The relationship between the action potential, intracellular calcium and force in intact phasic, guinea-pig uretic smooth muscle.

1. We investigated the relationship between the action potential, Ca2+ and phasic force in intact guinea-pig ureter, following physiological activation. 2. The action potential elicited a Ca2+ transient consisting of three components: a fast increment, associated with the first action potential spike, a slower increment, associated with subsequent spikes and the initial part of the plateau component, and a steady-state phase associated with the plateau. 3. Prolongation of the plateau, by agonists, prolonged the third component of the Ca2+ transient and increased force amplitude and duration. 4. The force-Ca2+ relationship during phasic contractions showed hysteresis; more force was produced as Ca2+ declined than when it rose. Paired pulse stimuli suggested that the delay between Ca2+ and force was not due to mechanical properties. Wortmannin, which has been shown to selectively inhibit force and myosin light chain (MLC) phosphorylation in the guinea-pig ureter, did not affect electrical activity or Ca2+ but significantly increased the delay, suggesting that myosin phosphorylation is a major contributor to it. 5. Prolongation of the duration of the [Ca2+]i transient, at unchanged amplitude, increased force. The rise of [Ca2+]i did not limit the rate of contraction. Slowing of the rate of [Ca2+]i rise abolished the hysteresis between Ca2+ and force. 6. Cooling reduced force, increased the delay and hysteresis between Ca2+ and force, but did not affect the rate of rise of Ca2+. The reduction in force could be compensated, by increasing the duration of the Ca2+ transient. 7. We suggest that in vivo, steady-state force-Ca2+ relationships are not applicable in phasic smooth muscles. Furthermore, agonists increase force mainly by prolonging the action potential, which increases the duration of the [Ca2+] signal.

The effect of cyclopiazonic acid on excitation-contraction coupling in guinea-pig ureteric smooth muscle: role of the sarcoplasmic reticulum.

1. We have investigated the effect of cyclopiazonic acid (CPA), an inhibitor of the sarcoplasmic reticulum (SR) Ca2+-ATPase on excitation-contraction (EC) coupling in guinea-pig ureter, by measuring membrane currents, action potentials, intracellular [Ca2+] and force. 2. CPA (20 micrometers) significantly enhanced the amplitude and duration of phasic contractions of ureteric smooth muscle associated with action potentials. This was accompanied by an increase in the duration of the intracellular Ca2+ transient in intact tissue and single cells but not their amplitude. However, CPA also slowed the rate of rise, and fall, of the force 1|1|Phiand1Phi Ca2+ transients. 3. Membrane potential recordings showed that CPA produced a small depolarization and a large increase in the duration of the plateau phase of the action potential. 4. Patch-clamp studies showed marked inhibition of outward potassium current in the presence of CPA and an inhibition of spontaneous transient outward currents (STOCs). CPA had no effect on inward Ca2+ current. 5. These data suggest that the SR plays a major role in modulating the excitability of the ureter, particularly via curtailing the action potential duration. This in turn will shorten the Ca2+ transient and decrease force. This negative action on developed force predominates over any small role it may play in initiating force in the guinea-pig ureter.

The effect of inhibition of myosin light chain kinase by Wortmannin on intracellular [Ca2+], electrical activity and force in phasic smooth muscle.

To investigate the role of myosin light chain kinase (MLCK) in phasic contractions of intact smooth muscle, we have applied Wortmannin, an MLCK inhibitor, to strips of guinea-pig ureter. Simultaneous measurements of electrical activity, intracellular [Ca2+] ([Ca2+]i) and phasic force showed that Wortmannin (1-4 microM) abolishes force with little or no change in [Ca2+]i and electrical activity. High-K+-induced force production was also abolished by Wortmannin. The effects of Wortmannin were dose dependent - at lower concentrations (100 nM) Wortmannin reduced phasic contractility by 40-50%. It also significantly increased the delay between the Ca2+ peak and force production. These data show that, in phasic smooth muscle, inhibition of MLCK causes contraction to fail, despite normal electrical activity and Ca2+ transients. Our results also indicate that Wortmannin has no secondary effects and that other means of producing force, independent of myosin phosphorylation, are negligible in this tissue. The increased lag between the rise of Ca2+ and force production when MLCK is inhibited was surprising and suggests that post-phosphorylation steps may play a larger role in the delay than was previously considered.

