Theodor Burdyga

Vimentin-Positive, c-KIT-Negative Interstitial Cells in Human and Rat Uterus: A Role in Pacemaking?

Abstract The mechanism underlying spontaneous pacemaker potential in the uterus is not clearly understood. Several spontaneously active smooth muscles have interstitial cells of Cajal (ICCs) or ICC-like cells. We therefore examined cells from freshly dispersed uterine muscle strips (from pregnant human and rat myometrium) and in situ uterine preparations to determine the cell types present. Both preparations revealed numerous ICC-like cells; they were multipolar, with spider-like projections and enlarged central regions. These cells were readily distinguished from uterine myocytes by their morphology and ultrastructure, i.e., no myofilaments, numerous mitochondria, caveolae, and filaments. In addition, the ICC-like cells were noncontractile. These cells were negative to c-kit, a classic marker for ICCs. They stained positive for the intermediate filament, vimentin, a marker for cells of mesenchymal origin but not differentiated myocytes. The ICC-like cells had a more or less stable resting membrane potential of −58 ± 7 mV compared with smooth-muscle cells, −65 ± 13 mV, and produced outward current in response to voltage clamp pulses. However, in contrast with uterine myocytes, inward currents were not observed. This is the first description of ICC-like cells in myometrium and their role in the uterus is discussed, as possible inhibitors of intrinsic smooth-muscle activity.

The Physiological Basis of Uterine Contractility: A Short Review

In this review we discuss our current understanding of the cellular basis of uterine contractility, highlighting those areas requiring further study. It is clear that the basic processes of excitation‐contraction coupling lie within the myometrial cell, and that these may be modified by agonists. Pacemaker acitivity, however, remains a mystery. The contribution of extracellular calcium entry to contraction is shown to be vital, whilst the role of the sarcoplasmic reticulum remains controversial. Much current experimental focus is on pathways controlling and regulating contraction, and we discuss sensitisation mechanisms and question their role in intact uterine preparations.

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.

The effects of inhibiting Rho-associated kinase with Y-27632 on force and intracellular calcium in human myometrium

Abstract. Recent work has indicated that smooth muscle force production may be influenced by pathways not dependent upon the Ca2+-calmodulin phosphorylation of light chains. Few studies, however, have examined the importance of these pathways in intact muscles that contract phasically rather than tonically. Therefore, to determine whether the Ca2+-independent Rho-A and associated kinase (ROK) pathway can affect contractions of the intact human myometrium, we used Y-27632 to inhibit ROK. Three types of contractile activity were examined: spontaneous and those elicited by oxytocin and by depolarisation by high K+. Y-27632 decreased force significantly under all three conditions, without changing intracellular [Ca2+]. However, the effects on force were only large when the uterus was producing force tonically rather than phasically. This suggests that the Rho-A-ROK pathway may not be a potent modulator of force in the human myometrium under physiological conditions.

Action potential refractory period in ureter smooth muscle is set by Ca sparks and BK channels

In excitable tissues the refractory period is a critical control mechanism preventing hyperactivity and undesirable tetani, by preventing subsequent stimuli eliciting action potentials and Ca2+ entry. In ureteric smooth muscle, peristaltic waves that occur as invading pacemaker potentials produce long-lasting action potentials (300–800 ms) and extraordinarily long (more than 10 s) refractory periods, which prevent urine reflux and kidney damage. For smooth muscles neither the mechanisms underlying the refractory period nor the link between excitability and refractoriness are properly understood. Here we show that a negative feedback process, which depends on Ca2+ loading the sarcoplasmic reticulum (SR) during the action potential and on the subsequent activation of local releases of Ca2+ from the SR (sparks), stimulating plasmalemmal Ca2+-sensitive K+ (BK) channels, determines the refractory period of the action potential. As sparks gradually reduce the Ca2+ load in the SR, electrical inhibition is released, the refractory period is terminated and peristaltic contractions occur again. The refractory period can be manipulated, for example from 10 s to 100 s, by altering the Ca2+ content of the SR or release mechanism or by inhibiting BK channels. This insight into the control of excitability and hence function provides a focus for therapies directed at pathologies of smooth muscle.

Rho-kinase inhibition and electromechanical coupling in rat and guinea-pig ureter smooth muscle: Ca2+-dependent and -independent mechanisms

