Giacomo Bruschi

Intracellular free [Ca2+] in circulating lymphocytes of spontaneously hypertensive rats

In the light of previous reports suggesting a common abnormality of Ca handling in most tissues of hypertensive humans and rats, we applied a novel technique using the fluorescent probe Quin 2 for measurement of cytosolic free Ca2+ in lymphocytes of spontaneously hypertensive rats (SHR). (Ca2+)i is increased in SHR (122.1 +/- 7.4 nM) versus normotensive Wistar-Kyoto (WKY) control rats (81.1 +/- 6.3 nM) Membrane exchange, as challenged by varying the extracellular Ca concentration over a 10(5)-fold range proved to be relatively unimportant in regulating (Ca2+)i and did not significantly affect the difference between SHR and WKY. Catecholamines and ouabain had no appreciable effect on (Ca2+)i. The mechanisms of increased (Ca2+)i in SHR lymphocytes remain to be fully elucidated.

Increased sensitivity to protein kinase C activation in aortas of spontaneously hypertensive rats.

The aortic muscle of spontaneously hypertensive (SHR) and normotensive control (WKY) rats was stimulated with phorbol esters and the contractile response was measured as isometric tension. Phorbol esters are known activators of protein kinase C. The aortas of SHR were characterized by the following distinct alterations in the response to phorbol myristate acetate: (1) increased sensitivity: half-maximal force was achieved at 62 ± 6 nmol/1 phorbol myristate acetate in SHR and 105 ± 8 nmol/l in WKY; (2) increased contractility: the maximal force developed by phorbol myristate acetate was greater in SHR aortas (1.9 ± 0.3 versus 1.6 ± 0.2 g in WKY) compared with the decreased contractility generated with noradrenaline and high levels of potassium; (3) decreased dependency on extracellular calcium for half-maximal tension: in the presence of 3 μmol/l phorbol myristate acetate 50% of maximal force was attained at 21 ± 8 μmol/1 extracellular calcium compared with 49 ± 9 μmol/l in WKY; (4) diminished relaxation in response to excess extracellular calcium: phorbol myristate acetate-precontracted WKY aortas began to relax when calcium was raised above 4 mmol/l in the bath and relaxation reached 51% at 8–10 mmol/l. Relaxation was almost absent in SHR (3–7%). Hence, there is an abnormality in the response to protein kinase C activation by phorbol esters in SHR vascular smooth muscle. Intracellular calcium appears to be involved. Studies of protein kinase C will prove important in understanding vascular smooth muscle function in normal and abnormal states.

Use of Fourier shape descriptors to improve the reproducibility of echographic measurements of arterial intima-media thickness.

Background A major source of error in the longitudinal assessment of the intima–media thickness (IMT) is the difficulty in retrieving the same echographic view of the vessel. Objective To present a method for increasing the reproducibility of IMT measurements by ultrasound in large arteries. Method The Fourier descriptor is a well-known means of describing an objects shape. By means of the discrete Fourier transform (DFT), the shape was represented in a frequency domain; the computational advantages of the DFT then permitted a measure of unlikeness between different shapes (the ‘distance’ measure; DM) to be defined and used as a criterion for reproducing the contour. When the sonographer compared successive images of a complex vascular segment, like the carotid bifurcation, the identity of the echographic cut was deduced from the identity of the vessels contour. The best match of the baseline image was the view that minimized the contour DM. Results Preliminary studies in the carotid artery bifurcations of eight subjects showed that the DM responds to systematic variations in the ultrasound interrogation angle and reveals minimal changes in transducer position. Duplicate scans of 12 subjects were performed by three sonographers with different strategies for acquisition of the same images: a low DM was associated with a low difference in pairs of IMT measurements. Data were classified into two groups (normal or borderline vessels with a pooled mean IMT of 0.62 mm and overtly thickened segments with a pooled mean IMT of 1.31 mm). When minimization of the DM was the criterion for the acquisition of replicate scans, the mean absolute difference of paired data for the mean IMT of the distal common carotid artery was 0.03 ± 0.02 mm for the first group and 0.06 ± 0.03 mm for the second group. This is a significant reduction in comparison with non-quantitative alternative criteria for image reproduction. For the maximum IMT of the same segments the mean absolute differences were 0.07 ± 0.03 and 0.13 ± 0.06 mm in the first and second groups, respectively. Conclusion This method can be applied to the serial assessment of single atherosclerotic segments. The computational time is negligible. By reducing the scatter in sequential IMT data, longitudinal investigations (e.g. of the results of antihypertensive therapy) with shorter durations and smaller sample groups may be rendered feasible.

The mechanism of Ca2+i increase in blood cells of spontaneously hypertensive rats.

