Partial contribution of the ATP-sensitive K+ current to the effects of mild metabolic depression in rabbit myocardium

The object of the study was to compare the capability of glibenclamide to block the effects of K⁺-ATP channel activators on action potential duration and steady state whole cell current to its efficiency in counteracting the effects of hypoxia or metabolic poisons in the presence of glycolytic substrate. The modulation of action potential duration by 30 M glibenclamide was tested in perfused hearts subjected to hypoxia or to the K⁺-ATP channel opener pinacidil. Similar protocols were used to study the modifications of the steady state whole cell current in isolated ventricular myocytes. It was found that glibenclamide did not prevent early action potential shortening induced by hypoxia but produced a partial recovery after 15 min of exposure. At the steady state the action potential duration had lengthened by 53±6% at plateau level and 42±3% at 95% repolarization. In contrast, action potential shortening induced by 100 M pinacidil was fully reversed by glibenclamide within 2 min. Freshly dispersed ventricular myocytes were characterized in control conditions as for the properties of the steady state current. This current, measured at the end of 450 ms long pulses showed typical inward rectification that was abolished by 50 M Ba²⁺. Cyanide (2 mM), carbonyl-cyanide m-chlorophenylhydrazone (CCCP, 200 nM) and BRL 38227 (30 M) produced characteristic increases in time independent outward currents. Glibenclamide abolished the outward current induced by BRL 38227 and the concomitant action potential shortening. Addition of cyanide in the presence of glibenclamide and BRL 38227 produced a new increase in outward current accompanied by action potential shortening. In the absence of K⁺-ATP channel activators, glibenclamide partly inhibited the CCCP induced current. Our data suggested that the delayed onset of glibenclamide action in hypoxic hearts is not due to diffusion barriers. They rather support the view that mechanisms other than K⁺-ATP channel activation could determine the early action potential shortening in whole hearts. The partial recovery observed under glibenclamide may be due, in part, to channel desensitization but also reflect the contribution of more than one current system to the action potential shortening because the glibenclamide insensitive fraction of the CCCP induced current is partly blocked by low concentrations of Ba²⁺. Differences with other data in the literature are attributed to the degree to metabolic blockade, to species differences, and to the inherent heterogeneities of the whole heart model where non-muscle cells may modulate the response to hypoxia.     

A Study on the Electrical Resistance of the Frog Sartorius Muscle

Four different methods of measuring the resistance of a muscle fiber have been applied to the frog sartorius muscle. The methods, in which the resistance of the microelectrode entered the calculation of the effective resistance of the fiber, resulted in values which were 8 times higher than the resistance values obtained with methods independent of the electrode resistance. A simple cable model of a muscle fiber could not account for the discrepancy in the effective resistance found in these measurements; therefore, an enlarged cable model for a muscle fiber has been proposed, and its biological implications have been discussed. The effective resistance (measured with the two different groups of methods) decreased when the potassium concentration in the bath increased. Using the proposed enlarged cable model for the interpretation of these results, it is shown that not only the membrane resistance but also the myoplasmic resistance decreases with an increasing potassium concentration in the Ringer solution.     

Magnesium: Effects on reperfusion arrhythmias and membrane potential in isolated rat hearts

