregulation in the elderly.

Demonstration of mercury in the human brain and other organs 17 years after metallic mercury exposure.

A male subject became exposed to metallic mercury vapor at work in 1973. He excreted 1,850 mg Hg/l urine initially. Controls of urine mercury excretion after D-penicillamine administration led to the assumption of a total body clearance of mercury latest since 1976. Subsequently he developed an organic psychosyndrome without detectable signs of classical mercurialism. He never returned to work again and died of lung cancer in 1990. In different organs (brain, kidney, and lung) which were sampled at autopsy elevated levels of mercury were documented by atomic absorption analysis. Histological examination of the tissue by the Danscher and Schroder method, which is specific for mercury, showed a highly positive staining in the majority of nerve cells and cells of other organs. Ultrastructurally mercury could be demonstrated by elemental x-ray analysis within lipofuscin deposits. The lipofuscin content was increased in the mercury positive nerve cells as demonstrated by a strong positive autofluorescence.

Potentiation of contraction as related to changes in free and total intracellular calcium.

In voltage-clamped guinea-pig ventricular myocytes, we studied the potentiation of contraction in dependence on the concentration of intracellular calcium; ionized calcium [Ca2+]c was measured by Indo-1 microfluospectroscopy and total calcium (sigma Ca) by electronprobe microanalysis (EPMA). After a 15 min rest period, [Ca2+]c was approx. 90 nM and sigma Ca was below the detection limit (80 microM) in myoplasm (sigma Ca(myo)), junctional sarcoplasmic reticulum (sigma CaSR) and mitochondria (sigma Ca(Mito)). Post rest, repetitive clamp steps (1 Hz) potentiated extent and rate of shortening by 300%. In the literature, post-rest potentiation is attributed to the replenishment of SR with releasable calcium; by EPMA the postulated increase in sigma CaSR was measured directly. Post-rest, the peaks of systolic [Ca2+]c transients increased, however only by 40%. In addition, a moderate increase of end-diastolic [Ca2+]c was measured. In an other series of experiments, contraction was potentiated by 800% increase by means of paired voltage-clamp pulses (1 Hz, 36 degrees C, 2 mM [Ca2+]o). In the potentiated state, end-diastolic [Ca2+]c was 180 nM and sigma Ca(myo) was 0.65 mM. During systole, [Ca2+]c peaked within 20 ms to 950 nM. sigma Ca(myo) rose within 20 ms to 1.4 mM and fell within 40 ms to 1.1 and within 90 ms to 0.8 mM. In contrast, the time course of contraction was slow and peaked at a time (130 ms) when the [Ca2+]c and sigma Ca(myo) transients were finished. We suggest that Ca2+ bound to troponin C (TnC) controls only the onset but not the time course of myofilament interaction. From [Ca2+]c and sigma Ca(myo) we estimated a Ca2+ buffering capacitance of 1.5 mmol sigma Ca(myo) per pCa change, only a fraction of which can be attributed to Ca2+ binding sites on TnC. A model explaining the results requires the assumption of 0.6 mM additional slow, high affinity Ca2+ sites and 2 mM fast, low affinity Ca2+ sites. We discuss that end-diastolic Ca2+ binding to these sites contributes to the potentiation of contraction. Junctional SR. At the end of diastole sigma CaSR was 2.4 mM which is 4 times larger than sigma Ca(myo). This difference disappeared 20 ms after depolarization (sigma CaSR 1.1 mM), within another 20 ms it largely recovered (sigma CaSR 2.0 mM). These properties suggest that the junctional SR is a compartment suitable not only for Ca2+ release but also for rapid Ca2+ reuptake. Mitochondria. Paired-pulse potentiation increased end-diastolic sigma Ca(Mito) significantly (0.4 mM).(ABSTRACT TRUNCATED AT 400 WORDS)

Hereditary hemorrhagic teleangiectasia (Rendu-Osler-Weber disease).

