Agonist-induced activation of smooth muscle involves a rise in intracellular Ca2+ concentration and sensitization of myosin light chain phosphorylation to Ca2+. Sr2+ can enter through Ca2+channels, be sequestered and released from sarcoplasmic reticulum, and replace Ca2+ in activation of myosin light chain phosphorylation. Sr2+ cannot replace Ca2+ in facilitation of agonist-activated Ca2+-dependent nonselective cation channels. It is not known whether Sr2+can replace Ca2+ in small G protein-mediated sensitization of phosphorylation. To explore mechanisms involved in α-receptor-activated contractions in smooth muscle, effects of replacing Ca2+ with Sr2+ were examined in rat portal vein. Norepinephrine (NE) at >3.0 × 10−7 M in the presence of Ca2+ resulted in a strong sustained contraction, whereas this sustained component was absent in the presence of Sr2+; only the amplitude of phasic contractions increased. Pretreatment with low (∼0.05 mM) free Ca2+followed by 2.5 mM Sr2+ resulted in a sustained component of the NE response. In β-escin-permeabilized preparations, phenylephrine in the presence of GTP or guanosine 5′-O-(3-thiotriphosphate) alone induced sensitization to Sr2+. In conclusion, a Ca2+-regulated membrane/channel process is required for the sustained component of NE responses in rat portal vein. Sensitization alone is not responsible for the sustained phase of the NE contraction.
- vascular smooth muscle
- calcium sensitization
agonist stimulation of smooth muscle initiates a complex series of events leading to activation of the contractile proteins. For most agonists, this involves both membrane depolarization, with an associated influx of Ca2+, and activation of membrane potential-independent processes [i.e., the electromechanical and pharmacomechanical coupling pathways (38)]. The main cellular event in activation is a rise in free Ca2+ concentration ([Ca2+]), initiating myosin light chain phosphorylation (14). However, agonist stimulation of smooth muscle also involves a sensitization of the contractile process to Ca2+(2). The sensitization of the contractile proteins to Ca2+ was originally described in experiments on intact smooth muscle (12, 23, 25, 34), where force was correlated with measured levels of free Ca2+. The mechanisms of Ca2+ sensitization have been further examined in skinned preparations, where the free [Ca2+] can be controlled. Sensitization to Ca2+ has substantial effects on force at low free [Ca2+] (18) and has been suggested to strongly contribute to the tonic (or sustained) phase of agonist-induced contractions via small G protein-associated pathways (37). Consistent with this view, inhibition of G protein (rhoA)-mediated Ca2+ sensitization results in loss of sustained force development in the rabbit portal vein activated by norepinephrine (9) and in the guinea pig ileum activated by carbachol (23, 28). Recently, a Ca2+-independent kinase, associated with myofilaments, was found and postulated to contribute to sensitization and Ca2+-independent contraction (41). This raises the following question: Are mechanisms associated with sustained [Ca2+] increase required, or is the sensitization alone sufficient for activation of the sustained contraction phase in the intact smooth muscle?
Sr2+ can replace Ca2+ in many biological systems. In smooth muscle, Sr2+ can activate myosin light chain phosphorylation and contraction of chemically skinned muscle fibers (11, 13, 35, 36), although the concentration-force relationship is shifted toward higher concentrations in the presence of Sr2+ than in the presence of Ca2+. In intact muscle, Sr2+ can support spontaneous contractile activity and contractions induced by membrane depolarization (1, 11,40). These results are consistent with electrophysiological studies showing that Sr2+ can enter through voltage-gated L-type channels (10) and thus replace Ca2+ in the electromechanical coupling.
It has been shown that one event in pharmacomechanical coupling is nonfunctional in the presence of Sr2+; the agonist-activated nonselective cation channel is facilitated by Ca2+, but not by Sr2+ (15); and Ca2+, as well as the agonist, is required for the channel to open. When open, the channel conducts several cations, including Sr2+ and Ca2+. This channel has been shown to have a very high conductance (200 pS) in the smooth muscle of the rat portal vein (22). In this tissue, norepinephrine-induced contractions, in contrast to high-K+ contractions, are greatly attenuated if Sr2+ is substituted for Ca2+ (1). This attenuation can reflect the effects of Sr2+ on the channel (see above). It can also indicate that sensitization, the main process in the pharmacomechanical coupling, is not functional in the presence of Sr2+.