The mechanism of Ca2+ release from the SR of permeabilised guinea-pig and rat ureteric smooth muscle.

Recent work has indicated that there is a major difference in the Ca2+ store of smooth muscle from rat and guinea-pig ureter; with the rat store being agonist-sensitive but ryanodine insensitive and the guinea-pig store being ryanodine sensitive but agonist insensitive [Th. V. Burdyga, M.J. Taggart, S. Wray, J. Physiol. 489 (1995) 327-335]. We have therefore examined directly the mechanism of Ca2+ release from the internal Ca2+ store (SR). Following permeabilisation with alpha-toxin or beta-escin the SR was Ca(2+)-loaded before application of carbachol or caffeine. Only carbachol evoked a transient contraction in rat ureter. The carbachol-induced contraction was blocked by heparin and cyclopiazonic acid (CPA) but not ryanodine. Only caffeine produced contraction in guinea-pig ureter, and this was blocked by ryanodine. Direct application of IP3 caused a small transient contraction in rat but not guinea-pig ureter. We conclude that rat ureter possesses only an IP3 sensitive store while guinea-pig ureter only has a ryanodine sensitive store.

Intracellular Na+ measurements in smooth muscle using SBFI--changes in [Na+], Ca2+ and force in normal and Na(+)-loaded ureter.

Our understanding of the control and effects of intracellular [Na+] ([Na+]i) in intact smooth muscle is limited by the lack of data concerning [Na+]i. The initial aim of this work was therefore to investigate the suitability of using the Na+-sensitive fluorophore SBFI in intact smooth muscle. We find this to be a good method for measuring [Na+]i in ureteric smooth muscle. Resting [Na+]i was found to be around 10 mM and rose to 25 mM when the Na+-K+-ATPase was inhibited by ouabain. This relatively low [Na+]i in the absence of Na+-K+-ATPase suggests that other cellular processes, such as Na+-Ca2+ exchange, play a role in maintaining [Na+]i under these conditions. Simultaneous measurements of [Na+]i or [Ca2+] i and force showed that Na+-Ca2+ exchange can play a functional role in ureteric smooth muscle. We found that the greater the driving force for Na+ exit and hence Ca2+ entry, the larger the contraction. In addition the Na+-Ca2+ exchanger activity under these conditions was found to be pH sensitive: acidification reduced the contraction and concomitant changes in [Ca2+] and [Na+]i. We conclude that SBFI is a useful method for monitoring [Na] in smooth muscle and that Na+-Ca2+ exchange may play a functional role in the ureter.

An investigation into the mechanism whereby pH affects tension in guinea-pig ureteric smooth muscle.

1. We have altered intracellular (pHi) and extracellular pH (pHo) in the smooth muscle of guinea-pig ureter and determined the effects on evoked phasic contractions. In order to investigate the mechanisms underlying the effects of pH alteration, intracellular Ca2+ ([Ca2+]i), pHi, electrical activity and force were measured. 2. Intracellular acidification, produced by the weak acid butyrate, application of CO2 at constant pHo or removal of weak bases, greatly increased phasic contractions. Alkalinization with weak bases or by removal of CO2 inhibited contractions. The results were similar whether Hepes or CO2-HCO3-buffered the solutions. 3. Phasic contractions were preceded by intracellular Ca2+ transients in the ureter. Acidification of the cytoplasm led to an increase in the amplitude of the Ca2+ transient, and alkalinization decreased its magnitude. 4. In the ureter the action potential leads to Ca2+ influx, therefore electrophysiological recordings of its configuration were made during alteration of pHi. Acidification led to the action potential duration and amplitude being increased, whereas alkalinization shortened the action potential and reduced its amplitude. 5. As the effects of acidification on the action potential resembled the effects of blocking of K+ channels, we investigated whether pHi alteration was able to alter tension when K+ channels were blocked by tetraethylammonium. Acidification was unable to potentiate force under these conditions nor did alkalinization decrease force. 6. External pH over the range 6.8-8.0 had little or no effect on pHi, phasic contractions and [Ca2+]i. Tonic contractions were enhanced, however, when pHo was increased. 7. These data suggest that pHi alteration in the guinea-pig ureter modulates the action potential, probably by alteration of K+ currents. Subsequent changes in [Ca2+]i and contraction then occur. A potentiating effect of acidic pH on force is not common in muscle, but may be a characteristic of the smooth muscle of the urinary tract. Changes of pHo had little effect on phasic force or pHi, but modulated tonic contractions. The possible physiological significance of these results is discussed.

