Cellular mechanism of the voltage-dependent change in slow potentials generated in circular smooth muscle of the guinea-pig gastric corpus.

The cellular mechanism of the voltage-dependent properties of slow potentials were investigated in single bundles of circular smooth muscle isolated from the gastric corpus of guinea-pig using conventional microelectrode recordings. Hyperpolarization of the membrane by current injection decreased the frequency and increased the amplitude of slow potentials linearly. At potentials negative of -80 mV, slow potential generation was abolished and a periodic generation of clustered unitary potentials was evident. Application of cyclopiazonic acid (CPA, 20 microM) or thapsigargin (1 microM; inhibitors of Ca(2+)-ATPase), carbonyl cyanide m-chlorophenyl hydrazone (CCCP, 0.1 microM; mitochondrial protonophore) or 2-aminoethoxydiphenyl borate (2-APB, 20 microM; inhibitor of IP(3) receptor-mediated Ca(2+) release) depolarized the membrane and reduced or inhibited the amplitude and frequency of slow potentials: repolarization of the membrane to the resting level by current injection resulted in a recovery of the amplitude of slow potentials in the presence of CPA or CCCP, but not 2-APB. The slow potentials abolished by thapsigargin did not recover upon membrane repolarization. The altered frequency of slow potentials by 2-APB, CPA or CCCP was not reversed by membrane repolarization to control potentials. Depolarization of the membrane by about 10 mV with high-potassium solution also reduced the amplitude and increased the frequency of slow potentials in a manner restored by repolarization to control potentials upon current injection, suggesting that membrane depolarization did not affect the voltage dependency of pacemaker activity. The results indicate that in corpus circular muscles the voltage dependency of the frequency and amplitude of slow potentials requires a functional Ca(2+) store and mitochondria.

An electrical description of the generation of slow waves in the antrum of the guinea-pig.

This paper provides an electrical description of the generation of slow waves in the guinea-pig gastric antrum. A short segment of a circular smooth muscle bundle with an attached network of myenteric interstitial cells of Cajal (ICC-MY) and longitudinal muscle sheet was modelled as three electrical compartments with resistive connexions between the ICC-MY compartment and each of the smooth muscle compartments. The circular smooth muscle layer contains a proportion of intramuscular interstitial cells of Cajal (ICC-IM), responsible for the regenerative component of the slow wave. Hence the equivalent cell representing the circular muscle layer incorporated a mechanism, modelled as a two stage reaction, which produces an intracellular messenger. The first stage of the reaction is proposed to be activated in a voltage-dependent manner as described by Hodgkin and Huxley. A similar mechanism was incorporated into the equivalent cell describing the ICC-MY network. Spontaneous discrete transient depolarizations, termed unitary potentials, are detected in records taken from either bundles of circular smooth muscle containing ICC-IM or from ICC-MY. In the simulation the mean rate of discharge of unitary potentials was allowed to vary with the concentration of messenger according to a conventional dose-effect relationship. Such a mechanism, which describes regenerative potentials generated by the circular muscle layer, also simulated the plateau component of the pacemaker potential in the ICC-MY network. A voltage-sensitive membrane conductance was included in the ICC-MY compartment; this was used to describe the primary component of the pacemaker potential. The model generates a range of membrane potential changes with properties similar to those generated by the three cell types present in the intact tissue.

An analysis of inhibitory junction potentials in the guinea-pig proximal colon.

Intracellular recordings were made from either sheets or isolated bundles of the circular muscle layer of guinea-pig proximal colon and the responses evoked by stimulating inhibitory nerve fibres were analysed. Inhibitory junction potentials (IJPs), evoked by single stimuli, had two components which could be separated on their pharmacological and temporal characteristics and their voltage sensitivities. The initial component, which was abolished by apamin and reduced in amplitude by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), had a brief time course: its amplitude was changed when the external concentration of potassium ions ([K+](o)) was changed. The second component of the IJP had a slower onset than the first component, was abolished by l-nitroarginine (NOLA) and oxadiazolo quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase: its amplitude was little affected by changing [K+](o) and was increased when the membrane potential of the circular layer was hyperpolarized. The observations suggest that the initial component of the IJP results from the release of ATP which triggers an increase in membrane conductance to K+ and that the second component results from the release of nitric oxide which suppresses a background inward current.