The aim of this study was to determine whether sensitization of the phosphorylation process is the only mechanism of physiological importance in control of the sustained phase of the agonist-induced contraction in intact tissue. We used the rat portal vein and examined norepinephrine-induced contractions in intact tissue in the presence of Sr2+ and Ca2+. To study the sensitization, we determined responses to guanosine 5′-O-(3-thiotriphosphate) (GTPγS) and norepinephrine/GTP in permeabilized preparations at fixed concentrations of Ca2+ and Sr2+.
Muscle fiber preparations.
Female Sprague-Dawley rats (150–200 g) were obtained from Möllegaard (M & B, Ry, Denmark). The animals were killed by cervical dislocation, and the portal veins were removed and dissected free of connective tissue.
Experiments on intact preparations.
The portal vein was opened longitudinally and used whole or split longitudinally into two equal parts. The strips, 7–9 mm long, were mounted in open organ baths at 37°C, using thin silk thread, between a stainless steel pin on an adjustable stand and a transducer (model FT03, Grass Instrument, Freeport, IL) for recording of isometric force. The preparations were equilibrated in physiological saline solution (PSS; see composition below) with 2.5 mM Ca2+ for 1 h at a passive tension corresponding to optimal length for active force (24). In each muscle preparation, a reference contraction was recorded after addition of 80 mM KCl from a 3 M stock solution to the PSS with 2.5 mM CaCl2. This activation method, which depolarizes in a nonisotonic solution, enables a fast change in KCl concentration without solution exchange and gives reproducible contractions. These contractions were stronger than those obtained using isosmolar substitution of Na+ for K+, probably due to cell swelling shown to occur after Na+/K+ isosmotic replacement (17). We used the plateau force of the 80 mM KCl in 2.5 mM CaCl2contractions as a reference for normalization of all later force responses.
We performed Sr2+ and Ca2+ experiments in varying order on the same fiber. Previous reports have shown that the sarcoplasmic reticulum is depleted after 5–6 min in Ca2+-free solution at 20°C (6). To ensure that all intracellular stores were emptied, preparations were kept in Ca2+/Sr2+-free PSS for 10 min, and thereafter the muscle was challenged three times with 10−5 M norepinephrine in this solution. The contractile responses were completely absent, showing that intracellular stores are emptied under these conditions.
The Ca2+ and Sr2+ dependence of force was examined at 60 and 128 mM K+ (isotonic replacement; see below). These experiments were performed in the presence of α- and β-blockers (10−6 M prazosin and 10−6 M propranolol). The muscle preparations were kept for 5 min in Ca2+/Sr2+-free PSS and then transferred to the respective high-K+ solution. CaCl2 or SrCl2 was added cumulatively at 5-min intervals, giving final [Ca2+] and Sr2+ concentration ([Sr2+]) of 0.1, 0.5, 1.0, 2.5, and 5.0 mM. Force was measured at the plateau of contraction at each [Ca2+]/[Sr2+] and expressed relative to the reference force.
Norepinephrine concentration-force relationships were examined in Ca2+ and Sr2+ solutions. The muscle preparations were held in PSS with 2.5 mM CaCl2 or SrCl2 and activated with different concentrations of norepinephrine. Force was recorded for 5–10 min, and the muscles were relaxed for 5 min in PSS between contractions. Peak and sustained force were measured and expressed relative to reference force.