Myocardial Na+,K+-ATPase was studied in patients with aortic valve disease, and myocardial Na+,K+- and Ca2+-ATPase were assessed in spontaneously hypertensive rats (SHR) and hereditary cardiomyopathic hamsters using methods ensuring high enzyme recovery. Na+,K+-ATPase was quantified by [3H]ouabain binding to intact myocardial biopsies from patients with aortic valve disease. Aortic stenosis, regurgitation and a combination hereof were compared with normal human heart and were associated with reductions of left ventricular [3H]ouabain binding site concentration (pmol/g wet weight) of 56, 46 and 60%, respectively (p < 0.01). Na+,K+ and Ca2+-ATPases were quantified by K+- and Ca2+-dependent p-nitrophenyl phosphatase (pNPPase) activity determinations in crude myocardial homogenates from SHR and hereditary cardiomyopathic hamsters. When SHR were compared to age-matched Wistar Kyoto (WKY) rats an increase in heart-body weight ratio of 75% (p < 0.001) was associated with reductions of K+- and Ca2+-dependent pNPPase activities (mol/min/g wet weight) of 42 (p < 0.01) and 27% (p < 0.05), respectively. When hereditary cardiomyopathic hamsters were compared to age-matched Syrian hamsters an increase in heart-body weight ratio of 69% (p < 0.001) was found to be associated with reductions in K+- and Ca2+-dependent pNPPase activities of 50 (p < 0.001) and 26% (p = 0.05), respectively. The reductions in Na+,K+- and Ca2+-ATPases were selective in relation to overall protein content and were not merely the outcome of increased myocardial mass relative to Na+,K+- and Ca2+-pumps. In conclusion, myocardial hypertrophy is in patients associated with reduced Na+,K+-ATPase concentration and in rodents with reduced Na+,K+- and Ca2+-ATPase concentrations. This may be of importance for development of heart f in hypertrophic heart disease.     

Liver plasma membrane calcium transport. Evidence for a Na+-dependent Ca2+ flux

Plasma membrane vesicles isolated from rat liver exhibited an azide-insensitive Mg2+-ATP-dependent Ca2+ pump which accumulated Ca2+ at a rate of 5.1 +/- 0.5 nmol of calcium/mg of protein/min and reached a total accumulation of 33.2 +/- 2.6 nmol of calcium/mg of protein in 20 microM Ca2+ at 37 degrees C. Equiosmotic addition of 50 mM Na+ resulted in a loss of accumulated calcium. Measurement of Mg2+-ATP-dependent Ca2+ uptake in the presence of 50 mM Na+ revealed no effect of Na+ on the initial rate of Ca2+ uptake, but a decrease in the total accumulation. The half-maximal effect of Na+ on Ca2+ accumulation was achieved at 14 mM. The Ca2+ efflux rate constant in the absence of Na+ was 0.16 +/- 0.01 min-1, whereas the efflux rate constant in the presence of 50 mM Na+ was 0.25 +/- 0.02 min-1. Liver homogenate sedimentation fractions from 1,500 to 105,000 X g were assayed for azide-insensitive Mg2+-ATP-dependent Ca2+ accumulation. Na+-sensitive Ca2+ uptake activity was found to specifically co-sediment with the plasma membrane-associated enzymes, 5'-nucleotidase and Na+/K+-ATPase, whereas Na+-insensitive Ca2+ uptake was found to co-sediment with the endoplasmic reticulum-associated enzyme, glucose-6-phosphatase. The plasma membrane Ca2+ pump was also distinguished from the endoplasmic reticulum Ca2+ pump by its sensitivity to inhibition by vanadate. Half-maximal inhibition of plasma membrane Ca2+ uptake occurred at 0.8 microM VO4(3-), whereas half-maximal inhibition of microsomal Ca2+ uptake occurred at 40 microM.     

Voltage control during inward current flow in rat ventricular muscle using a double sucrose gap technique.

In rat ventricular muscle, measurements of the membrane potential with microelectrodes during depolarizing voltage steps showed that deviation of the membrane potential from the command signal were never larger than 15 mV during flow of the fast inward current and that voltage control was regained within 15 ms after the beginning of the voltage step. During the flow of the slow inward current, tail currents elicited by interrupting the time course of the slow current at different time intervals returned exponentially to the steady-state level, thus indicating acceptable voltage control. It is concluded that rat ventricular muscle is a rather favorable preparation for voltage-clapm experiments and this is attributed mainly to the geometry of the preparation.     

Lead inhibits 1,25-dihydroxyvitamin D-3 regulation of calcium metabolism in osteoblastic osteosarcoma cells (ROS 17/2.8).