Rendu-Osler-Weber disease, or hereditary hemorrhagic telangiectasia (HHT), is an autosomal dominant disorder with incomplete penetrance, characterized by vascular anomalies which may virtually develop in many organs. The prevalence varies and may range from 1/3,500 to 1/5,000 in specific regions. Two chromosal sites have been at least identified: in HHT1, mutations at chromosome 9 alter the protein endoglin and in HHT2, mutations at chromosome 12 alter the protein activine or ALK-1. Clinical manifestations include recurrent epistaxis, mucocutaneous telangiectases that bleed easily, and larger arteriovenous malformations in parenchymatous organs. Epistaxis is the first symptom, occurring in the vast majority of affected persons. The lung is the most common site for arteriovenous malformations. Brain abscess, transient ischemic attack and ischemic stroke occur exclusively in patients with pulmonary arteriovenous malformations and right-to-left shunt, which facilitates the passage of emboli into the cerebral circulation. Transcatheter embolotherapy with detachable balloons or stainless-steel coils has been used in order to occlude such malformations and to prevent such complications. At present a genetic diagnosis is possible in only a few families. The clinical diagnosis is based on 4 criteria: family history, epistaxis, mucocutaneous telangiectases and arteriovenous malformations. The diagnosis will be definite if 3 criteria are present, suspected if 2 criteria are present, unlikely if fewer than 2 criteria are present. In conclusion, the authors examine clinical features of 28 HHT patients observed in the HHT University Centre of Bari from September 2000 to May 2001.

Stretch induced accumulation of total Ca and Na in cytosol and nucleus: a comparison between cardiac trabeculae and isolated myocytes.

By means of electron probe microanalysis (EPMA), we quantified changes in total sodium [Na] and calcium [Ca] concentration owing to the following: (i) local axial stretch (LAS) of isolated rat myocytes and (ii) end-to-end stretch (ETES) of rat ventricular trabeculae. For LAS, the distance between patch pipette and a cell-attached stylus was increased by maximally 20%; this activated a nonselective cationic current I(SAC) of approximately -0.5 nA, which was blocked by streptomycin. Trabeculae were stretched end-to-end from 85% L(max) to L(max). Stretch increased cytosolic [Na](total) by 34% in isolated myocytes (p < 0.001) and by 43% in trabeculae (p < 0.001). The increment in nuclear [Na](total) was 21% in myocytes (p < 0.01) and 20% in trabeculae (p < 0.001). Stretch increased [Ca](total) in isolated myocytes, in both cytosol (from 0.63 +/- 0.09 to 1.09 +/- 0.20 mmol/L, p < 0.05) and nucleus (from 0.33 +/- 0.05 to 0.64 +/- 0.13 mmol/L, p < 0.05). In trabeculae, the stretch-induced increment of 51% in cytosolic [Ca](total) remained nonsignificant (p < 0.15). In the nucleus, [Ca](total) did not change. We interpret the difference of stretch on nuclear calcium in myocytes vs. trabeculae with the assumption that LAS, but not ETES, produces shear-stress components that translate the mechanical stimulus deeply into the cell where it may modulate [Ca](total) by signals independent of I(SAC).

Twitch-potentiation increases calcium in peripheral more than in central mitochondria of guinea-pig ventricular myocytes.

1. The mitochondrial total calcium content ([Ca]mt) was studied with electron probe microanalysis (EPMA) in isolated guinea-pig ventricular myocytes in order to answer the question of whether electrical stimulation increases [Ca]mt in subsarcolemmal and central mitochondria to a different extent. 2. In unstimulated myocytes subsarcolemmal [Ca]mt was (mean +/- s.e.m.) 535 +/- 229 micromol (kg dry weight (DW))-1 and central [Ca]mt was 513 +/- 162 micromol (kg DW)-1. These values do not differ and correspond to approximately 180 micromol calcium per litre of mitochondria or 180 microM. 3. Contractions were potentiated to an optimum by stimulation with trains of 12 paired stimuli. After potentiation with 12 paired action potentials, cells were shock-frozen 120 ms after the start of the first action potential of the 13th pair. Subsarcolemmal [Ca]mt was 1.3 +/- 0.4 mmol (kg DW)-1 (433 microM) and central [Ca]mt was 227 +/- 104 micromol (kg DW)-1 (76 microM). The difference was significant. 4. After potentiation with 12 paired voltage-clamp pulses, cells were shock-frozen 120 ms after the start of the first pulse of the 13th pair. Subsarcolemmal [Ca]mt was 2.2 +/- 1.0 mmol (kg DW)-1 (733 microM) and central [Ca]mt was 630 +/- 180 micromol (kg DW)-1 (210 microM). After removal of extracellular K+, five paired voltage-clamp pulses increased subsarcolemmal [Ca]mt to 2.1 +/- 0.8 mmol (kg DW)-1 (700 microM), which was significantly higher than the central [Ca]mt of 389 +/- 88 micromol (kg DW) -1 or 130 microM. 5. In unstimulated cells, [Na] and [K] in subsarcolemmal and central mitochondria were not different. In potentiated myocytes, subsarcolemmal [Na]mt was 236 +/- 20 mmol (kg DW)-1 or 79 mM, which is significantly higher than the central [Na]mt of 50 +/- 5 mmol (kg DW)-1 or 16 mM. 6. The differences in [Ca]mt and [Na]mt are attributed to subsarcolemmal cytosolic microdomains of elevated [Ca2+] and [Na+] generated during contractile potentiation by transmembrane Ca2+ and Na+ fluxes.

