ryanodine and 2--4--dichlorobenzamil-amiloride

Ca2+ mobilization elicited by simulation with brief pulses of high K+ were monitored with confocal laser scanned microscopy in intact, guinea pig cardiac myocytes loaded with the calcium indicator fluo-3. Single wavelength ratioing of fluorescence images obtained after prolonged integration times revealed non-uniformities of intracellular Ca2+ changes across the cell, suggesting the presence of significant spatial Ca2+ gradients. Treatment with 20 microM ryanodine, an inhibitor of Ca2+ release from the SR, and 10 microM verapamil, a calcium channel blocker, reduced by 42% and 76% respectively the changes in [Ca2+]i elicited by membrane depolarization. The overall spatial distribution of [Ca2+]i changes appeared unchanged. Ca2+ transients recorded in the presence of verapamil and ryanodine (about 20% of the size of control responses), diminished in the presence of 50 microM 2-4 Dichlorbenzamil (DCB) or 5 mM nickel, two relatively specific inhibitors of the Na+/Ca2+ exchange mechanism. Conversely, when the reversal potential of the Na+/Ca2+ exchange was shifted to negative potentials by lowering [NA+]o or by increasing [Na+]i by treatment with 20 microM monensin, the amplitude of these Ca2+ transients increased. Ca2+ transients elicited by membrane depolarization and largely mediated by reverse operation of Na(+)-Ca2+ exchange could be recorded in the presence of ryanodine, verapamil and monensin. These finding suggest that in intact guinea pig cardiac cells, Ca2+ influx through the Na+/Ca2+ exchange mechanism activated by a membrane depolarization in the physiological range can be sufficient to play a significant role in excitation-contraction coupling.

Topics: Amiloride; Animals; Biological Transport; Calcium; Calcium Channel Blockers; Calcium Channels; Carrier Proteins; Cells, Cultured; Guinea Pigs; Heart; Heart Ventricles; Membrane Potentials; Microscopy, Confocal; Microscopy, Fluorescence; Monensin; Muscle Proteins; Myocardium; Nickel; Ouabain; Potassium; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Signal Transduction; Sodium-Calcium Exchanger; Verapamil

1. The relationship between intracellular pH (pHi) and contractile activity was investigated in papillary muscles isolated from right ventricle of normal and streptozotocin (STZ)-induced diabetic rats. pHi changes induced by 20 mM-NH4Cl were recorded with H(+)-sensitive microelectrodes. 2. An increase in pHi of approximately 0.20 pH units on exposure to NH4Cl led to an increase of the maximum developed tension, which was 707.8 +/- 57.5% (mean +/- S.E. of mean, n = 10) of control in normal muscles and 271 +/- 16.3% (n = 10) in diabetic muscles. On the other hand, acidosis induced by NH4Cl withdrawal was associated with a fall in developed tension to 48.2 +/- 6.7% of control in diabetic muscles, as compared to 79.2 +/- 8% in normal muscles. 3. The decrease in tension associated with acidosis was rapidly followed (in approximately 2 min) by a transient redevelopment of force, which peaked at 80.2 +/- 8.6% of control in the diabetic muscles as compared to 153.5 +/- 11.7% in normal papillary muscles. The peak of this secondary positive inotropy coincided in both groups of muscles with the maximum decrease of pHi, i.e. -0.40 +/- 0.02 and -0.28 +/- 0.04 pH units in diabetic and normal muscles, respectively. 4. Caffeine (10 mM), which had a marked positive inotropic effect in both groups of muscles, abolished the transient recovery of tension occurring after NH4Cl withdrawal. Ryanodine (2 microM) which had a marked negative inotropic effect on both normal and diabetic papillary muscles, also suppressed the transient recovery of tension. 5. The presence of amiloride (1 mM) during acidosis induced by NH4Cl withdrawal abolished the observed differences in developed tension, in particular the transient recovery of tension, between normal and diabetic muscles, as it abolished the differences in the amplitude of pHi decrease and in the time course of pHi recovery. 6. The presence of 2',4'-dichlorobenzamil amiloride (40 microM) significantly and similarly delayed and reduced the amplitude of transient recovery of tension in both normal and diabetic papillary muscles. 7. We conclude that STZ-induced diabetes induces a decrease in pHi sensitivity of contractile force. This may be the consequence of a change in sarcoplasmic reticulum (SR) composition and function, and may also indirectly result from changes in Na(+)-H+ exchange activity, particularly during intracellular acidosis.

Topics: Amiloride; Ammonium Chloride; Animals; Caffeine; Diabetes Mellitus, Experimental; Hydrogen-Ion Concentration; In Vitro Techniques; Male; Muscle Contraction; Papillary Muscles; Rats; Rats, Inbred Strains; Ryanodine