Autoregulation and local control theory Flashcards
What drugs can modulate Ca regulation?
Caffeine and tetracaine
What does caffeine do?
• Caffeine, which is known to increase the sensitivity of RyR2 to activating Ca2+.
What does tetracaine do?
• Tetracaine, which decreases the sensitivity of the RyR to activating Ca2+.
Describe the effect of caffeine on the regulation of calcium
Addition of caffeine increases the sensitivity of the RyRs to activating Ca2+, the Ca2+ transient increases.
However, competition between SERCA and Na/Ca exchange means that more Ca2+ is then removed from the cell during each release
This results in a progressive decrease in the SR Ca2+ content, and therefore the amount of Ca2+ released, until the transient returns to control levels
In this new steady state, the same Ca2+ transient amplitude is achieved (despite a lower SR Ca2+ content) by an increase in fractional release. i.e. a greater fraction of the total SR Ca2+ content is released during each activation.
What effect does tetracaine have?
The opposite
Decreased Po of RyR channels leads to less Ca2+ transient
Less removal of Ca from the cell via the NCX
Leads to progressive increase in SR Ca content and therefore the amount of Ca released until transient returns to control levels
In new steady state, same Ca tranient amplitude with a decrease in fractonal release
What evidence is there for the use of tetracaine on Ca transient?
What happens upon adding and removing tetracaine?
What happens to the L-type channel and NCX current? Why?
What does tetracaine cause?
Is the response steady state or transient?
What is the limitation with the method?
Overend et al., 1998
In rat ventricular myocytes, perforated patch clamped cells using Amphotericin B.
Small transient increase in the integral of the L-type Ca2+ current and a larger transient decrease of the Na+-Ca2+exchange current on repolarization. These are attributed to decreased systolic release of Ca2+. On adding tetracaine, efflux was transiently less than influx.
Tetracaine produced an initial decrease of contraction before a recovery towards the control level. Removal of tetracaine produced a transient overshoot of contraction to levels greater than the control.
There is no steady-state effect on the magnitude of the systolic Ca2+ transient.
SR Ca2+ content calculated by integrated current records which is rather indirect and makes no allowance for changes between pulses.
What is the local control theory?
Once started, interrupting Ca2+ influx via ICa can lead to termination of SR Ca2+ release. The discovery of Ca2+ sparks led to “local control” theories
What evidence is there for the local control theory?
Wier et al., 1994
Single rat ventricular cells underwent whole cell patch clamp and calcium transient was calculated from the fluorescence of Indo-1.
The rising Ca transients could be interrupted by stopping Ca influx rapidly by increasing/decreasing the voltage clamp to potentials which inactivates the L-type channel.
This suggests that SR calcium release is controlled locally by Ca influx via the L-type channel and neighbouring RyR channels.
What does the evidence by Wier et al., 1994 contradict?
What is the limtation?
Rios & Pizarro, (1989)
Found SR release was directly activated by depolarisation in amphibian skeletal muscle cells. The difference in findings could be the different voltage dependence in ECC in the different species tested.
It was studied in cells without Na which can lead to a highly loaded SR and unstable Ca waves
To combat this, they excluded any cells exhibiting waves – can they be sure to exclude every one?
What are calcium sparks?
The Ca spark is observed in mature heart cells and is a local increase in Ca transient.
It arises as a cluster of RyRs are activated by [Ca2+]i.
When the AP occurs, the L-type Ca2+ channels are gated open by the depolarization and permit a large local increase in the [Ca2+]i signal between SR and T tubule and the spark rate increases by 103 fold.
What is the evidence for Calcium sparks?
Cheng et al., 1996
Confocal microscopy and fluorescent calcium imaging detected spontaneous local increases in the concentration of intracellular calcium, called “calcium sparks,” in rat heart cells.
Spontaneous Ca2+ sparks propagate Ca waves in CICR Becomes more sensitive to an increase in ICa2+]i during Ca2+ overload and depends on the close anatomical proximity of the sarcolemmal Ca2+ channels
One problem with confocal imaging method described here was that it only views approximately 10% of the RyR receptors which leads to a large assumption for the whole cell. A greater resolution would give the study more weight.
What do calcium sparks occur? What do they reflect?
What does the calcium transient actually reflect and when?
Ca2+ sparks occur spontaneously in intact cells.
ʀeflects the spontaneous firing of a ‘cluster’ of RyRs (perhaps 20-30) present in the junctional region of the SR, which faces the t-tubule (and the L-Type Ca2+ channels)
More importantly, if Ca2+ influx is reduced it can be seen that the spatially averaged Ca2+ transient actually reflects the summation of numerous, localised Ca2+ sparks, each triggered directly by Ca2+ influx via opposing L-type Ca2+ channels in the t-tubule membrane
What evidence is there for calcium sparks and how are they linked to L type calcium current?
Is a single channel sufficient to trigger SR release?
Collier et al., 1999
Single rat ventricular myocytes were voltage clamped with patch pipettes.
Confocal microscopy produced line scan images which showed individual Ca2+ sparks by reducing Ca2+ current density with nifedipine.
The time course of ICa is similar to the rate of occurrence of Ca2+ sparks during a voltage-clamp depolarization when the probability of Ca2+ spark occurrence is low. Suggests that the opening of a single L-type Ca2+ channel is sufficient to trigger localized SR Ca2+ release at all membrane potentials.
ʟopez ʟopez et al. (1995) reported similar results, except that ICa was not measured in their experiments which gives them less weight to draw conclusions.
What happens when you increase Ical?
Under normal physiological conditions, what is the sensitivity of the RyR receptors?
An increase in ICa,L will recruit more Ca2+ sparks, increasing the amplitude of the spatially averaged Ca2+ transient
The anatomical positioning of the L-type Ca2+ channel and the RyRs in the junctional face of the SR is, therefore, a critical factor, which allows CICR to be a stable process, smoothly graded in response to changes in ICa
Under normal physiological conditions, the sensitivity of the RyRs is such that release does not spread among neighbouring clusters.
Does autoregulation affect sparks?
Autoregulation of Ca2+ spark frequency can be explained by the interdependent effects of modulators, which bind to cytosolic sites on the RyR and feedback from luminal Ca2+ on the RyR