Functional properties of Salmonella minnesota Rb-bound and Rb-unbound cell fractions in elderly donors.

The capacity of Salmonella minnesota R345 (Rb) binding to human peripheral blood lymphocytes allows the recovery of Rb-bound and Rb-unbound cell populations that elicit different functions. Here, we have applied this method to lymphocytes from aged individuals to evaluate the possibility that such an approach could reverse the senescence-related impaired immune responsiveness. In this regard we show that Rb binding augments either spontaneous or T-dependent plaque-forming cell generation in Rb-unbound fraction. By contrast, Rb-bound cells are enriched for lymphocytes releasing several lymphokines. This experimental approach seems to represent a useful tool to elucidate better the age-related alterations of the immune function.

Inhibition by antibiotics of Rb Salmonella binding to human peripheral blood lymphocytes.

Several lines of evidence point out that Salmonella minnesota R345 (Rb) possesses the capacity to adhere spontaneously to human peripheral blood lymphocytes. The binding is mediated via the lipopolysaccharide moiety of the bacterial outer membrane. In this report, we have evaluated the effects of various antibiotics on bacterial binding. Our data show that trimethoprim/sulphamethoxazole, chloramphenicol and erythromycin significantly decrease Rb binding, while gentamicin and sisomicin are without effect. Antibiotics display their inhibitory effect by acting on peripheral blood lymphocytes likely by competing with lipopolysaccharide for receptor binding capacity on lymphocyte surface.

Presystolic calcium-loading of the sarcoplasmic reticulum influences time to peak force of contraction. X-ray microanalysis on rapidly frozen guinea-pig ventricular muscle preparations.

Guinea-pig ventricular small papillary muscles and trabeculae were rapidly frozen presystolically after prolonged rest following positive inotropic interventions which strongly influenced peak of force and time to peak force. The possible sources of activator calcium for the different types of contraction were investigated. After rest in the presence of noradrenaline (10(-5)mol/l) the first post-rest contraction showed a retarded activation and a "late" peak of force. Muscle strips frozen after a rest period of 5 min in a bath solution containing noradrenaline were cryosectioned and analyzed with X-ray microanalysis for elemental distribution: although at this time an applied stimulus would induce a potentiated contraction, intracellular membrane systems such as sarcoplasmic reticulum and mitochondria failed to reveal any accumulation of calcium. After rest in a low sodium Tyrode the first post-rest contraction showed an "early" peak of force. Muscles frozen after rest in a low sodium solution revealed intracellular Ca accumulation on the sarcoplasmic reticulum, in the network at the level of the Z-lines. The results support the hypothesis that 1. the sarcoplasmic reticulum (SR) accumulates calcium presystolically when "early" contractions follow stimulation; 2. the network of sarcoplasmic reticulum at the level of the Z-lines is a crucial source of activator calcium; 3. the activator calcium for late contractions is probably of extracellular origin.

Ca-pools involved in the regulation of cardiac contraction under positive inotropy. X-ray microanalysis on rapidly-frozen ventricular muscles of guinea-pig.

Electron probe microanalysis of rapidly-frozen small ventricular trabeculae of guinea-pig demonstrates that the distribution of total intracellular calcium varies under positive inotropy depending on the type of inotropic intervention. The sarcoplasmic reticulum (SR) (or part of it) localized at the level of the z-lines reveals high calcium accumulation at the end of diastole whenever a stimulus is followed by a contraction with a short time to peak of force. After paired pulse stimulation, only this cell compartment accumulates calcium at the end of diastole. Since this cell compartment is "Ca-empty" in muscles frozen during contraction, SR is considered to be the source of activator Ca. In several cases of inotropy (after application of ARL, caffeine or after lowering the extracellular Na+ concentration), calcium is also detectable on the mitochondria, suggesting that these organelles participate in slow regulation of cytosolic calcium. In some cases, total calcium located on the sarcomeres is increased. The interpretation of this finding is intriguing and requires the assumption of supplementary cytosolic Ca-sinks as yet unknown.

