RAPID COMMUNICATION
Force relaxes before the fall of cytosolic calcium in the photomechanical response of rat sphincter pupillae

Andrew P. Krivoshik1,2 and Lloyd Barr1

1 Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801; and 2 Mayo Clinic, Rochester, Minnesota 55905


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the rat sphincter pupillae, as in other smooth muscles, the primary signal transduction cascade for agonist activation is receptor right-arrow G protein right-arrow phospholipase C right-arrow inositol trisphosphate right-arrow intracellular Ca2+ concentration ([Ca2+]i) right-arrow calmodulin right-arrow myosin light chain kinase right-arrow phosphorylated myosin right-arrow force development. Light stimulation of isolated sphincters pupillae can be very precisely controlled, and precise reproducible photomechanical responses (PMRs) result. This precision makes the PMR ideal for testing models of regulation of smooth muscle myosin phosphorylation. We measured force and [Ca2+]i concurrently in sphincter pupillae following stimulation by light flashes of varying duration and intensity. We sampled at unusually short (0.01-0.02 s) intervals to adequately test a PMR model based on the myosin phosphorylation cascade. We found, surprisingly, contrary to the behavior of intestinal muscle and predictions of the phosphorylation model, that during PMRs force begins to decay while [Ca2+]i is still rising. We conclude that control of contraction in the sphincter pupillae probably involves an inhibitory process as well as activation by [Ca2+]i.

contractile force; light; contraction; myosin phosphorylation; contraction models


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ABSORPTION OF LIGHT BY photosensitive receptor molecules in sphincter pupillae smooth muscle myocytes evokes contraction by triggering the release of calcium ions (Ca2+) from intracellular stores. This photosensitive receptor, which has an action spectrum similar to the absorption spectra of rhodopsin, is located in the plasma membrane and has antigenic sites, which bind antibodies for rhodopsin (1). The photomechanical response (PMR) signal transduction cascade couples an opsin receptor via a G protein to the typical smooth muscle phosphorylation contraction cascade. The member of the opsin family in the cell membrane of the sphincter pupillae myocytes that triggers the PMR is probably either rhodopsin (2) or melanopsin (22). It has been demonstrated that the PMR of the isolated pupillary sphincter is independent of the retinal pretectal reflex pathways, which mediate the pupillary reflex of humans and other mammals. In frog sphincter pupillae, the PMR and the neural pupillary reflex are equally strong (1). However, in mammals, the PMR is much weaker than the contraction elicited by muscarinic receptor activation. Nonetheless, forces developed during mammalian PMRs, 1-50 µN, are easily measured. The PMR of the frog sphincter pupillae is a fast relatively simple smooth muscle contraction. It shows none of the "latch" properties commonly found in other smooth muscles. Most significantly, the force time courses of frog PMRs to light stimuli of different durations or intensities are quantitatively predicted by a simple model of the phosphorylation cascade (3). We report here for the first time, additionally, the time courses of cytosolic Ca2+ concentration ([Ca2+]i) during PMRs. We studied the PMR of sphincter pupillae from albino (4, 17) rats to minimize any interference with our fluorescence measurements by melanin, present in the myocytes of the frog sphincter pupillae. We found the first direct evidence of a very fast regulatory component in addition to the Ca2+-calmodulin-phosphorylation cascade in smooth muscle.

