Having left the field a while ago, for reasons that I won’t go into, suffice it to say I no longer have access to the relevant literature, I’ve been drawn quite by accident to consider the recent proposal that the inhibitory junction potential (IJP) recorded in the gastrointestinal smooth muscle has its origin in cells that are not smooth muscle in nature. This challenges the accepted wisdom that IJPs, or more specifically, “fast” IJPs, result from an increase in potassium conductance in smooth muscle cells bearing receptors for ATP released at en passant synapses with enteric inhibitory motor nerves coursing through the muscle layer, and that the attendant hyperpolarization of the membrane potential of the affected smooth muscle cells spreads electrotonically to other coupled smooth muscle cells in the bundle, opposing any concomitant depolarizing inputs and force generation. The new scheme proposed by Sanders et al. posits that non-smooth cells (PDGFα positive) in fact are the transducers of the ATP induced hyperpolarization which is transmitted electrotonically to coupled smooth muscle cells via gap junctions.

The attractiveness of this proposal is that it compartmentalizes the response and obviates the apparent paradox that an inhibitory neurotransmitter that induces muscle relaxation by stimulating the release of stored calcium inside smooth muscle cells to activate a potassium conductance, does so without simultaneously activating contractile proteins. Although this mechanism can be accommodated within the existing framework involving only smooth muscle cells and enteric inhibitory neurons by invoking localized calcium domains affecting membrane channels and not contractile proteins, and different calcium release mechanisms and coupling to calcium entry, the compartmentalization of the electrical component of the inhibitory response effectively insulates the smooth muscle cells from any possible contrary effects of calcium spill over onto the contractile apparatus.

One caveat that comes to mind, however, that may argue against an intermediary cell relay between the inhibitory nerves and smooth muscle cells is the fact that in many types of cells, notably cardiac cells that are also coupled electrically via gap junctions, the increase in intracellular calcium that precedes contraction has the effect of drastically reducing the conductance of gap junction channels (connexins). Therefore, if in the new scheme proposed by Sanders et al. the transducing element for the IJP is the PDGFa positive cell which is electrically coupled to smooth muscle cells, then any increase in intracellular calcium in the former upon activation of P2 purinoreceptors by nerve-released ATP or related molecules, to induce a robust hyperpolarization by activating SK channels; this raises the question of what fraction of the hyperpolarization in the PDGFα positive cells is transmitted to the smooth muscle if the conductance of gap junction channels is blocked or substantially diminished by the rise in intracellular calcium?

In the defence of this new proposal, however, the gap junctional conductance between the PDGFα positive cells and smooth muscle may not be entirely blocked during the rise in intracellular calcium, and although the intercellular resistance may be increased, it may still be sufficiently low for a significant fraction of the hyperpolarization in the PDGFα positive cells to spread to the smooth muscle and hyperpolarize it. In this regard, it has been shown by me that in electrically coupled supporting cells in the olfactory epithelium that when neighboring cells are stimulated with ATP to activate BK channels via intracellular release of calcium, a transjunctional current can be still be recorded from the patch-clamped supporting cell, although the latter needs to be dialyzed internally with an unnaturally high concentration of calcium buffer to prevent a rise in calcium, and has multiple inputs as far as electrotonically conducted events are concerned from the many surrounding supporting cells to which it’s coupled. Thus it remains to be seen whether in the case of PDGFα positive cells coupled to smooth muscle cells, to what extent a rise in intracellular calcium in the former decreases trans-junctional resistance at a time when current flow needs to be uncompromised for the hyperpolarization to spread with minimal decrement to the smooth muscle cells.

So much for electrical coupling between PDGFα positive cells and smooth muscle cells and the role of the former as the effector cell for the IJP response recorded in the smooth muscle. But another issue raised by this new proposal is the role of purinoceptors and SK channels in smooth muscle cells themselves, and the extent of their contribution to the generation of the IJP. Both P2 purinoceptors and apamin-sensitive SK channels are found in smooth muscle cells of the circular muscle layer in a smooth muscle tissue known to generate IJPs, that is, the mouse ileum. But the question is, are they expressed in sufficient abundance, if not to generate the IJP, then to contribute to it? If they were expressed at a sufficient density in smooth muscle cells to generate the IJP then it would seem that the hyperpolarization generated by PDGFα positive cells may not be necessary and could simply be an epiphenomenon that occurs at the same time and has a similar timecourse to the IJP following nerve stimulation, and subserves and entirely different function. This could be to mediate in the release of other substances, given that SK channels are expressed at high densities in many types of secretory cell. Could it be that the large sustained hyperpolarization induced by ATP in PDGFα positive cells underlies capacitative calcium entry to support slow vesicular release of whatever substance(s) these cells secrete?

In circular smooth muscle cells isolated from the mouse ileum, if one extrapolates the magnitude of the ATP-evoked apamin-sensitive current recorded from cell-attached patches to its size over the entire cell membrane, based on rough estimates of patch area and cell surface area from cell capacitance, then there are grounds to believe that a current of sufficient magnitude can indeed be generated to hyperpolarize smooth muscle cells without invoking the mediation of other cells types. But the question is, is the concentration of ATP used experimentally to induce this current a fair mimic of that released from nerves in the IJP response, and is the timecourse of activation of the SK potassium conductance on the timescale of the IJP? Without knowing the answer to these questions, it would be reasonable to conclude that smooth muscle cells, or at least a fraction of circular smooth muscle cells of the mouse ileum that survive enzymatic treatment during the cell isolation, are capable of hyperpolarizing in response to ATP through the opening of SK channels. If these cells do not contribute directly or significantly to the generation of the IJP itself, then they may participate in the overall inhibitory response in a “volume transmission” manner as a result of spill-over of ATP released from enteric inhibitory nerves diffusing to circular smooth muscle cells in the vicinity. This scenario invokes the concept of junctional and extrajunctional receptors as in vascular smooth muscle and elsewhere. In this regard, it was noted in my experiments that a prominent effect of stimulation of the circular smooth muscle cells by exogenous ATP was to enhance the transient outward current component that was activated by calcium current-dependent calcium entry/release and which was sensitive to apamin. Enhancement of this current by extrajunctional ATP alone would suppress the excitability of the circular smooth muscle cells by increasing the interval between bursts of action potentials and contractions in situ. Moreover, the sustained rise in intracellular calcium occasioned by release of stored calcium by extrajunctional ATP would be expected to inactivate voltage-gated calcium channels in smooth muscle cells, thereby adding to a refractoriness of the muscle to further excitation and contraction.

In any case, here’s something to remind us of the physiological relevance of the IJP and its role in gastrointestinal motility broadly speaking.

Fivos Vogalis PhD