The Ca2+ dependence of the sustained phase after norepinephrine-induced activation was examined using the following protocol. The preparations were equilibrated in Ca2+/Sr2+-free PSS with 1 mM EGTA for 10 min. Then EGTA concentration was decreased to 0.1 mM, and 10−5M norepinephrine was added. After 30 s, 2.5 mM SrCl2was added. One set of experiments was performed at a low free [Ca2+] (0.15 mM CaCl2 added to the 0.1 mM EGTA solution) and one set at zero free [Ca2+] (no CaCl2 added). The free [Ca2+] at 0.15 CaCl2 and 0.1 EGTA is estimated to be 50 μM. This would change to a maximum of 55 μM when 2.5 mM SrCl2 was added, since the binding constant for Sr2+ to EGTA is low (11). As a control experiment to determine the force responses at maximal [Ca2+], the procedure described above was performed with 2.5 mM CaCl2, instead of 2.5 mM SrCl2.
Experiments on β-escin-permeabilized preparations.
The portal vein was prepared as described above, and then four to six 5- to 7-mm-long longitudinal strips were cut from each vein. The strips were mounted horizontally on small metal clips between the extended arm of a force transducer (Scientific Instruments, Heidelberg, Germany) and a steel pin, enabling length adjustment. The experiments were carried out at ambient temperature (∼23°C) in 1-ml cups. The strips were equilibrated for ∼45 min in HEPES-buffered PSS (see below) before a norepinephrine (10−5 M) test contraction was made. The preparations were chemically permeabilized using β-escin essentially as described previously (19, 28). The strips were thoroughly relaxed in Ca2+-free HEPES-buffered PSS for 10 min. After the preparations were transferred to relaxing solution for 5 min, they were treated with relaxing solution plus 50 μM β-escin for 35 min. Finally, the preparations were kept for 10 min in β-escin-free relaxing solution.
After the β-escin skinning procedure, a reference contraction was made in the contracting solution (pCa 4.9). Each strip was placed in relaxing solution for 10 min before being contracted at intermediate [Ca2+] or [Sr2+]. Each strip was then placed in a solution of the same composition containing, in addition, 100 μM GTPγS. We evaluated force at intermediate [Ca2+] or [Sr2+] without and with GTPγS and expressed the values relative to force of the reference contraction at pCa 4.9. In separate experiments, the muscles were transferred to a relaxing solution containing 100 μM GTP after the initial reference contraction. A contraction was then elicited at intermediate [Ca2+] or [Sr2+] in the presence of GTP. At the plateau of contraction, 10−4 M phenylephrine was added. The increase in force induced by phenylephrine was expressed relative to force of the reference contraction at pCa 4.9.
Solutions for intact preparations.
The PSS contained (in mM) 122 NaCl, 4.7 KCl, 1.2 MgCl2, 25 NaHCO3, 1.2 KH2PO4, and 11.5 glucose. Ca2+- and Sr2+-containing solutions were made by addition of CaCl2 or SrCl2, respectively. High-K+ solutions, for K+dependence experiments, were made by isosmotic substitution of K+ for Na+. Two different isosmolar high-K+ solutions, with 60 and 128 mM K+, were used. The reference contraction in 80 mM K+ was made by addition of KCl to the PSS with 2.5 mM CaCl2. All solutions were continuously gassed with 95% O2-5% CO2during the experiments, giving a pH of 7.4 at 37°C.
Solutions for chemically permeabilized preparations.
The HEPES-buffered PSS contained (in mM) 118 NaCl, 5 KCl, 1.2 Na2HPO4, 1.2 MgCl2, 1.6 CaCl2, 24 HEPES, and 10 glucose, with pH adjusted to 7.4. The Ca2+-free PSS had the same composition, except CaCl2 was replaced by 2 mM EGTA. Double-concentrated solutions were used for skinned preparations and were stored at −20°C until use. The relaxing solution contained (in mM) 20 imidazole, 7.5 Na2ATP, 10 EGTA, 10 magnesium acetate, 10 phosphocreatine, 2 dithioerythritol, 5 NaN3, 0.001 leupeptin, and 0.0015 calmodulin. Ionic strength was adjusted to 150 mM with potassium methanesulfonate, and pH was adjusted to 7.0 at room temperature. The Sr2+ and Ca2+ contraction solutions were made by addition of 10 mM CaCl2 or, alternatively, 10 mM SrCl2. Solutions with intermediate free [Sr2+] and [Ca2+] were made by mixing the relaxing and contracting solutions to achieve different ratios of total SrCl2/CaCl2 to EGTA. Free [Ca2+] and [Sr2+] were calculated as described previously (8) using the apparent binding constants for Ca2+ and Sr2+ to EGTA provided elsewhere (11).