Major difference between rat and guinea-pig ureter in the ability of agonists and caffeine to release Ca2+ and influence force.

1. We have investigated the internal Ca2+ store and its ability to affect contraction by simultaneously measuring force and Ca2+ in the ureter from guinea-pig and rat. Both species responded in a similar manner to electrical stimulation and depolarization with high-K+, generating plateau-type action potentials and increasing intracellular calcium ([Ca2+]i) and force. 2. In the guinea-pig, carbachol had no effect on [Ca2+]i and force in the resting ureter. In contrast, resting rat ureter always responded with a large [Ca2+]i rise and maintained force to carbachol in Ca(2+)-containing solution, and in Ca(2+)-free solution it showed a transient increase in [Ca2+]i and force. This Ca2+ release and force development was also present in both polarized and high-K(+)-depolarized preparations and was insensitive to nifedipine, suggesting the presence of a receptor-coupled pathway of Ca2+ release in rat ureter. 3. Caffeine was able to produce a release of Ca2+ from the internal store of guinea-pig ureter and elicit contraction. However, rat ureter failed to respond to caffeine. In the presence of La3+, the caffeine response in the guinea-pig ureter and carbachol response in the rat ureter, elicited in Ca(2+)-free solutions, were always increased and prolonged and could be repeatedly evoked, suggesting similarity in Ca2+ uptake behaviour of the store in both species. 4. Ryanodine blocked the caffeine responses of the guinea-pig ureter elicited both in Ca(2+)-containing and Ca(2+)-free solutions, both in the absence and presence of La3+. However, ryanodine failed to prevent the rat ureter responding to carbachol, suggesting that carbachol was releasing Ca2+ from a ryanodine-insensitive channel in the sarcoplasmic reticulum (SR). 5. Cyclopiazonic acid, which inhibits the SR Ca(2+)-ATPase, abolished the effects of both caffeine and carbachol in Ca(2+)-free solutions in guinea-pig and rat, respectively. 6. We conclude that there is a major difference in the mechanisms of Ca2+ release in the internal Ca2+ store of smooth muscle from guinea-pig and rat ureter. The data suggest that the guinea-pig store is purely a calcium-induced calcium release (CICR)-type store and that the rat store is a pure receptor-operated Ca2+ store.

Effects of temperature and Na0+ on the relaxation of phasic and tonic tension of guinea-pig ureter muscle.

Effects of temperature and Na0+ on the relaxation of guinea-pig ureter smooth muscle were studied. Relaxation of phasic contraction was found to be highly temperature-dependent, practically independent of Na0+ and Ca02+, and resistant to vanadate. The relaxation of the tonic tension of both high-K and low-Na contracture was less temperature-dependent and affected by Na0+. The relaxation of tonic tension produced by introduction of Na0+ was about 3-5 times faster than that produced by Ca-free solution. La3+ ions were found to block the relaxation of the tonic component of the Na+-free contracture initiated by removal of Ca02+. Three systems of regulation of cell calcium are suggested to be operative in the ureter muscle: a fast one which is highly temperature-dependent and responsible for the relaxation of the phasic contraction (probably the sarcoplasmic reticulum), and two slow membrane-linked carriers, one of which is dependent on Na0+ (probably Na-Ca exchange) and another one which is independent of Na0+ and inhibited by La3+ (probably Ca-pump).

Effects of caffeine on the electrical and mechanical activity of guinea-pig ureter smooth muscle.

The effects of caffeine on the electrical and mechanical activity of the guinea-pig ureter smooth muscle were studied. Under untreated conditions caffeine mainly showed inhibitory action on the ureter, inhibiting the evoked action potentials and phasic contractions as well as potassium contracture. Caffeine was also found to suppress the low-Na contracture of Na-loaded ureter muscle. It is established that Na-loaded tissue is able to generate transient contracture in response to caffeine application at 37 degrees C. These caffeine contractures could be evoked under completely removed [Ca2+]0 and in the presence of high doses of Ca-channel blockers (nifedipine, diltiazem, Mn ions) and could be reversibly blocked by tetracaine, procaine and benzocaine. Caffeine contractures could also be produced by the ureter muscle placed in isotonic K-solution. Cooling significantly potentiated low-Na, potassium and caffeine contractures of the ureter muscle. Filling of the store is totally dependent on the entry of Ca ions from the extracellular Ca2+ store sites which sequester Ca ions entering the cell on either Na-Ca exchange or via voltage operated Ca channels.