Properties of unitary potentials generated by intramuscular interstitial cells of Cajal in the murine and guinea-pig gastric fundus.

Intracellular recordings were made from isolated bundles of the circular muscle layer of mouse and guinea-pig gastric fundus. These preparations displayed an ongoing discharge of membrane noise (unitary potentials), similar to that recorded from similar preparations made from the circular layer of the antrum. Bundles of muscle from the fundus of W/W(V) mice, which lack intramuscular interstitial cells of Cajal (ICC(IM)) lacked the discharge of membrane noise observed in wild-type tissues. When the membrane potential was changed by passing depolarizing or hyperpolarizing current pulses, the discharge of membrane noise was little changed. The membrane noise was unaffected by adding chloride channel blockers; however, agents which buffered the internal concentration of calcium ions reduced the discharge of membrane noise. Treatment of tissues with CCCP, which interferes with the uptake of calcium ions by mitochondria, also reduced the membrane noise and caused membrane hyperpolarization. Similar observations were made on bundles of tissue isolated from the circular layer of the guinea pig antrum. Together the observations indicate that membrane noise is generated by a pathway located in ICC(IM). The properties of this pathway appear to vary dramatically within a given organ. The lack of voltage sensitivity of the discharge of membrane noise in the fundus provides a possible explanation for the lack of rhythmic electrical activity in this region of the stomach.

Role of interstitial cells of Cajal in neural control of gastrointestinal smooth muscles.

Specialized cells known as interstitial cells of Cajal (ICC) are distributed in specific locations within the tunica muscularis of the gastrointestinal tract and serve as electrical pacemakers, active propagation pathways for slow waves, and mediators of enteric motor neurotransmission. Recent morphological studies have provided evidence that motor neurotransmission in the gut does not occur through loosely defined synaptic structures between nerves and smooth muscle, but rather via synaptic-like contacts that exist between varicose nerve terminals and intramuscular ICC (ICC-IM). ICC-IM are coupled to smooth muscle cells via gap junctions and electrical responses elicited in ICC are conducted to muscle cells. Electrophysiological studies of the stomach of wild-type and mutant animals that lack ICC-IM have provided functional evidence for the importance of ICC in cholinergic and nitrergic motor neurotransmission. The synaptic-like contacts between nerve terminals and ICC-IM facilitate rapid diffusion of transmitters to specific receptors on ICC. ICC-IM also play a role in generating unitary potentials in the stomach that contribute to the excitability of the gastric fundus and antrum.

Propagation of pacemaker activity in the guinea-pig antrum.

Cyclical periods of depolarization (slow waves) underlie peristaltic contractions involved in mixing and emptying of contents in the gastric antrum. Slow waves originate from a myenteric network of interstitial cells of Cajal (ICC-MY). In this study we have visualized the sequence and propagation of Ca(2+) transients associated with pacemaker potentials in the ICC network and longitudinal (LM) and circular muscle (CM) layers of the isolated guinea-pig gastric antrum. Gastric antrum was dissected to reveal the ICC-MY network, loaded with Fluo-4 AM and activity was monitored at 37 degrees C. Ca(2+) waves propagated throughout the ICC-MY network at an average velocity of 3.24 +/- 0.12 mm s(-1) at a frequency of 4.87 +/- 0.16 cycles min(-1) (n= 4). The propagation of the Ca(2+) wave often appeared 'step-like', with separate regions of the network being activated after variable delays. The direction of propagation was highly variable (Delta angle of propagation 44.3 +/- 10.9 deg per cycle) and was not confined to the axes of the longitudinal or circular muscle. Ca(2+) waves appeared to spread out radially from the site of initiation. The initiating Ca(2+) wave in ICC-MY was correlated to secondary Ca(2+) waves in intramuscular interstitial cells of Cajal, ICC-IM, and smooth muscle cells, and the local distortion (contraction) in a field of view. TTX (1 microm) had little effect on slow wave or pacemaker potential activity, but 2-APB (50 microm) blocked all Ca(2+) waves, indicating a pivotal role for intracellular Ca(2+) stores. Nicardipine (2 microm) eliminated the Ca(2+) transient generated by smooth muscle, but did not affect the fast upstroke associated with ICC-MY. These results indicate that slow waves follow a sequence of activation, beginning with the ICC-MY and ICC-IM network, followed later by a sustained Ca(2+) transient in the muscle layers that is responsible for contraction.