Values are means ± SE, with the number of observations in parentheses.
Spontaneous contractile activity and high-K+-induced force in Ca2+ and Sr2+ solutions in intact muscle.
All effects of Sr2+ and Ca2+ in intact muscle were fully reversible and independent of whether Sr2+experiments were performed before or after the Ca2+controls. The rat portal vein exhibited spontaneous contractile activity in PSS with 2.5 mM CaCl2. In PSS containing 2.5 mM SrCl2, the spontaneous contractile activity was also present, although with a slightly more regular pattern (Fig.1, left). In SrCl2, the frequency of contractions was 1.0 ± 0.1 min−1 and the force amplitude was 69 ± 6% (n = 6) of the “reference force” (seemethods) in response to activation with 80 mM K+ in 2.5 mM CaCl2. The corresponding values for spontaneous activity in CaCl2 were 2.2 ± 0.3 min−1 and 54 ± 10% (n = 6).
Activation with 80 mM K+ resulted in sustained contractions in CaCl2 and SrCl2 (Fig. 1, left andmiddle). The force induced by 80 mM K+ was lower in the presence of SrCl2 (50 ± 3% of the response in CaCl2, n = 8), and the effects of Ca2+/Sr2+ substitutions were reversible.
We determined the active force in 2.5 mM Ca2+ and Sr2+ at 60 and 128 mM K+. Preliminary experiments (data not shown) revealed that the active force in 2.5 mM Ca2+ was maximal at 60 mM KCl and decreased at higher concentrations. In 2.5 mM Sr2+, force increased gradually at increasing KCl and was maximal at 128 mM KCl. To characterize the depolarization-induced contraction in the presence of Sr2+and Ca2+, we compared Ca2+ and Sr2+dependencies of force at 60 and 128 mM KCl in the presence of α- and β-blockers (Fig. 2). The magnitude of depolarization-induced force responses at optimal KCl is similar in the presence of Sr2+ and Ca2+.
Norepinephrine-induced force responses in the presence of Ca2+ and Sr2+ in intact muscle.
In the presence of Ca2+, 10−8–10−7 M norepinephrine increased the amplitude and frequency of the spontaneous activity. After activation at higher norepinephrine concentrations (above ∼3.0 × 10−7 M), a sustained component appeared. If Sr2+ was substituted for Ca2+, responses at lower norepinephrine concentrations were essentially unaltered. However, no sustained component appeared at high norepinephrine concentrations; instead, the amplitude and frequency of the phasic contractions increased (Fig. 1, right). In experiments not shown here, we increased the SrCl2 concentration to 5 and 10 mM and observed no sustained contraction. In Fig.3, summarizing the results at 2.5 mM CaCl2 and SrCl2, the amplitude of the phasic (i.e., baseline to peak force) and sustained (i.e., baseline to sustained force) components are plotted against the norepinephrine concentration. The phasic component reached half-maximal amplitude at ∼3 × 10−8 M norepinephrine in the presence of Ca2+ and at 1 × 10−7 M norepinephrine in the presence of Sr2+. The sustained component reached half-maximal amplitude at ∼1 × 10−6 M norepinephrine in the presence of Ca2+. It was absent in the presence of Sr2+.
The apparent rate constant of relaxation (determined as the natural logarithm of 2 divided by the time to relax to 50% of peak) was determined during spontaneous contractile activity in PSS. The rate of relaxation of the spontaneous contractions was slightly, although not significantly, faster in the presence of Ca2+ than in the presence of Sr2+: 0.50 ± 0.05 (n = 12) and 0.36 ± 0.03 s−1 (n = 6), respectively. In the presence of Sr2+, the rate of relaxation of the phasic contractions at 10−5 M norepinephrine was lowered to 25% of that during spontaneous activity (0.0825 ± 0.007 s−1, n = 12,P < 0.001 compared with spontaneous activity in Ca2+ or Sr2+), whereas frequency and amplitude were increased (Figs. 1 and 3).