We conclude that, while for some other mammalian smooth muscles alternative activating pathways have been proposed, a fast Ca2+-independent inhibition is most likely responsible for the termination of contraction in the sphincter pupillae.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Albino Sprague-Dawley rats were maintained in the dark for at least 10 days to increase their sensitivity to light. Sphincter pupillae preparations were isolated under a dissecting microscope in far red light (Wratten Filters 1A) at 0°C. The annular bundles of smooth muscle were loaded with indo 1 by exposing them for 40 min to 2 µM indo 1-acetoxymethyl (AM) (9, 29, 30). To augment loading, 10 µl of 20% (wt/vol) Pluronic F-127 dissolved in dimethyl sulfoxide was added to the 2.5-ml mammalian isotonic phosphate-buffered physiological bathing solution [136 mM NaCl, 1 mM MgSO4· 7H2O, 0.1 mM EDTA, 4 mM propionic acid (hemicalcium salt), 5 mM glucose, 2 mM sodium succinate·7H2O, 1.6 mM NaH2PO4·H2O, 6.8 mM K2HPO4·3H2O] in the tissue chamber. EDTA is used as a chelating agent to remove contaminant heavy metals from solution. To minimize extracellular hydrolysis of AM ester, a phosphate buffer is used instead of an amine buffer. The isolated sphincter's pupillae in a 37°C chamber on the stage of a Nikon Optiphot microscope were attached to a Cambridge 400A tension transducer lever. The microscope system utilizes two Hamamatsu R374 head-on type photomultiplier tubes and custom band-pass filters. The relative dead time of the electronics system (including photomultiplier tubes) dedicated to the fluorescence measurements was experimentally determined to be on the order of milliseconds and estimated to be no greater than 10 ms. The microscope setup was extensively modified with light traps to minimize stray light, since the background contractile activity of the sphincter pupillae is proportional to the logarithm of background light intensity (2). Filters were used to allow excitation of the indo 1 without stimulating the sphincter pupillae and also to stimulate the sphincter pupillae to produce a PMR without interfering with the fluorescence measurements. Light that stimulated the preparation passed through a long-pass dark orange filter (OG515) and was strongly attenuated by the filters in the photomultipler light paths. To excite the indo 1 without appreciably eliciting a PMR, shorter wavelength of light outside of the action spectra of the PMR was used to excite indo 1. This was done by strongly attenuating visible light in the exciting light path with a 310-nm filter with half-band-pass width of 50 nm in series with a 347-nm band-pass filter 40 nm wide at half band pass. Both the force signal and fluorescence signals are digitally sampled and processed through digital Savitsky-Golay smoothing filters (12, 24). The fluorescence signals are subsequently manipulated through a ratiometric data analysis to generate an apparent Ca2+ measurement (9). Digital data sampling began 0.5 s before the beginning of an experimental flash stimulus to sample the dark steady state before flash stimulation.

Because the structural apparatus for force transmission in smooth muscle occupies between 80 and 90% of the cell volume (8), contraction reflects the spatial average of [Ca2+]i (20). In fact, the spatial average of [Ca2+]i and Ca2+ subjacent to the cell membrane may even be anticorrelated (32). Therefore, fast time resolution of a spatial averaged [Ca2+]i and force were used to test quantitative signal transduction model predictions (15, 16).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The effects on the PMR of varying the intensity of light with the same durations of illumination were tested. Figure 1 shows that the PMR has several unusual kinetic properties. First, Fig. 1, A and B, shows the similarities of concurrent time courses of developed force and [Ca2+]i in a sphincter stimulated by two stimuli of 0.5 s duration, but different intensities. Intensity increases the amplitude of the response but does not change the time course. Second, the latency period of the rat PMR is much shorter than that of the amphibian PMR (2) or fish PMR (1). After brief flashes of light, the peak force of a PMR occurs after the stimulus light flash has ended, i.e., the flash of light triggers a cascade perturbation that continues to activate Ca2+ and force after the light stimulus is terminated. Finally, maximal peak force occurs before maximal peak [Ca2+]i, and the force decays faster than does the [Ca2+]i.


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 1.   Force and intracellular Ca2+ time courses during 2 photomechanical responses (PMRs) of an albino rat sphincter pupillae stimulated by 2 light flashes of same duration but different intensities. A: 2 force time courses following 0.5-s flashes whose intensities were attenuated by neutral density filters: 0.2 optical density (OD) or 0 OD (no filter), respectively. Very large forces (>10 mg), occurring during peak of largest PMR were out of dynamic range of analog-to-digital converter and were digitized manually from parallel records obtained via an analog strip chart recorder. Peaks of responses occur with same latency after each stimulus. B: 2 intracellular Ca2+ time courses corresponding to force time courses in A. Peaks of intracellular Ca2+ time courses all occur at same interval after stimulus, but a little later than the time to peak for the force trajectory. Similarly, the decay of intracellular Ca2+ from its peak is slower than the decay of force from its peak. Amplitudes of force and Ca2+ time courses increase less than linearly with increasing stimulus intensity. From studies with slower sampling rates over longer periods of data acquisition, Ca2+ for comparable stimuli returns entirely to baseline on the order of 2 min.