Recent data have shown Ca2+‐dependent activation of Rho‐kinase by sustained depolarization of arterial smooth muscle. Visceral smooth muscles, however, contract phasically in response to action potentials and it is unclear whether Ca2+‐dependent or ‐independent Rho‐kinase activation occurs. We have therefore investigated this, under physiologically relevant conditions, in intact ureter. Action potentials, ionic currents, Ca2+ transients, myosin light chain (MLC) phosphorylation and phasic contraction evoked by action potentials in guinea‐pig and rat ureter were investigated. In rat, but not guinea‐pig ureter, three Rho‐kinase inhibitors, Y‐27632, HA‐1077 and H‐1152, significantly decreased phasic contractions and Ca2+ transients. Voltage‐ and current‐clamp data showed that Rho‐kinase inhibition reduced the plateau component of the action potential, inhibited Ca2+‐channels and, indirectly, Ca2+‐activated Cl− channels. The Ca2+ channel agonist Bay K8644 could reverse these effects. The K+ channel blocker TEA could also reverse the inhibitory effect of Y‐27632 on the action potential and Ca2+ transient. Ca2+ transients and inward current, activated by carbachol‐induced sarcoplasmic reticulum Ca2+release, were not affected by Rho‐kinase inhibition. Rho‐kinase inhibition produced a Ca2+‐independent increase in the relaxation rate of contraction, associated with acceleration of MLC dephosphorylation, which was sensitive to calyculin A. These data show for the first time that: (1) Rho‐kinase has major effects on Ca2+ signalling associated with the action potential, (2) this effect is species dependent and (3) Rho‐kinase controls relaxation of phasic contraction of myogenic origin. Thus Rho‐kinase can modulate phasic smooth muscle in the absence of agonist, and the mechanisms are both Ca2+‐dependent, involving ion channels, and Ca2+‐independent, involving MLC phosphorylation activity.

Membrane cholesterol regulates smooth muscle phasic contraction.

The regulation of contractile activity in smooth muscle cells involves rapid discrimination and processing of a multitude of simultaneous signals impinging on the membrane before an integrated functional response can be generated. The sarcolemma of smooth muscle cells is segregated into caveolar regions-largely identical with cholesterol-rich membrane rafts—and actin-attachment sites, localized in non-raft, glycerophospholipid regions. Here we demonstrate that selective extraction of cholesterol abolishes membrane segregation and disassembles caveolae. Simultaneous measurements of force and [Ca2+]i in rat ureters demonstrated that extraction of cholesterol resulted in inhibition of both force and intracellular Ca2+ signals. Considering the major structural reorganization of cholesterol-depleted sarcolemma, it is intriguing to note that decreased levels of membrane cholesterol are accompanied by a highly specific inhibition of phasic, but not tonic contractions. This implies that signalling cascades that ultimately lead to either phasic or tonic response may be spatially segregated in the plane of the sarcolemma. Replenishment of cholesterol restores normal contractile behavior. In addition, the tissue function is re-established by inhibiting the large-conductance K+-channel. Sucrose gradient ultracentrifugation in combination with Western blotting analysis demonstrates that its α-subunit is associated with detergent-resistant membranes, suggesting that the channel might be localized within the membrane rafts in vivo. These findings are important in understanding the complex signalling pathways in smooth muscle and conditions such as premature labor and hypertension.

A review of recent insights into the role of the sarcoplasmic reticulum and Ca entry in uterine smooth muscle

The uterine sacroplasmic reticulum (SR) takes up and stores calcium [Ca], using an ATPase (SERCA) and the Ca-buffering proteins, calsequestrin and calreticulin. This stored Ca can be released via IP(3)-gated Ca channels. Decreases in luminal Ca concentration [Ca] have been directly measured following agonist stimulation. During spontaneous contractions however, there appears to be no involvement of the SR, as Ca entry and efflux across the plasma membrane account for these phasic contractions. After over-viewing current knowledge concerning SR structure and function, we highlight three areas of research which suggest new ways of looking at the role of the SR in the uterus, although they may be controversial or speculative at the moment. Firstly, we review the evidence for the function, if any, of Ca-induced SR Ca release channels, the ryanodine receptor (RyR) and the lack of Ca sparks (the elemental release events from RyRs), in the uterus. Secondly, we ask does regulation of SERCA by the accessory protein, phospholamban, occur in the uterus and what is the effect of knocking out phospholamban on uterine activity? Thirdly, we address the question of when and how store-operated Ca entry occurs in the myometrium. By analogy with other, usually less excitable tissues, is there a mechanism that links store Ca depletion to plasma membrane Ca entry in smooth muscle cells within intact uterus and is it physiologically relevant and regulated? Are the recently described proteins ORAI and STIM-1 involved in uterine store-operated Ca entry? We end the review by integrating these new insights with previous data to present a new working model of the SR in the uterus.

A new technique for simultaneous and in situ measurements of Ca2+ signals in arteriolar smooth muscle and endothelial cells

We report here the first local and global Ca(2+) measurements made from in situ terminal arterioles. The advantages of the method are that there is minimal disturbance to the vessels, which retain their relationship to the tissue they are supplying (rat ureter) and the small size of vessel that can be studied. Good loading with the Ca(2+) indicator, Fluo-4 was obtained, and confocal sectioning through the tissue enabled vascular smooth muscle and endothelial cells to be clearly seen, along with red blood cells, nerve endings and the ureteric smooth muscle cells. We find the terminal arterioles to be extremely active, both spontaneously and in response to nor-adrenaline stimulation, with Ca(2+) sparks occurring in the vascular myocytes and Ca(2+) puffs in the endothelial cells. Even under resting conditions, endothelial cells produced oscillations and waves, which could pass from cell to cell, whereas the vascular myocytes only produced waves in response to agonist stimulation, and with no increase in the frequency of Ca(2+) sparks, and no spread from cell to cell. We compare our data to those obtained in dissected intact vessels and single cells. We conclude that this approach is a convenient and useful method for studying inter- and intracellular Ca(2+) signalling events and communication between cell types, particularly in very small vessels.

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.