Ca2+ dependence of the sustained phase during norepinephrine-induced contraction in intact muscle.
The experiments described above to study activation with norepinephrine were performed in the continuous presence of Sr2+ or Ca2+. In an attempt to provoke a sustained contraction phase in the presence of Sr2+, we added 2.5 mM Ca2+ 3 min after activation with 10−5 M norepinephrine in 2.5 mM Sr2+. This induced irregular contractions but without a sustained phase. This suggests a competition between Sr2+ and Ca2+ in a step involved in the activation of the sustained norepinephrine force. To overcome this interaction but still examine the Ca2+ dependence of the sustained contraction, we performed experiments where the muscle preparation initially was depleted of Ca2+ and Sr2+ using 1 mM EGTA for 10 min. Thereafter, the muscle was exposed to a low concentration of EGTA (0.1 mM) and challenged with 10−5 M norepinephrine. This did not cause a contraction. If 2.5 mM Sr2+ was introduced, phasic contractions without any sustained force gradually developed in a manner similar to that described above (Fig. 4,left). If, instead, 2.5 mM Ca2+ was used, a sustained contraction developed rapidly (Fig. 4, right). In the presence of low [Ca2+] (0.1 mM EGTA and 0.15 mM CaCl2), norepinephrine did not cause a contraction, showing that this low free [Ca2+] (∼50 μM) did not support contraction by itself (Fig. 4, middle). If 2.5 mM Sr2+ was added in this situation (Fig. 4,middle), a strong contraction developed and was sustained for ≥1 min. Thereafter, the sustained force returned to baseline and a phasic contraction pattern, similar to that observed in the presence of Sr2+ alone, appeared. These results show that a very low free [Ca2+], which by itself cannot support contraction, can act as a switch permitting a sustained phase in combination with Sr2+. In addition, Sr2+ appears to compete with Ca2+ and inhibit this process.
Ca2+ and Sr2+ dependence of G protein-mediated sensitization of contraction in β-escin-permeabilized preparations.
To examine whether the lack of sustained force in the norepinephrine-induced contraction in the presence of Sr2+was due to a failure of G protein-mediated sensitization of the contractile machinery, we performed experiments on β-escin-permeabilized portal veins. Figure5, top, shows contractile responses to Ca2+ and Sr2+ in a thin permeabilized preparation of a portal vein. At intermediate [Sr2+], addition of 100 μM GTPγS induced a pronounced increase in force. Transfer to saturating [Sr2+] resulted in a force equaling that induced at maximal [Ca2+]. Similar sensitization effects of GTPγS were observed at intermediate [Ca2+]. Figure 5, bottom, summarizes the data from permeabilized muscles. The force responses were dependent on free [Ca2+] and [Sr2+], and GTPγS caused a leftward shift (∼0.2 pCa and 0.3 pSr unit) of the Ca2+- and Sr2+-force relationship, i.e., sensitization. To examine whether the receptor-mediated responses were present in Sr2+-containing solution, we examined the phenylephrine-induced increase in force in the presence of GTP in β-escin-skinned preparations. The muscles were activated with Ca2+ or Sr2+ in the presence of GTP to give ∼30% of maximal Ca2+-induced force: 29 ± 7% (n = 8) and 32 ± 7% (n = 6) in the presence of Ca2+ and Sr2+, respectively. In the presence of Ca2+ and Sr2+, addition of 10−4 M phenylephrine resulted in an increase in force [14 ± 4% (n = 8) and 21 ± 3% (n = 6) in Ca2+ and Sr2+, respectively], showing that receptor-coupled pathways are functional in the presence of Ca2+ and Sr2+.
A rise in intracellular free [Ca2+] and sensitization of the contractile machinery to Ca2+ are key features in receptor-mediated agonist activation of smooth muscle (27). Clear evidence has been presented that the sustained phase of responses to norepinephrine and carbachol is influenced by sensitization (18, 23, 28). We show here that the sustained phase of norepinephrine-induced contraction in a vascular smooth muscle is dependent also on Ca2+-specific regulation of a membrane-associated process and that the agonist-induced sensitization of the contractile proteins is not the only mechanism determining the sustained phase.