If the stimulus flash duration and quantal contents are small enough, there is reciprocity between intensity and duration, and the responses to such constant quanta, variable duration flashes, are indistinguishable. As seen in the amphibian PMR, the force of any particular sphincter's response to a short flash of light is proportional to the logarithm of intensity times duration, namely the quantal content (3).

Therefore, PMRs to 0.5-s flashes of different intensities differ only in amplitude, with the shape of the time courses of the PMRs appearing independent of the light intensity. Therefore, the force time courses normalized to the peak force are nearly identical and superimpose (Fig. 2A). Thus the time it takes to reach any given fraction of the peak force of the twitch is independent of stimulus intensity. Therefore, also, the exponential time constants for the evolution and decay of the PMR vary little with intensity. In Fig. 2A, the corresponding [Ca2+]i time courses normalized to peak [Ca2+]i may be seen to also collapse to a single curve. The exponential time constants for the evolution and decay of the [Ca2+]i time course therefore also vary little with intensity. The semilogarithmic scale for the normalized force and [Ca2+]i time courses makes the time constants of the signal transduction processes more apparent. The rise of force is nearly a single exponential, but the relaxation involves at least two exponential components. As is also shown in Fig. 2A, there are both very fast and slow components to the relaxation of [Ca2+]i as well. The complex relationship in the dynamic state between [Ca2+]i and force are apparent in the phase relationship of [Ca2+]i with respect to force as shown in Fig. 2B. During the rapid falling phase of force in a PMR, there is a clear decoupling from still slowly rising [Ca2+]i. The family of phase loops generated by increasing flash intensity with constant duration are clockwise in time and also when normalized to peak collapse to a single canonical phase loop.


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 2.   Relaxation of force precedes fall of intracellular Ca2+ PMRs of sphincter pupillae smooth muscle of albino rat. Moreover, trajectories of PMRs to short flashes of increasing intensity elicit responses whose amplitudes increase but whose time courses remain the same. Peaks of force and intracellular Ca2+ occur at times that are characteristic of stimulus duration, not stimulus strength. Phase relations between force and intracellular Ca2+ are therefore the same for all PMRs resulting from same duration stimuli. A: overlay of semilog plots of normalized force and normalized Ca2+ during PMRs after 0.5-s flashes of different intensities (data from Fig. 1). Normalized force time courses superimpose as do normalized Ca2+ time courses. Both force and Ca2+ time courses have 2 time constants apparent in relaxation phases. The 2 dominant relaxation time constants for force are in the neighborhood of 2.8 and 9.8 s. The 2 dominant relaxation time constants for Ca2+ are in the neighborhood of 7.7 and 14.7 s. B: phase relationship between force and intracellular Ca2+ shows time increases clockwise around the hysteresis loop (data from Fig. 1). Because relaxation is slower than contraction, there are many more data points in the relaxation portion of the hysteresis loop. Clearly, force is falling in a period, upper right hand quadrant, where intracellular Ca2+ is still rising. Thus, in time, intracellular Ca2+ changes first lead, and then lag behind force changes during a photomechanical response. Return to baseline force and baseline Ca2+ is represented as completion of hysteresis loop in phase diagram. C: longer durations of stimulating flashes elicit larger PMR responses up to at least 10 s, but responses evolve toward a plateau. Three time courses of force during PMRs in response to light flashes of 2, 5, and 10 s. Quanta of light arriving later during a flash longer than ~0.3 s have less effect on increasing force and intracellular Ca2+. During very long flashes force slowly declines due to use decline (see Fig. 5). D: similarly, 3 time courses of intracellular Ca2+ during PMRs (shown in C) in response to light flashes of 2, 5, and 10 s. In each case, decay of intracellular Ca2+ is slower than decay of force. Initial spurious points at beginning of PMRs attributable to stimulating light flash (shutter) artifact were deleted. E: phase relationship between force and intracellular Ca2+ shows time increases clockwise around the hysteresis loop (data from C and D).