Cytoplasmic free calcium (Ca2+i) is increased in platelets and lymphocytes of spontaneously hypertensive rats (SHR) and, to a lesser extent, in essential hypertensive patients. In this study, a method was devised to evaluate cellular Ca fluxes and the cellular Ca buffering power by means of the intracellular Ca indicator Quin-2. Ca influx was measured under conditions of Ca-pump inhibition, either by vanadate or ATP depletion. Lymphocytes of 5-month-old SHR were compared with those of normotensive Wistar-Kyoto rats (WKY). In both strains, the Ca entry rate and the cellular buffering capacity were higher in vanadate-treated than in ATP-depleted cells. SH rats exhibited a higher Ca influx and a greater intracellular Ca buffering power when vanadate was used to stop the Ca pump; these differences, however, were abolished in ATP-depleted lymphocytes. It is suggested that Ca entry in lymphocytes is ATP-dependent. Accelerated Ca entry in SHR can account for the previously reported higher levels of intracellular free Ca.

Adrenoceptor Alterations in Different Tissues in Human and Animal Genetic Hypertension

It is scarcely necessary to stress the importance of adrenoceptor regulation in arterial hypertension. In this disease an altered response to adrenergic agonists has been described in different organs, and particularly in the blood vessels [1–6]. Moreover, antiadrenergic drugs, and particularly alpha and beta blockers, are effective antihypertensive agents.

The vasodilator effect of human atrial natriuretic factor (99–126) on human omental arteries

The effect of synthetic human atrial natriuretic factor [ANF-(99–126)] on human omental arteries was investigated. The compound produced concentration-dependent relaxation, 10, 50 and 90% of maximum effect being observed at about 1, 10 and 100 nmol/l, respectively. These concentrations are considerably higher than those measured by radio-immunoassay in human plasma, even under extreme conditions (0.001–0.3 nmol/l). Therefore, ANF can dilate human resistance-size arteries, but whether it does so under physiological conditions has not been established.

The Relationship between Myoplasmic Calcium and Force Developed during Stimulation of Arterial Smooth Muscle: A Study with Fura 2

Calcium is believed to play a primary role in vascular contraction, but quantitative simultaneous measurements of free intracellular calcium (hereafter: Ca;+) and force have not been reported. Novel intracellular calcium indicators, like Fura 2, provide this opportunity.’ Rat (Wistar-Kyoto, 8-9 weeks old) aortic medial rings were prepared by removal of adventitia and intima. The smooth muscle rings (1.5 mm wide, about 50 Fm thick) were light-microscopically intact and developed about 100% of force developed by intact whole aortic rings. They were mounted between two parallel pins in a 20 ml chamber with quartz windows. The lower pin was immovable, while the upper one was connected to a very rigid (0.02 mm displacement per gram force) tension transducer. The rigidity of the transducer ensured that little geometrical changes took place during stimulation and contraction of smooth muscle samples. The chamber was held in the sample compartment of a Perkin-Elmer MPF44A spectrofluorimeter in the position normally occupied by ordinary cuvettes. The smooth muscle tissue was loaded with 10-20 r M Fura2-AM (resulting in 100-200 p M intracellular free dye, with signals 8-1 0 times higher than basal autofluorescence). Preparations were excited alternately at 340-380 nm by motorizing the shaft of the excitation monochromator. The emitted fluorescence was recorded at 510 nm. The 340/380 ratios were used to calculate Cat+ as previously described,’ using a calibration curve obtained by exposing the samples to 10 FM ionomycin in the presence of 1 nM-1 mM extracellular calcium (Ca:+). The results of this study suggest the following conclusions: (1) the level of myoplasmic calcium is crucial for contraction, but (2) factors that increase the force:CaT+ ratio are equally important. Conclusion (1) is suggested by the fact that under any condition (including norepinephrine (NE) and high K stimulation) the level of force was proportional to CaT+ (FIGS. 1 and 2 ) and that raising Ca:+ “forcefully” with a ionophore (ionomycin) evoked Ca:’-dependent contractions. On the other hand conclusion (2) is supported by the findings that in the initial, phasic portion of high K and NE contraction, one observes a rapid increase in force without any major increase in Cat+; the phasic onset of stimulation is characterized by an increase of the force:CaT+ ratio, which is maintained as long as stimulation is conserved; that a wash (which removes N E and high K) and EGTA addition (in the presence of the above agents) produce similar effects on force (i.e. relaxation), but a t very different Ca;+ levels (FIGS. 1 and 2); and that ionophore-induced contractions require much higher levels of CaT+ than N E and high K to produce the same amount of force. NE-induced contractions in Ca-free media have been attributed to intracellular