Interaction between excitatory and inhibitory metabotropic pathways in the guinea-pig antrum.

Intracellular recordings were made from isolated bundles of the circular muscle layer of guinea-pig gastric antrum and the responses evoked by stimulating nitrergic nerve fibres were examined. Nitrergic inhibitory junction potentials (nitrergic-IJPs), evoked by trains of stimuli, had small amplitudes and were associated with a reduction in the rate of occurrence and amplitude of spontaneously occurring depolarizing potentials, termed unitary potentials. Nitrergic-IJPs were abolished either by membrane hyperpolarization or by 4, 4'-diisothiocyano-2, 2'-stilbene disulfonic acid (DIDS); both of these abolished the discharge of unitary potentials. Membrane depolarization increased the rate of discharge of unitary potentials so that they summed to give rise to are generative potential. Nitrergic nerve stimulation abolished regenerative potentials; this inhibition did not result from a change in threshold for the initiation of regenerative potentials,rather it occurred at some stage after the gating process. Inhibitory nitrergic nerve responses were blocked by L-nitroarginine (NOLA) and oxadiazolo quinoxalin-l-one (ODQ), an inhibitor of soluble guanylate cyclase. The observations suggest that the inhibition of regenerative potentials results from an interaction between an inhibitory and an excitatory metabotropic pathway.

Electrical coupling between the myenteric interstitial cells of Cajal and adjacent muscle layers in the guinea-pig gastric antrum.

Intracellular recordings were made from short segments of the muscular wall of the guinea-pig gastric antrum. Preparations were impaled using two independent microelectrodes, one positioned in the circular layer and the other either in the longitudinal layer, in the network of myenteric interstitial cells of Cajal (ICCMY) or in the circular layer. Cells in each layer displayed characteristic patterns of rhythmical activity, with the largest signals being generated by ICCMY. Current pulses injected into the circular muscle layer produced electrotonic potentials in each cell layer, indicating that the layers are electrically interconnected. The amplitudes of these electrotonic potentials were largest in the circular layer and smallest in the longitudinal layer. An analysis of electrical coupling between the three layers suggests that although the cells in each layer are well coupled to neighbouring cells, the coupling between either muscle layer and the network of ICCMY is relatively poor. The electrical connections between ICCMY and the circular layer did not rectify. In parallel immunohistochemical studies, the distribution of the connexins Cx40, Cx43 and Cx45 within the antral wall was determined. Only Cx43 was detected; it was widely distributed on ICCMY and throughout the circular smooth muscle layer, being concentrated around ICCIM, but was less abundant in the circular muscle layer immediately adjacent to ICCMY. Although the electrophysiological studies indicate that smooth muscle cells in the longitudinal muscle layer are electrically coupled to each other, none of the connexins examined were detected in this layer.

Mathematical description of regenerative potentials recorded from circular smooth muscle of guinea pig antrum.

Regenerative potentials evoked by intracellular current injection in single bundles of circular smooth muscle taken from guinea pig antrum have the characteristics of the secondary regenerative component of the slow wave occurring in the same muscle layer. Such regenerative depolarizations might result from a mechanism that responds to membrane polarization with a delayed increase in the rate of production of unitary potentials detected in this tissue. To test this possibility, a two-stage reaction leading to the formation of an intracellular messenger was proposed. The first forward reaction was voltage-dependent, in the manner described by the Hodgkin-Huxley transient Na conductance formalism, allowing simulation of anode break excitation, stimulus threshold strength-duration characteristics, and refractory behavior. A conventional dose-effect relationship was proposed to describe the dependence of the mean rate of discharge of unitary potentials on messenger concentration. Unitary potentials were modeled as unitary membrane conductance modulations with an empirically derived amplitude distribution and Poisson-distributed intervals. The model reproduces a range of spontaneous and evoked membrane potential changes characteristic of antral circular muscle bundles.