Figure 2, C and D, shows the time courses of PMRs to stimuli of constant intensity and varying duration. Responses to long flashes tend toward a plateau after ~15 s. The family of phase loops generated by increasing flash duration with constant intensity are clockwise in time and follow similar initial rise trajectories and final relaxation trajectories, except that the maximal Ca2+ maximal force shoulder is displaced to higher values for longer durations. Moreover, the phase relationships follow similar initial and final trajectories and are all clockwise in time (Fig. 2E). Qualitatively similar clockwise phase loops are found in stimulating flashes as long as 1 min.

To check whether this response of the rat sphincter pupillae is peculiar, we did similar experiments on the frog sphincter pupillae. Figure 3 is a semilogarithmic plot of force and [Ca2+]i time courses for the average of five frog PMRs to flashes of 0.1 s duration. In contrast to the rat PMR, both the rise and relaxation of force in the frog appear to be simple exponential functions of time (2). Nonetheless, as shown in Fig. 3B, in the frog PMR, the phase relationship of [Ca2+]i with respect to force follows a trajectory similar to that of the rat but with slower kinetics. In the phase diagrams of Fig. 2, B and E, and Fig. 3B, time increases clockwise around the hysteresis loop, emphasizing that force falls before [Ca2+]i. By using very short (0.1-s) flashes of light, the shutter artifact could be separated in time from the recordings of [Ca2+]i and force. The initial rise of [Ca2+]i is seen to precede the rise of force (Fig. 3B).


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 3.   Relaxation of force precedes fall of intracellular Ca2+ PMRs of sphincter pupillae smooth muscle of frog. Averaged normalized intracellular Ca2+ and normalized force time courses from 5 frog PMRs after 0.1-s flashes of constant intensity are slightly slower than rat. Note however, frog sphincter pupillae were investigated at 25°C. Due to intrinsic pigments in the frog preparation, in addition to signal averaging, it was necessary to increase loading time, concentration of indo 1-AM, and intensity of short-wavelength light used to excite indo 1. A: PMRs of frog sphincter pupillae are qualitatively similar to those of rat. In frog, as well as rat, force begins to fall while Ca2+ is still increasing. In frog PMR, there is only 1 apparent time constant in relaxation of both force and Ca2+ and is, therefore, kinetically less complex than rat PMR. B: for frog PMRs, phase relationship between force and intracellular Ca2+ also shows time increases clockwise around the hysteresis loop (data from A).

To test whether or not force relaxation generally precedes fall of [Ca2+]i in other smooth muscles, we measured [Ca2+]i and force concurrently in spontaneously active rat duodenal muscle rings. These data show that force lags [Ca2+]i by a nearly constant phase angle. In particular and in contrast to the PMRs, a fall in [Ca2+]i always precedes the fall in force (Fig. 4A). Figure 4B shows the phase relationship in which time increases counterclockwise around the hysteresis loop and the phase plot is close to a circle. Ca2+ and force data from similar experiments described in the literature (21) transform into qualitatively similar phase diagrams. Similar counterclockwise phase diagrams have been reported for vascular smooth muscle (10). The differences between sphincter pupillae and intestine predict a qualitatively different signal transduction system in the sphincter pupillae, which allows the pupil to relax rapidly to decreases in ambient light.


