Fredric S. Fay

Mitochondria contribute to Ca2+ removal in smooth muscle cells.

Recent evidence, from a variety of cell types, suggests that mitochondria play an important role in shaping the change in intracellular calcium concentration ([Ca2+]i,) that occurs during physiological stimulation. In the present study, using a range of inhibitors of mitochondrial Ca2+ uptake, we have examined the contribution of mitochondria to Ca2+ removal from the cytosol of smooth muscle cells following stimulation. In voltage-clamped single smooth muscle cells, we found that following a 8-s train of depolarizing pulses, the rate of Ca2+ extrusion from the cytosol was reduced by more than 50% by inhibitors of cytochrome oxidase or exposure of cells to the protonophore carbonyl cyanideP-trifluoromethoxy-phenylhydrazone. Using the potential-sensitive indicator tetramethyl rhodamine ethyl ester, we confirmed that the effect of these agents was associated with depolarization of the mitochondrial membrane. Since, the primary function of the mitochondria is to provide the cells ATP, it could be argued that it is the ATP supply to the ion pumps which is limiting the rate of Ca2+ removal. However, experiments carried out with the mitochondrial Ca2+ uniporter inhibitor ruthenium red produced similar results, while the ATP synthetase inhibitor oligomycin had no effect, suggesting that the effect was not due to ATP insufficiency. These results establish that mitochondria in smooth muscle cells play a significant role in removing Ca2+ from the cytosol following stimulation. The uptake of Ca2+ into mitochondria is proposed to stimulate mitochondrial ATP production, thereby providing a means for matching increased energy demand, following the cells rise in [Ca2+]i;, with increased cellular ATP production.

Mitochondrial Ca2+ homeostasis during Ca2+ influx and Ca2+ release in gastric myocytes from Bufo marinus

The Ca2+‐sensitive fluorescent indicator rhod‐2 was used to monitor mitochondrial Ca2+ concentration ([Ca2+]m) in gastric smooth muscle cells from Bufo marinus. In some studies, fura‐2 was used in combination with rhod‐2, allowing simultaneous measurement of cytoplasmic Ca2+ concentration ([Ca2+]i) and [Ca2+]m, respectively. During a short train of depolarizations, which causes Ca2+ influx from the extracellular medium, there was an increase in both [Ca2+]i and [Ca2+]m. The half‐time (t½) to peak for the increase in [Ca2+]m was considerably longer than the t½ to peak for the increase in [Ca2+]i. [Ca2+]m remained elevated for tens of seconds after [Ca2+]i had returned to its resting value. Stimulation with caffeine, which causes release of Ca2+ from the sarcoplasmic reticulum (SR), also produced increases in both [Ca2+]i and [Ca2+]m. The values of t½ to peak for the increase in [Ca2+] in both cytoplasm and mitochondria were similar; however, [Ca2+]i returned to baseline values much faster than [Ca2+]m. Using a wide‐field digital imaging microscope, changes in [Ca2+]m were monitored within individual mitochondria in situ, during stimulation of Ca2+ influx or Ca2+ release from the SR. Mitochondrial Ca2+ uptake during depolarizing stimulation caused depolarization of the mitochondrial membrane potential. The mitochondrial membrane potential recovered considerably faster than the recovery of [Ca2+]m. This study shows that Ca2+ influx from the extracellular medium and Ca2+ release from the SR are capable of increasing [Ca2+]m in smooth muscle cells. The efflux of Ca2+ from the mitochondria is a slow process and appears to be dependent upon the amount of Ca2+ in the SR.

Sodium/calcium exchange regulates cytoplasmic calcium in smooth muscle

The sodium/calcium (Na+/Ca2+) exchanger is often considered to be a key regulator of the cytoplasmic calcium concentration ([Ca2+]) in smooth muscle but neither its precise role in Ca2+ homeostasis nor even its existence in smooth muscle are generally agreed upon. Here we directly assessed the role Na+/Ca2+ exchange plays in regulating [Ca2+] in single voltage-clamped smooth muscle cells. Following an elevation of [Ca2+], its decline was found to have both voltage-dependent and voltage-independent components. The voltage-dependent component was abolished when Na+ was removed from the external bathing solution. During the fall of [Ca2+] a small and declining Na+-dependent inward current was observed of a magnitude predicted by 3∶1 Na+/Ca2+ exchange stoichiometry. At [Ca2+] above 400 nM the principal efflux of Ca2+ above rest was attributed to this Na+-dependent removal mechanism. These results establish that a Na+/Ca2+ exchanger exists in smooth muscle and argue that it can regulate [Ca2+] at physiological Ca2+ concentrations.

Role of Na+‐Ca2+ Exchanger in β‐Adrenergic Relaxation of Single Smooth Muscle Cellsa