Interstitial cells: involvement in rhythmicity and neural control of gut smooth muscle.

Many smooth muscles display spontaneous electrical and mechanical activity, which persists in the absence of any stimulation. In the past this has been attributed largely to the properties of the smooth muscle cells. Now it appears that in several organs, particularly in the gastrointestinal tract, activity in smooth muscles arises from a separate group of cells, known as interstitial cells of Cajal (ICC), which are distributed amongst the smooth muscle cells. Thus in the gastrointestinal tract, a network of interstitial cells, usually located near the myenteric plexus, generates pacemaker potentials that are conducted passively into the adjacent muscle layers where they produce rhythmical membrane potential changes. The mechanical activity of most smooth muscle cells, can be altered by autonomic, or enteric, nerves innervating them. Previously it was thought that neuroeffector transmission occurred simply because neurally released transmitters acted on smooth muscle cells. However, in several, but not all, regions of the gastrointestinal tract, it appears that nerve terminals, rather than communicating directly with smooth muscle cells, preferentially form synapses with ICC and these relay information to neighbouring smooth muscle cells. Thus a set of ICC, which are distributed amongst the smooth muscle cells of the gut, are the targets of transmitters released by intrinsic enteric excitatory and inhibitory nerve terminals: in some regions of the gastrointestinal tract, the same set of ICC also augment the waves of depolarisation generated by pacemaker ICC. Similarly in the urethra, ICC, distributed amongst the smooth muscle cells, generate rhythmic activity and also appear to be the targets of autonomic nerve terminals.

Involvement of intramuscular interstitial cells in nitrergic inhibition in the mouse gastric antrum.

Intracellular recordings were made from isolated bundles of the circular muscle layer of mouse gastric antrum and the responses evoked by stimulating intrinsic nerve fibres were examined. Transmural nerve stimulation evoked a fast inhibitory junction potential (fast-IJP) which was followed initially by a smaller amplitude long lasting inhibitory junction potential (slow-IJP) and a period of excitation. The excitatory component of the response was abolished by atropine, suggesting that it resulted from the release of acetylcholine and activation of muscarinic receptors. Fast-IJPs were selectively reduced in amplitude by apamin and slow-IJPs were abolished by N(omega)-nitro-L-arginine. Slow-IJPs were associated with a drop in membrane noise, suggesting that inhibition resulted from a reduced discharge of unitary potentials by intramuscular interstitial cells of Cajal (ICC(IM)). The chloride channel blocker, anthracene-9-carboxylic acid, reduced the discharge of membrane noise in a manner similar to that detected during the slow-IJP. When recordings were made from the antrum of W/W(V) mice, which lack ICC(IM), the cholinergic and nitrergic components were absent, with only fast-IJPs being detected. The observations suggest that neurally released nitric oxide selectively targets ICC(IM) causing a hyperpolarization by suppressing the discharge of unitary potentials.

Pacemaker shift in the gastric antrum of guinea-pigs produced by excitatory vagal stimulation involves intramuscular interstitial cells.