View larger version (34K):
[in this window]
[in a new window]
 
Fig. 4.   Intracellular Ca2+ changes precede force changes throughout contractile cycles of rhythmic contractions of rat duodenum. Strips of rat duodenal circular muscle with longitudinal layer attached were mounted and loaded in same manner as sphincters pupillae. A: during spontaneous rhythmic contractions, force clearly lags behind intracellular Ca2+ changes. In absence of appropriate calibrations, ratio of emitted fluorescent light at 405-485 nm corrected for background fluorescence is reported instead of actual Ca2+ concentration. B: in contrast to results from sphincters pupillae, intestinal muscle relaxes only after intracellular Ca2+ falls. For these rhythm contractions, phase relationship between force and fluorescence ratio shows time increases counterclockwise around the hysteresis loop (data from Fig. 3A). , Fluorescence ratio and force of native data averaged over the 6 cycles. Because actual Ca2+ concentration is a monotonic function of fluorescence ratio, the shape of the hysteresis loop would be changed by calibration but not the direction that time takes around the loop. From these observations for duodenal muscle, there is no need to posit any modulator of force beyond intracellular Ca2+ concentration.

Given the rapid response time of the experimental apparatus and the rapid reaction kinetics of indo 1, it is unlikely that such a phase relationship between Ca2+ and force is a measurement artifact attributable to the system response time or kinetic properties of the fluorescent Ca2+-sensitive probe. Indo 1 is well suited to measure rapid changes in concentration of intracellular Ca2+; stopped flow data for indo 1 indicate that the association rate constant approach the diffusion-controlled limit, and the measurement of a transient decay in Ca2+ with a half-time on the order of 15 ms is possible in a system with a dead time on the order of 1 ms (11). Furthermore, when ultrashort duration flashes of stimulating light are used (Fig. 3B), the latency in the initial rise of force after the initial rise in Ca2+ is consistent with the rapid response time of the experimental apparatus. Although both rate constants and dissociation constant (Kd) are essentially invariant between pH 7.0 and 8.0 (11), undetected transient changes in pH below that range may distort the apparent Ca2+ kinetics (19). Although indo 1 (9, 29) is rather selective for Ca2+, other potential factors such as Mg2+ and Zn2+ transients and fluorescent probe protein interactions may also confound the interpretation of the Ca2+ kinetics. As an experimental instrument control, it is notable that measurements in the intestinal preparation (Fig. 4) compare well with those in the literature (10, 21).

