Control of uterine Ca by membrane potential: role of the T-type calcium channels.

Human parturition is one of the most important physiological processes ensuring continuation of mankind. The regulation of parturition is immensely complex and poorly understood. Powerful contractions of the myometrium (uterine smooth muscle) at the end of pregnancy are vital for the successful childbirth. During labour, coordinated cycles of myometrial contractions and relaxations expel the foetus and the placenta through the birth canal. Abnormal (premature or too weak) contractions of the myometrium may lead to serious and sometimes life threatening complications (e.g. preterm labour, uterine dystocia, etc). It is therefore important to elucidate the mechanisms responsible for the initiation and timing of uterine contractions. The contraction of any type of smooth muscle (including the myometrium) is triggered by an increase in the concentration of calcium ions in cytoplasm of the cell. This rise in cytoplasmic calcium, referred to as 'calcium signal', is thought to be responsible not only for triggering the contraction, but also for regulating many other intracellular regulatory pathways. Calcium ions enter the cytoplasm from outside the cell through specialised membrane proteins called voltage-dependent calcium channels. Previous studies on human myometrium have identified two classes of voltage-dependent calcium channels: high-voltage activated L-type and low-voltage activated T-type. Despite many years of research, the mechanisms of calcium signalling and especially, the role of T-type calcium channels are not fully understood. Considering the importance of calcium signalling for the initiation and regulation of myometrial contractions, this project will focus on the role of the T-type calcium channels in calcium signalling and electrogenesis of human uterine smooth muscle cells. Previous studies have found that the biophysical characteristics of the T-type calcium channels are such that they might be active at resting conditions (low membrane potential, relaxed cell). Our hypothesis is that the activity of the T-type calcium channels at resting conditions contributes to the accumulation of calcium ions by the intracellular organelle called sarcoplasmic reticulum (SR) and provides inward current for the initiation of the action potential (slow depolarisation). During activation of the myometrial cell (fast depolarisation phase of the action potential), a large inward calcium current through the L-type calcium channels triggers the SR calcium release into the bulk cytoplasm thereby initiating the contraction. We will test this hypothesis using a combination of modern experimental techniques applied to isolated human myometrial cells. Myometrial tissue will be obtained from women undergoing Caesarean section. Individual cells will be isolated from samples of human myometrium using established collagenase digestion method. Membrane potential and ionic currents will be recorded using a patch clamp technique. The cytoplasmic and SR calcium concentrations will be measured using digital imaging of voltage clamped cells loaded with calcium-sensitive indicator. We will investigate the contribution of the T-type and L-type calcium channels to the increase in cytoplasmic calcium concentration in response to voltage clamp pulses resembling the shape of the natural action potential of uterine smooth muscle cell (the action potential waveform voltage clamp). The data obtained in this study will help us understand how the uterine contractions are triggered, which will ultimately lead to a better understanding of the process of normal labour and delivery in women.

Control of uterine contractility is essential for the health of the foetus and successful childbirth. In recent years we have learnt much about the basic events underlying uterine contraction. Spontaneous myometrial contractions are of myogenic origin, i.e. neural or hormonal stimuli are not required for the contraction to occur but can be modulated by them. The mechanism of pacemaking in the myometrium has not been elucidated yet. Many spontaneously active cells (including human myometrium) express low voltage activated (T-type) Ca channels. The proposed study will characterise the distribution and biophysical properties of the human myometrial T-type Ca channels and elucidate their role in Ca signalling and pacemaking. Our hypothesis is that in human myometrium, a subset of uterine myocytes can perform the role of the pacemaker due to rhythmic releases of Ca from the SR and subsequent activation of Ca-dependent channels carrying inward current. The T-type Ca channels contribute to the regulation of the SR Ca by providing a steady-state influx of Ca necessary for the maintenance of the SR Ca content. This hypothesis will be tested by combining patch clamp and fast digital imaging of cytoplasmic and SR calcium in freshly isolated and cultured human myometrial cells. The results of this study will increase our understanding of normal and abnormal uterine Ca signalling and inform further research into the control of uterine contractility.