j3-Adrenergic agents cause smooth muscle of many tissues to relax. Despite the importance of this process for the function of many of our organs, we still do not fully understand the mechanism underlying this basic physiological response. Considerable progress has been recently achieved by analyzing the response to P-adrenergic stimulation at the single-cell level, using isolated smooth muscle cells.-3 Our own work on smooth muscle has focused on single cells enzymatically isolated from the stomach of the toad Bufo marinus. These cells are relatively large and retain their s t r~c tura l ,~ physiological,5 pharmacological,6 and biochemical2 properties following isolation, thereby facilitating studies at the single-cell level. They have been the subject of intense study for about 20 years and the knowledge base that has been developed is extremely powerful both for planning and interpreting studies. Finally, mechanisms uncovered in this cell system have in general been shown to be present in many other smooth muscle cell types, making it a generally useful model. Previous studies directed at understanding the events underlying 6-adrenergic relaxation of these smooth muscle cells have focused on changes in radioactive ion fluxes,J changes in membrane electrical proper tie^,^ as well as changes in the 35-cyclic adenosine monophosphate (CAMP)-dependent protein kinase pathwayv2 that accompanies the inhibitory effect of P-adrenergic agents like isoproterenol on contractility. Those studies led to the view of j3-adrenergic relaxation of smooth muscle shown in FIGURE 1. According to the model, isoproterenol (ISO), a P-adrenergic agonist, acts via a rise in CAMP and activation of CAMP-dependent protein kinase to stimulate the Na+-K+ pump. The consequent steepening of the transmembrane a+] gradient in turn would be expected to place the Na+-Ca2+ exchange into Ca2+-extrusion mode, thereby diminishing [Ca2+] changes and contractile force induced by excitatory stimuli. In addition I S 0 also hyperpolarizes the membrane through an increase in potassium cond~ctance,~ and this too would be expected to diminish contractile responsiveness by decreasing the extent of depolarization and consequently decreasing the activation of CaZ+ channels by excitatory ~ t imul i .~ The notion that the Na+-K+ pump is stimulated by IS0 has rested principally on radioactive ion-flux studies that revealed that IS0 stimulated 42K+ influx and 24Na+ efflux and that these effects could be mimicked by dibutyryl CAMP and blocked by ouabain.2*7 Furthermore, the notion that stimulation

Calcium-calmodulin-dependent mechanisms accelerate calcium decay in gastric myocytes from Bufo marinus.

1 [Ca2+]i was recorded in voltage‐clamped gastric myocytes from Bufo marinus. Repolarization to ‐110 mV following a 300 ms depolarization to +10 mV led to triphasic [Ca2+]i decay, with a fast‐slow‐fast pattern. After a conditioning train of repetitive depolarizations the duration of the second, slow phase of decay was shortened, while the rate of decay during the third, faster phase was increased by 34 ± 6 % (mean ± s.e.m., n= 21) when compared with unconditioned transients. 2 [Ca2+]i decay was biphasic in cells injected with the calmodulin‐binding peptide RS20, with a prolonged period of fast decay followed by a slow phase. There was no subsequent increase in decay rate during individual transients and no acceleration of decay following the conditioning train (n= 8). Decline of [Ca2+]i in cells injected with the control peptide NRS20 was triphasic and the decay rate during the third phase was increased by 50 ± 19 % in conditioned transients (n= 6). 3 Cell injection with CK3AA, a pseudo‐substrate inhibitor of calmodulin‐dependent protein kinase II, prevented the increase in the final rate of decay following the conditioning train (n= 6). In cells injected with an inactive peptide similar to CK3AA, however, there was a 45 ± 17 % increase after the train (n= 5). 4 Inhibition of Ca2+ uptake by the sarcoplasmic reticulum with cyclopiazonic acid or thapsigargin did not prevent acceleration of decay. 5 These results demonstrate that [Ca2+]i decay is accelerated by Ca2+‐calmodulin and calmodulin‐dependent protein kinase II. This does not depend on Ca2+ uptake by the sarcoplasmic reticulum but may reflect upregulation of mitochondrial Ca2+ removal.

Computerized analysis of TV images for ultrasensitive monitoring of the reaction of fluorochrome with protein

Preparation of protein-specific fluorescent probes with the desired degree of fluorochrome can be greatly facilitated by a technique that combines thin-layer chromatography (TLC) with quantitative image analysis (QIA). Using TLC/QIA, the investigator can determine the fluorochrome/protein ratio on-line with only a few micrograms of protein as fluorochrome is conjugated to protein. In addition, this technique allows rapid quantitation of dye noncovalently adsorbed to fluorochrome-labeled protein.

Calcium homeostasis in single intact smooth muscle cells

We have demonstrated that ISO produces part of its negative inotropic action through activation of the plasmalemmal Na+/K+ pump, and reduction of [Na+]i. This action is mediated by the beta-adrenergic receptor through activation of adenylate cyclase. The reduction of [Na+]i is most probably translated to a change in the contractile state of the cell through activation of the Na+/Ca2+ exchanger. While the exchanger is at equilibrium when the cell is at rest, after ISO it would extrude Ca2+ at the expense of the increased Na+ gradient, resulting in a decrease Ca2+ availability and a reduction in the magnitude of subsequent contractions. We have also seen that the previous calcium history of the myoplasm can influence the time course of future calcium transients. Prolonged large increases in [Ca2+]i can accelerate the rate of its removal and depress basal [Ca2+]i levels. This action is most probably mediated through a Ca2+/calmodulin dependent protein kinase. We have observed that MLCK is both necessary and sufficient to produce contraction of Bufo marinus stomach smooth muscle. There is also evidence that an as yet unidentified Ca(2+)-calmodulin dependent protein kinase is acting to limit the magnitude and the duration of the Ca2+ transient by feeding back on processes involved in Ca2+ signal generation.

High-speed stroboscopic multispectral imaging of fluorescent probes in living cells

Studies using fluorescent probes to determine the distribution of physiologically important ions and molecules have been the focus of much interest. Some of these probes may be used to measure the actual concentration of chemical species within cells if measurements are made at two or more wavelengths. However, because of the low photon levels of fluorescent emission at the single cell level, current intensified cameras yield useful information at about 1 second time resolution. Many biologically relevant change; in ionic composition in cells take place much more rapidly than this, therefore requiring a faster imaging system to study these processes in a localized manner. A fast digital fluorescence microscope capable of obtaining dual wavelength images at 30 images per second is described. It can record up to six minutes of data while switching excitation wavelength between image frames.