During control experiments in the rat sphincter pupillae, it was noted that PMRs decayed in amplitude as the experiments proceeded. The maximal peak amplitude in the PMRs of a preparation follows a nearly perfect exponential decay as a function of number of light stimuli of fixed duration and intensity. The decay of peak response is essentially independent of between flash recovery time and hence also independent of total time of survival (Fig. 5A). The decay is a function of both the intensity and duration of stimuli. The greater the duration of the flash stimulus, the greater the rate of decay (Fig. 5B). After many flash stimuli, although the maximal peak PMR is attenuated, the response to an acetylcholine challenge remains essentially unchanged, so that the decline with frequency of stimulation is related to the early light receptor-like portion of the signal transduction pathway. Therefore, small duration and low intensity flashes were typically used during most Ca2+ measuring experiments. Long exposure to mercury light used to excite indo 1 causes tissue damage, and therefore Ca2+ could not be meaningfully measured concurrently with force for these decay experiments. Also, since PMRs normalized to peak tension have the same time courses, myosin phosphatase is probably not involved. Attempts to treat the preparation with all-trans-retinol were not protective. Preliminary experiments show that both caffeine and okadaic acid increased this decay, suggesting that phosphorylation of the light transduction system may be responsible for this decay, in that phosphorylation may inactivate rhodopsin (5). Moreover, perhaps this decay phenomenon is related to a regulatory protein such as arrestin (18), since transgenic mouse rods that lack arrestin exhibit prolonged photoresponses (31). So, although interesting, this usage decay phenomenon is probably independent of the second regulatory process of the PMR.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 5.   Responsiveness of rat sphincter pupillae decays in vitro according to usage. A: force amplitudes of PMRs decay exponentially with number of flash stimuli. Two sphincter pupillae from the same rat were stimulated with 0.5-s duration, constant intensity flashes. For 1 sphincter the recovery time between flashes was first 3 min for 10 flashes and then 5 min for three flashes. For the other sphincter the recovery time between flashes was first 5 min for 9 flashes and then 3 min for 10 flashes. On a semilog plot all the force amplitudes fall on parallel straight lines; the slopes of the 4 best fit lines for each subgroup are not distinguishable by Chi square (P > 0.95). Varying recovery time between flashes does not alter decrement in responsiveness following a PMR; instead it is proportional to the PMR amplitude. B: decrement in responsiveness after a PMR increases with duration of the stimulus and response. A sphincter pupillae was stimulated with flashes of the same intensity but whose durations first 0.1 s and then 0.2 s with a 3-min interflash recovery delay. Doubling flash duration increases the decrement per PMR in addition to increasing initial peak force generated. These 2 slopes are statistically different to 99% confidence by homoscedastic t-test for mean differences.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The relation between force and Ca2+ in smooth muscle varies during a contraction-relaxation cycle, and the apparent Ca2+ sensitivity of force depends on the mode of activation (26,27). Clearly, the relative activities of myosin light chain kinase and the myosin phosphatases determine the level of myosin phosphorylation. Therefore, a decrease in force at unaltered Ca2+ may be attributable to factors that alter the activities of myosin light chain kinase or the phosphatases, such as calmodulin (CaM) kinase II, telokin, and cGMP-activated kinases. Relaxation disproportionate to the decline in [Ca2+]i has been previously related to rapid dephosphorylation of regulatory myosin light chains, due to either inactivation of myosin light chain kinase by CaM kinase II (28) or enhanced phosphatase activity referred to as "desensitization to [Ca2+]i" (25). Decreased myosin phosphorylation accompanying a decline in force during maintained high [Ca2+]i has been observed in permeabilized smooth muscle (13). Moreover, CaM kinase II has been shown in vitro to decode the frequency of Ca2+ spikes into distinct kinase activity periods (6). Thin filament proteins, such as calponin and caldesmon, may also influence the relation between force and Ca2+. Mitogen-activated protein kinase in vascular smooth muscle may be related to Ca2+-independent smooth muscle contraction and caldesmon phosphorylation (7). In uterine smooth muscle, oxytocin receptor stimulation coupled via a G protein activates phospholipase C, generating inositol trisphosphate that triggers release of Ca2+ from intracellular stores; this oxytocin receptor stimulation of phospholipase C is inhibited by cAMP involving the action of protein kinase A (23). In general, if force is proportional to myosin phosphorylation, then the clockwise in time phase diagram between Ca2+ and force implies the existence of an inhibitory pathway in the contraction cycle. Concurrent measurements of myosin light chain phosphorylation might discriminate between such putative mechanisms, but current methods are not adequate to resolve such fast time courses.

Preliminary experiments with rat PMR show that cAMP but not cGMP attenuates the PMR as well as increases the rate of PMR relaxation (14). However, cAMP collapses the phase loops to the right (14, 15). If there is only a Ca2+-dependent activating process, then the direction of time in the phase diagram would be counterclockwise. If there is a strong enough Ca2+-independent inhibition during a relaxation, then, however, the phase diagram may run clockwise. A constant increase in the relationship of Ca2+ to force may shift the phase diagram to the right. If cAMP is present in an inhibitory pathway, then added cAMP should collapse phase loops progressively (i.e., if the cAMP process is saturated, cAMP release during a cycle would have no effect). Although cAMP may activate myosin phosphatases, cAMP may also increase phosphorylation of myosin light chain kinase. Although the appearance of an inhibitory process (e.g., via a cAMP event) to initiate the fall of force seems possible (15), other possibilities exist. If light in parallel to triggering Ca2+ release also turned on a faster pathway that increased force development or the efficacy of Ca2+ (e.g., via a GTP mechanism), then, when light turns off, that activating system's quick collapse might cause force to fall before the slower Ca2+ cascade collapses. A third possibility also might involve cAMP but in a manner similar to cGMP's role in the retinal rod. If there is a significant concentration of cAMP causing a tonic inhibition of the contractile cascade and if also light quickly turned on a disinhibiting phosphodiesterase, then, when the light is turned off, a cAMP-induced inhibition might reappear before the slower Ca2+ cascade collapses and [Ca2+]i begins to fall. Some of these alternatives can be tested by investigating the pharmacological aspects of GTP and cAMP behavior in rat sphincter pupillae smooth muscle.