Binding of calcium to myoplasmic buffers contributes to the frequency-dependent inotropy in heart ventricular cells.

In guinea-pig ventricular cells, the Ca2+ buffer capacity of the myoplasm was estimated from the ratio of ionized calcium (from Indo-1 fluorescence) through total calcium (ionized plus bound calcium, from x-ray microprobe analysis). During post-rest potentiation (1 Hz paired-pulses in voltage-clamp), where diastolic sarcomere length remained nearly constant, Ca2+ buffer capacity slowly fell from 5500:1 to 700:1 suggesting that slow Ca2+ binding sites became saturated. We discuss that frequency-inotropy depends not only on the replenishment of intracellular stores with Ca2+, but also on binding of Ca2+ to these slow sites; the slow Ca2+ sites could complete with the fast activator sites on troponin C for systolic Ca2+, or they could enhance the Ca2+ affinity of the fast Ca2+ sites on troponin C by cooperative interaction.

X-ray microanalysis of single cardiac myocytes frozen under voltage-clamp conditions.

By means of a patch pipette, an isolated ventricular myocyte was transferred into the taper of a silver holder covered by pioloform film. Once the cell was on the film, the cell was voltage clamped (pulses from -45 to +5 mV at 0.5 Hz). The amount of Ca entry was estimated from the Ca current. When contractility (cell shortening) was potentiated with either five pulses of 0.2 s or four pulses of 1 s, shock freezing was timed 116 or 816 ms after start of the clamp pulse. Electron micrographs from freeze-substituted cells revealed the good preservation of the intracellular compartments. The myocytes were cut at -150 degrees C, and the cryosections were freeze dried. In representative examples, the amount of Ca entry is compared with the subcellular Ca distribution as it is analyzed with energy dispersive X-ray microprobe analysis in cytoplasm, junctional sarcoplasmic reticulum (SR), mitochondria, and the subsarcolemmal space (sarcolemma, peripheral SR, fringe of cytosol).

Total and free myoplasmic calcium during a contraction cycle: x-ray microanalysis in guinea-pig ventricular myocytes.

1. At 36 degrees C and 2 mM [Ca2+]o single guinea-pig ventricular myocytes were voltage clamped with patch electrodes. With a paired-pulse protocol applied at 1 Hz, a first pulse to +5 mV was followed by a second pulse to +50 mV. When paired pulsing had potentiated the contraction to the maximum, the cells were shock-frozen for electron-probe microanalysis (EPMA). Shock-freezing was timed at the end of diastole (-80 mV) or at different times during systole (+5 mV). 2. The same paired-pulse protocol was applied to another group of myocytes from which contraction and [Ca2+]i was estimated by microfluospectroscopy (50 microM-Na5-Indo-1). Potentiation moderately reduced diastolic sarcomere length from 1.85 to 1.82 microns and increased diastolic [Ca2+]i from about 95 to 180 nM. In potentiated cells, during the first pulse, contraction peaked within 128 +/- 25 ms after start of depolarization. [Ca2+]i peaked within 25 ms to 890 +/- 220 nM (mean +/- S.E.M.) and fell within 100 ms to about 450 nM. 3. Sigma Camyo, the total calcium concentration in the overlapping myofilaments (A-band), was measured by EPMA in seventeen potentiated myocytes. During diastole, sigma Camyo was 2.6 +/- 0.4 mmol (kg dry weight (DW]-1 which can be converted to 0.65 mM (mmoles per litre myofibrillar space). Since [Ca2+]i was 180 nM, we estimate that 99.97% of total calcium is bound. 4. A time course for systolic sigma Camyo was determined by shock-freezing thirteen cells at different times after start of depolarization to +5 mV. Sigma Camyo was 5.5 +/- 0.3 mmol (kg DW)-1 (1.4 mM) after 15-25 ms, 4.6 +/- 0.5 mmol (kg DW)-1 (1.1 mM) after 30-45 ms, and 3.1 mmol (kg DW)-1 (0.8 mM) after 60-120 ms. The fast time course of sigma Camyo suggests that calcium binds to and unbinds from troponin C at a fast rate. Hence, it is the slow kinetics of the cross-bridges that determines the 130 ms time-to-peak shortening. 5. Mitochondria of potentiated cells contained during diastole a total calcium concentration, sigma Camito, of 1.3 +/- 0.2 mmol (kg DW)-1 (0.4 mM). During the initial 15-25 ms of systole, sigma Camito did not change, however, during 30-45 ms sigma Camito rose to 3.7 +/- 0.5 mmol (kg DW)-1 (1.2 mM). The data suggest that sigma Camito can follow sigma Camyo with some delay, thereby participating in both slow diastolic and fast systolic changes in total calcium (sigma Ca), at least under the given conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of non-toxic doses of ouabain on sodium, potassium, calcium distribution in guinea pig papillary muscle. Electronprobe microanalysis.