Evidence for sodium-calcium exchange in the guinea-pig ureter.

The effects of high-K and low-Na solutions on the smooth muscle of the guinea-pig ureter have been examined in both normal tissues, and tissues in which the Na pump had been blocked by exposure to K-free solutions or ouabain (high-Na tissues). Tension recording, membrane potential measurements and ion analysis were used. High-K solutions depolarize normal tissues, leading to action potential generation and phasic contractions followed, at concentrations greater than 20-30 mM, by cessation of action potentials and the development of a biphasic contracture which declines slowly during continuous exposure. The contracture is abolished by Ca-antagonist drugs, procaine and Ca-free solutions. Short exposures of normal tissues to Na-free solutions do not result in tension development. Longer exposures may initiate tension, depending on the Na substitute used. Sucrose causes depolarization of the cells and spike development associated with phasic contractions, superimposed on a small contracture; Li depolarizes the cells but causes no tension generation; Tris hyperpolarizes the cells and a small increase in basal tone may be seen. On exposure to K-free solutions or ouabain, the tissues do not develop significant tone but their response to short application of high-K solutions grows with time. The tissues also develop the ability to contract on short applications of low-Na solutions. The low-Na contractures are resistant to concentrations of Ca antagonists that abolish the K responses of normal tissues, but are abolished in Ca-free solutions. The ability of the tissues to contract in Na-free solutions is accompanied by an increase in intracellular Na and loss of intracellular K. Even after several hours' exposure to ouabain, however, the tissues still contain significant amounts of K and the membrane potential is the same as, or more negative than that in normal tissues. Therefore it appears that another mechanism, apart from the Na pump, can regulate intracellular Na. On continuous exposure to Na-free solutions, the contracture declines rapidly. The decline is associated with a loss of intracellular Na. The Na-free contracture is larger when K rather than Tris is used as the substitute. This difference persists in the presence of a concentration of Mn that abolishes the K contracture of normal tissues but is abolished by high concentrations (10 mM) of procaine.(ABSTRACT TRUNCATED AT 400 WORDS)

The effects of papaverine on the electrical and mechanical activity of the guinea-pig ureter.

1. The effects of papaverine (10(-5)-2 X 10(-4) M) were studied on the evoked electrical and mechanical activity of the guinea-pig ureter smooth muscle. In normal conditions the action potential consists of an initial spike followed by further spikes superimposed on a plateau phase. Papaverine reversibly decreased the duration of the plateau of the action potential, blocked the associated spikes, greatly reduced the amplitude of the contraction but enhanced the initial component of the action potential. 2. Papaverine did not change the membrane potential and had little effect on the membrane resistance. 3. Tetraethylammonium (5 mM), which blocks the delayed outward K current, did not prevent the decrease in the duration of the plateau nor the decrease of the contractile response caused by papaverine. 4. In Na-free solution the duration of the action potential was decreased until only a single spike was seen, due to suppression of the plateau. An effect of papaverine could not be observed under these conditions. 5. Mn2+ ions (1 mM) completely suppressed the spike component and tension while the plateau component was substantially increased. Papaverine in the presence of Mn2+ reversibly blocked the generation of the action potential. When Mn2+ ions were added to Na-free solution the duration as well as the amplitude of the spike was increased. Again, papaverine reversibly blocked the generation of the action potential. 6. Noradrenaline (10(-4) M) and histamine (10(-5) M) in normal conditions prolonged the duration of the action potential plateau and increased both the duration and amplitude of the concentration. Papaverine again blocked the plateau and greatly reduced the contractile response. 7. Papaverine caused the relaxation of KCl-induced contractures, preferentially blocking the tonic component. 8. It is suggested that the inhibitory action of papaverine on ureter smooth muscle results from its specific blockade of the 'slow' Na/Ca channels responsible for the generation of the plateau component of the action potential.