    ACKNOWLEDGEMENTS

This work was supported in part by National Science Foundation Grant DCB-41526, by the University of Illinois at Urbana-Champaign Research Board, and by National Institute of Drug Abuse National Research Service Award Grants F30-DA-05574 and T32-GM-08276.


    FOOTNOTES

Address for reprint requests and other correspondence: A. P. Krivoshik, Pediatrics-Baldwin 3B, Mayo Clinic, 200 First St. SW, Rochester, MN 55905 (E-mail: krivoshik.andrew{at}mayo.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 14 February 2000; accepted in final form 4 April 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Barr, L. Photochemical coupling in the vertebrate sphincter pupillae. CRC Crit Rev Neurobiol 4: 325-366, 1989.

2.   Barr, L, and Alpern M. Photosensitivity of the frog iris. J Gen Physiol 46: 1249-1265, 1963[Abstract/Free Full Text].

3.   Barr, L, and Gu F. A quantitative model of myosin phosphorylation and the photochemical response of the response of the isolated sphincter pupillae of the frog iris. Biophys J 51: 895-904, 1987[Abstract].

4.   Bito, L, and Turansky D. Photoactivation of pupillary constriction in the isolated in vitro iris of a mammal (Mesocricetus auratus). Comp Biochem Physiol A Physiol 50A: 407-413, 1975.

5.   Chen, J, Makino C, Peachey N, Baylor D, and Simon M. Mechanisms of rhodopsin inactivation in vivo as revealed by a COOH-terminal truncation mutant. Science 267: 374-377, 1995[ISI][Medline].

6.   De Koninck, P, and Schulman H. Sensitivity of CaM kinase II to the frequency of Ca2+ oscillations. Science 279: 227-230, 1998[Abstract/Free Full Text].

7.   Dessy, C, Kim I, Sougnez CL, Laporte R, and Morgan KG. A role for MAP kinase in differentiated smooth muscle contraction evoked by alpha -adrenoceptor stimulation. Am J Physiol Cell Physiol 275: C1081-C1086, 1998[Abstract/Free Full Text].

8.   Gabella, G. Structural apparatus for force transmission in smooth muscle. Physiol Rev 64: 455-475, 1984[Free Full Text].

9.   Grynkiewicz, G, Poenie M, and Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440-3450, 1985[Abstract].

10.   Himpens, B, Kitazawa T, and Somlyo AP. Agonist-dependent modulation of Ca2+ sensitivity in rabbit pulmonary artery smooth muscle. Pflügers Arch 417: 21-28, 1990[ISI][Medline].

11.   Jackson, AP, Timmerman MP, Bagshaw CR, and Ashley CC. The kinetics of calcium binding to Fura-2 and Indo-1. FEBS Lett 216: 35-39, 1987[ISI][Medline].

12.   Kargacin, M, and Kargacin G. Methods for determining cardiac sarcoplasmic reticulum Ca2+ pump kinetics from fura 2 measurements. Am J Physiol Cell Physiol 267: C1145-C1151, 1994[Abstract/Free Full Text].

13.   Kitazawa, T, and Somlyo AP. Desensitization and muscarinic re-sensitization of force and myosin light chain phosphorylation to cytoplasmic Ca2+ in smooth muscle. Biochem Biophys Res Commun 88: 741-744, 1990.

14.   Krivoshik, AP, and Barr L. Regulation of intracellular calcium during the photomechanical response of albino rat sphincter pupillae smooth muscle (Abstract). Biophys J 74: A381, 1998[ISI].

15.   Krivoshik, AP, and Barr L. Kinetics of [Ca2+]i and force during the photomechanical response of albino rat sphincter pupillae (Abstract). Biophys J 72: A183, 1997.

16.   Krivoshik, AP, and Barr L. Kinetics of [Ca++]i during the photomechanical response of rat sphincter pupillae (Abstract). FASEB J 8: A570, 1994[ISI].