We have determined the dose-response of 1,25-dihydroxyvitamin D-3 (1,25-(OH)2D3) on the intracellular free calcium-ion concentration ([Ca2+]i) in the osteoblastic osteosarcoma cells, ROS 17/2.8, using 19F-NMR and the intracellular divalent cation indicator, 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (5F-BAPTA). The dose-response demonstrated an inverted U-shaped relationship with maximal elevation of [Ca2+]i at doses of 1 to 10 nM 1,25-(OH)2D3. At 10 nM, 1,25-(OH)2D3 elevated the [Ca2+]i from a control level of 118 +/- 4 nM to a peak value of 237 +/- 8 nM within 40 min. 1,25-(OH)2D3 also increased the initial rate of Ca2+ influx into ROS 17/2.8 cells, measured by 45Ca uptake, with a dose-response relationship which paralleled its effect on [Ca2+]i. Treatment of ROS 17/2.8 cells with Pb2+ at 1 and 5 microM significantly increased [Ca2+]i but significantly reduced the 1,25-(OH)2D3-induced elevation of [Ca2+]i. Simultaneous treatment of naive cells with 1,25-(OH)2D3 and Pb2+ produce little reduction of 1,25-(OH)2D3-induced 45Ca uptake while 40 min treatment with Pb2+ before addition of 1,25-(OH)2D3 significantly reduced the 1,25-(OH)2D3-induced increase in 45Ca influx. These findings suggest that Pb2+ acts by inhibiting 1,25-(OH)2D3-activation of Ca2+ channels and interferes with 1,25-(OH)2D3 regulation of Ca2+ metabolism in osteoblastic bone cells.     

Oxidative stress-induced calcium signalling in Aspergillus nidulans

The effects of oxidative stress on levels of calcium ion (Ca(2+)) in Aspergillus nidulans were measured using strains expressing aequorin in the cytoplasm (Aeq(cyt)) and mitochondria (Aeq(mt)). When oxidative stress was induced by exposure to 10-mM H(2)O(2), the mitochondrial calcium response (Ca(mt)(2+)) was greater than the change in cytoplasmic calcium (Ca(c)(2+)). The Ca(mt)(2+) response to H(2)O(2) was dose dependent, while the increase in [Ca(c)(2+)] did not change with increasing H(2)O(2). The increase in both [Ca(c)(2+)] and [Ca(mt)(2+)] in response to oxidative stress was enhanced by exposure of cells to Ca(2+). The presence of chelator in the external medium only partially inhibited the Ca(mt)(2+) and Ca(c)(2+) responses to oxidative stress. Reagents that alter calcium fluxes had varied effects on the Ca(mt)(2+) response to peroxide. Ruthenium red blocked the increase in [Ca(mt)(2+)], while neomycin caused an even greater increase in [Ca(mt)(2+)]. Treatment with ruthenium red and neomycin had no effect on the Ca(c)(2+) response. Bafilomycin A and oligomycin had no effect on either the mitochondrial or cytoplasmic response. Inhibitors of both voltage-regulated calcium channels and intracellular calcium release channels inhibited the Ca(2+)-dependent component of the Ca(mt)(2+) response to oxidative stress. We conclude that the more significant Ca(2+) response to oxidative stress occurs in the mitochondria and that both intracellular and extracellular calcium pools can contribute to the increases in [Ca(c)(2+)] and [Ca(mt)(2+)] induced by oxidative stress.     

Monitoring dynamic collagen reorganization during skin stretching with fast polarization‐resolved second harmonic generation imaging

The mechanical properties of biological tissues are strongly correlated to the specific distribution of their collagen fibers. Monitoring the dynamic reorganization of the collagen network during mechanical stretching is however a technical challenge, because it requires mapping orientation of collagen fibers in a thick and deforming sample. In this work, a fast polarization‐resolved second harmonic generation microscope is implemented to map collagen orientation during mechanical assays. This system is based on line‐to‐line switching of polarization using an electro‐optical modulator and works in epi‐detection geometry. After proper calibration, it successfully highlights the collagen dynamic alignment along the traction direction in ex vivo murine skin dermis. This microstructure reorganization is quantified by the entropy of the collagen orientation distribution as a function of the stretch ratio. It exhibits a linear behavior, whose slope is measured with a good accuracy. This approach can be generalized to probe a variety of dynamic processes in thick tissues.