The mechanisms and the cellular structures which are definitely involved in the accumulation and release of calcium in heart muscle treated with cardiac glycosides are not yet known. The distribution of sodium, potassium and calcium in small papillary muscles of the guinea pig right ventricle was examined with the aid of energy dispersive x-ray microanalysis and cryotechniques. The primary aim of the present study was twofold: firstly, to determine whether an increase in intracellular sodium concentration is detectable in muscles showing positive inotropy resulting from treatment with non-toxic doses of ouabain; and secondly, whether at the end of diastole cellular stores are detectable accumulating Ca which could be responsible for the pronounced contraction which normally would follow. Analyses on interstitium, cell membrane, sarcomeres, Z-lines and mitochondria of 7 muscles strips treated with non-toxic doses of ouabain and frozen at the end of diastole showed the following: sodium concentration in the sarcoplasm was significantly higher than over the mitochondria; it was also higher than over the sarcoplasm of non-treated muscles frozen at the end of diastole. High calcium concentrations were also measured over the cell membrane. These calcium concentrations were higher than that detected in sarcomeres, Z-lines and mitochondria. Over the sarcomeres, the calcium concentration was higher than in experiments on non-treated muscles which were also frozen at the end of diastole. Mitochondria did not accumulate any detectable concentration of calcium.(ABSTRACT TRUNCATED AT 250 WORDS)

Extra- and intracellular lanthanum: modified calcium distribution, inward currents and contractility in guinea pig ventricular preparations.

In guinea pig ventricular strips and isolated cells, 0.1 mM LaCl3 blocks contractility and shortens the action potential (AP) in less than 2 min ("early La-effect"). After 30 min, it prolongs the APs which trigger slow contractions ("late La-effect"). These results confirm earlier reports. X-ray microprobe analysis shows that La initially displaces only a small fraction of that Ca which is superficially bound to the sarcolemma. But, since this Ca is completely removed by Ca-free solutions within 2 min, we suggest that La blocks contractility not by displacing superficial Ca but by blocking the Ca inward current iCa. Blocking of iCa is analyzed with voltage clamp experiments. It is not La-specific, and can also be observed with other calcium channel blockers as well. When iCa has been blocked, the membrane can still generate 100-200 ms long plateaus via the sodium inward current iNa. During the late La-effect, the cells internalize La. Intracellular La is detected by x-ray microprobe analysis in cryosections of frozen muscles and as La-precipitates in EM images from freeze substituted preparations. Simultaneously, the cytosol gains Na and Ca, but the plasmalemmal and sarcoplasmic reticulum (SR) membranes are no longer occupied by Ca but by La. The late La-effect on the prolongation of the AP is La-specific. In the absence of extracellular La, it can be induced by pressure injection of La into the cytosol. The long APs are based on an additional La, it can be induced by pressure injection of La into the cytosol. The long APs are based on an additional inward current which is insensitive to Ca-removal, is inactivated by holding potentials of -40 mV, and is TTX-sensitive. We suggest that the current flows through a fraction of original Na-channels that is modified by i.c. La with respect to inactivation and selectivity. We attribute the late re-occurrence of contractility to activator Ca entering from the bath. Ca-entry might be mediated via enhanced Na/Ca-exchange whose rate is increased by the i.c. Na-load. In addition, Ca may enter through the La-modified Na-channels due to their impaired selectivity. Since i.c. La is known to interfere with the Ca-sequestration by the SR, it is expected to impair relaxation.