17.   Lau, K, So K-F, Campbell G, and Lieberman A. Pupillary constriction in response to light in rodents, which does not depend on central neural pathways. J Neurol Sci 113: 70-79, 1992[ISI][Medline].

18.   Lefkowitz, RJ. G protein-coupled receptors. III. New roles for receptor kinases and beta -arrestins in receptor signaling and desensitization. J Biol Chem 273: 18677-18680, 1998[Free Full Text].

19.   Morris, SJ, Wiegmann TB, Welling LW, and Chronwall BM. Rapid simultaneous estimation of intracellular calcium and pH. Methods Cell Biol 40: 183-220, 1994[ISI][Medline].

20.   Nelson, MT, Cheng H, Rubart M, Santana LF, Bonev AD, Knot HJ, and Lederer WJ. Relaxation of arterial smooth muscle by calcium sparks. Science 270: 633-637, 1995[Abstract].

21.   Ozaki, H, Stevens RJ, Blondfield DP, Publicover NG, and Sanders KM. Simultaneous measurement of membrane potential, cytosolic Ca2+, and tension in intact smooth muscle. Am J Physiol Cell Physiol 260: C917-C925, 1991[Abstract/Free Full Text].

22.   Provencio, I, Jiang G, De Grip W, Hayes W, and Rollag M. Melanopsin: an opsin in melanophores, brain, and eye. Proc Natl Acad Sci USA 95: 340-345, 1998[Abstract/Free Full Text].

23.   Sanborn, BM, Dodge K, Monga M, Oian A, Wang W, and Yue C. Molecular mechanisms regulating the effects of oxytocin on myometrial intracellular calcium. Adv Exp Med Biol 449: 277-286, 1998[ISI][Medline].

24.   Savitzky, A, and Golay M. Smoothing and differentiation of data by simplified least squares procedures. Anal Chem 36: 1627-1639, 1964[ISI].

25.   Somlyo, AP, and Somlyo AV. Signal transduction and regulation in smooth muscle. Nature 372: 231-236, 1994[ISI][Medline].

26.   Somlyo, AP, and Somlyo AV. From pharmacological coupling to G-proteins and myosin phosphatase. Acta Physiol Scand 164: 437-448, 1999[ISI].

27.   Somlyo, AP, Wu X, Walker LA, and Somlyo AV. Pharmacolmechanical coupling: the role of calcium, G-proteins, kinases and phosphatases. Rev Physiol Biochem Pharmacol 134: 201-234, 1999[Medline].

28.   Tansey, MG, Luby-Phelps K, Kamm KE, and Stull JT. Ca2+ dependent phosphorylation of myosin light chain kinase decreases the Ca2+ sensitivity of light chain phosphorylation within smooth muscle. J Biol Chem 269: 9912-9920, 1994[Abstract/Free Full Text].

29.   Tsien, RY. New calcium indicators and buffers with high selectivity against magnesium and protons: design, synthesis, and properties of prototype structures. Biochemistry 19: 2396-2404, 1980[ISI][Medline].

30.   Tsien, RY. A non-disruptive technique for loading calcium buffers and indicators into cells. Nature 290: 527-528, 1981[ISI][Medline].

31.   Xu, J, Dodd R, Makino C, Simon M, Baylor D, and Chen J. Prolonged photoresponses in transgenic mouse rods lacking arrestin. Nature 389: 505-509, 1997[ISI][Medline].

32.   Yamaguchi, H, Kajita J, and Madison JM. Isoproterenol increases peripheral [Ca2+]i and decreases inner [Ca2+]i in single airway smooth muscle cells. Am J Physiol Cell Physiol 268: C771-C779, 1995[Abstract/Free Full Text].


Am J Physiol Cell Physiol 279(1):C274-C280
0363-6143/00 $5.00 Copyright © 2000 the American Physiological Society




This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Search for citing articles in:
ISI Web of Science (2)
Google Scholar
Articles by Krivoshik, A. P.
Articles by Barr, L.
Articles citing this Article
PubMed
PubMed Citation
Articles by Krivoshik, A. P.
Articles by Barr, L.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online