The sustained phase of norepinephrine-induced contraction was selectively removed when Sr2+ was substituted for Ca2+. In the presence of Ca2+, the peak and sustained components of the norepinephrine contraction in the portal vein had different norepinephrine dependencies (cf. Fig. 2). On replacement of Ca2+ with Sr2+, the phasic responses at low norepinephrine concentration were similar. Although the concentration-response relationship of the phasic component was shifted slightly toward higher norepinephrine concentrations in the presence of Sr2+, the response at saturating norepinephrine concentration reached the same amplitude as in the presence of Ca2+. In contrast, no sustained response appeared, even at very high norepinephrine concentrations (≤3 × 10−5M). Thus the lack of sustained contraction in the presence of Sr2+ is not due to a shift in the norepinephrine dose-response relationship but, rather, reflects a failure in the adrenergic activation pathway in the presence of Sr2+.
Norepinephrine-induced activation involves Ca2+ release from the sarcoplasmic reticulum (30). The role of the released Ca2+ is dual: to activate the contractile machinery directly and to regulate Ca2+-sensitive channel activity, which alters the membrane potential and Ca2+channel influx (16, 27, 31). The sarcoplasmic reticulum in phasic muscle, such as the portal vein, is poorly developed (7). Ca2+ release from the sarcoplasmic reticulum primarily contributes to the fast initial phase of contraction (30). Sr2+ can be sequestered by the sarcoplasmic reticulum (33, 39) in smooth muscle and released (4). Sr2+ can induce Ca2+release from the sarcoplasmic reticulum (10). These results suggest that impaired release from the sarcoplasmic reticulum is not responsible for the loss of sustained contraction in the presence of Sr2+.
Previous studies on chemically permeabilized smooth muscle have shown that Sr2+ can activate the contractile proteins and induce myosin light chain phosphorylation (11, 13, 36), although the concentration dependence is shifted toward higher concentrations in the presence of Sr2+ than in the presence of Ca2+. Our data from the β-escin-treated portal veins are consistent with these results. Because [Ca2+] and [Sr2+] dependencies of force were similar in high-K+-activated muscle and because spontaneous activity was present in Sr2+-containing PSS, depolarization of the membrane can lead to sufficient activation of L-type channels, ion influx, and activation of the contractile machinery in the presence of Sr2+ and Ca2+. This is consistent with previous results from K+-depolarized swine carotid media (11) showing that Sr2+ can substitute for Ca2+ in the activation of intact smooth muscle. The phosphorylation-stress relationships of depolarized intact smooth muscle have been shown to be similar in the presence of Sr2+ and Ca2+ (11). These results, together with our finding that the spontaneous activity (Fig. 1) and high-K+ induced force (Fig. 2) were of high amplitude in the presence of Sr2+, show that the level of phosphorylation can reach the same levels in the presence of Sr2+ and Ca2+. The presence of a very small [Ca2+] can activate the sustained phase in the presence of Sr2+ (Fig. 4). We find that the effect of Ca2+ in the presence of Sr2+ depended on the order in which the ions were introduced; adding Ca2+, even at 2.5 mM, after Sr2+ did not result in a sustained phase. In addition, the increase in [Ca2+] that resulted in a sustained phase in the presence of Sr2+ (Fig. 4) was very low, on the order of 50 μM, which, by itself, was too low to induce force, even in fully depolarized muscle (Fig. 2). We can therefore exclude the possibility that a difference in concentration dependence for activation of the contractile proteins between Sr2+ and Ca2+ is responsible for the lack of sustained force after norepinephrine activation in the presence of Sr2+. Instead, we propose that Ca2+ has a switchlike action at the membrane level, allowing [Sr2+] to be high for a sufficient amount of time to support a sustained phase.
We show, using GTPγS and GTP/phenylephrine in permeabilized preparations, that the receptor-coupled G protein-mediated sensitization pathway is present in Sr2+-containing solutions. The increase in force of the phasic component (cf. Figs. 2and 3) and the slowed relaxation after activation with norepinephrine in the presence of Sr2+ are consistent with an inhibition of the phosphatase. Sensitization via G protein-mediated inhibition of the phosphatase is an important mechanism after receptor activation (20, 28). The sensitization is considered a major mechanism for the induction of the sustained phase (21,37). Theoretically, it is possible that norepinephrine-induced sustained force could be the result of the inhibition of the myosin light chain phosphatase alone, independently of an increase in free Ca2+. If a constitutively active Ca2+-independent light chain kinase is present, inhibition of the phosphatase alone would be sufficient. In a recent study (41), a Ca2+-independent light chain kinase, distinct from the myosin light chain kinase, was demonstrated in rat caudal arterial smooth muscle strips and shown to be associated with myofilaments in isolated chicken gizzard. It was suggested that this kinase could have a role in Ca2+ sensitization and Ca2+-independent contraction of smooth muscle in response to stimuli that act via Ca2+-independent pathways and inhibition of the phosphatase. If this kinase is operating in the rat portal vein, we can exclude the possibility that it has any physiological significance in preserving the sustained phase in norepinephrine-induced contraction, since we can induce G protein-mediated sensitization without eliciting a sustained component in the presence of Sr2+.
Because the responses of the permeabilized preparations were similar in the presence of Ca2+ and Sr2+, the lack of a sustained phase in the presence of Sr2+ must be attributed to a membrane-associated mechanism. Two Ca2+-regulated channels have been suggested to contribute to membrane depolarization after norepinephrine activation in the portal vein (5): the agonist-activated, Ca2+-facilitated nonspecific cation channel and the Ca2+-activated Cl− channel. As a consequence of the depolarization, L-type channels would open and permit a large inflow of Ca2+. A failure to open the agonist-activated nonselective Ca2+-facilitated cation channel and/or the Ca2+-activated Cl− channel could be responsible for the lack of a sustained phase after norepinephrine activation in the presence of Sr2+.
The nonselective Ca2+-facilitated cation channel is sensitive to intracellular Ca2+ and possibly also to external Ca2+ (5). That the agonist-activated nonselective Ca2+-facilitated cation channel is Ca2+ specific [i.e., could not be facilitated by Sr2+ (15, 29)], is active in the right time scale for the sustained phase (29), and has a substantial conductance (200 pS) in the portal vein (22) suggests that this channel may be important for the sustained phase of the norepinephrine-induced contraction in the intact portal vein. The sustained phase after activation with norepinephrine is mainly dependent on prolonged depolarization activating the L-type Ca2+ channels (26). This depolarization is postulated to be maintained primarily by the agonist-activated cation channel, possibly in cooperation with the Ca2+-activated Cl− channel (32).
In conclusion, norepinephrine causes sensitization of the myosin phosphorylation in the presence of Ca2+ and Sr2+. The sensitization, via small G proteins and inhibition of phosphatase, is activated without the appearance of a sustained contraction in the presence of Sr2+. Our interpretation of the lack of sustained phase is that the activities of myosin light chain kinase and/or other myosin phosphorylating kinases are insufficient to reach the contractile threshold of light chain phosphorylation, even in the sensitized myofilaments. The results show that sensitization is not sufficient for the sustained phase of agonist-induced contraction.
This study was supported by Swedish Medical Research Council Grants 04x-8248 and 04x-12584 (A. Arner and U. Malmqvist) and Deutsche Forschungsgemeinschaft Grant Pf 226/4-2 (G. Pfitzer). Collaboration between Lund University and the University of Köln was supported by grants from the Deutsche Akademische Austauchdienst. H. Nilsson is a Nordisk Forskerutdanningsakademi guest professor at Lund University.
Part of these results has been presented in preliminary form (3).
Address for reprint requests and other correspondence: A. Arner, Dept. of Physiological Sciences, Lund University, BMC F11, Tornavägen 10, SE-221 84 Lund, Sweden (E-mail:).
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- Copyright © 2002 the American Physiological Society