Intracellular recordings were made from isolated bundles of the circular muscle layer of guinea-pig gastric antrum and the responses produced by stimulating intrinsic nerve fibres were examined. After abolishing the effects of stimulating inhibitory nerve terminals with apamin and L-nitroarginine (NOLA), transmural nerve stimulation often evoked a small amplitude excitatory junction potential (EJP) and invariably evoked a regenerative potential. Neurally evoked regenerative potentials had similar properties to those evoked in the same bundle by direct stimulation. EJPs and neurally evoked regenerative potentials were abolished by hyoscine suggesting that both resulted from the release of acetylcholine and activation of muscarinic receptors. Neurally evoked regenerative potentials, but not EJPs, were abolished by membrane hyperpolarization, caffeine and chloride channel blockers. In the intact antrum, excitatory vagal nerve stimulation increased the frequency of slow waves. Simultaneous intracellular recordings of pacemaker potentials from myenteric interstitial cells (ICC(MY)) and slow waves showed that the onset of each pacemaker potential normally preceded the onset of each slow wave but vagal stimulation caused the onset of each slow wave to precede each pacemaker potential. Together the observations suggest that during vagal stimulation there is a change in the origin of pacemaker activity with slow waves being initiated by intramuscular interstitial cells (ICC(IM)) rather than by ICC(MY).

Regenerative component of slow waves in the guinea-pig gastric antrum involves a delayed increase in [Ca(2+)](i) and Cl(-) channels.

Regenerative potentials were initiated by depolarizing short segments of single bundles of circular muscle isolated from the gastric antrum of guinea-pigs. When changes in [Ca(2+)](i) and membrane potential were recorded simultaneously, regenerative potentials were found to be associated with an increase in [Ca(2+)](i), with the increase starting after a minimum latency of about 1 s. Although the increase in [Ca(2+)](i) was reduced by nifedipine, the amplitudes of the regenerative responses were little changed. Regenerative responses and associated changes in [Ca(2+)](i) were abolished by loading the preparations with the Ca(2+) chelator MAPTA-AM. Regenerative potentials were abolished by 2-aminoethoxydiphenyl borate (2APB), an inhibitor of IP(3) induced Ca(2+) release, by N-ethylamaleimide (NEM), an alkylating agent which blocks activation of G-proteins and were reduced in amplitude by two agents which block chloride (Cl(-))-selective channels in many tissues. The observations suggest that membrane depolarization triggers IP(3) formation. This causes Ca(2+) release from intracellular stores which activates Ca(2+)-dependent Cl(-) channels.

Regional variation in contribution of myenteric and intramuscular interstitial cells of Cajal to generation of slow waves in mouse gastric antrum.

When intracellular recordings were made from the antral region of murine stomach, cells with three different patterns of electrical activity were detected. One group of cells generated follower potentials, the second group generated pacemaker potentials and the third group generated slow waves that consisted of primary and secondary components. Slow waves recorded in different regions of the gastric antrum had similar amplitudes but different characteristic shapes. At the greater curvature, slow waves had large initial components. Midway between the greater and lesser curvature, the amplitude of the initial component was reduced and at the lesser curvature an initial component was difficult to detect. When the distributions of myenteric (ICC-MY) and intramuscular interstitial cells of Cajal (ICC-IM) were determined, using an antibody to Kit, ICC-MY were found to be present at the greater curvature but were greatly reduced in density at the lesser curvature. In contrast, ICC-IM were found in the circular layer of each region. When recordings were made from the antrum of W/W(V) mice, which lack ICC-IM, incomplete slow waves were detected and their amplitudes fell from the greater to the lesser curvature. Again, a corresponding fall in the density of ICC-MY was detected. The observations indicate that the contribution of ICC-MY and ICC-IM to the generation of slow waves varies in different regions of the mouse gastric antrum.

Voltage independence of vasomotion in isolated irideal arterioles of the rat.

The cellular mechanisms underlying vasomotion of irideal arterioles from juvenile rats have been studied using electrophysiological methods, ratiometric calcium measurements and video microscopy. Vasomotion was not affected by removal of the endothelium. Spontaneous contractions were preceded by spontaneous depolarizations. Both were abolished by the intracellular calcium chelator, BAPTA AM (20 microM), but not by ryanodine (10 microM), suggesting a dependence on the cyclical release of calcium from intracellular stores, other than those operated by ryanodine receptors. Oscillations were little changed when the membrane potential of short segments of arteriole was either depolarized or hyperpolarized. When the segments were voltage clamped, oscillating inward currents were recorded, indicating that the changes in membrane potential were voltage independent. Vasomotion was preceded by intracellular calcium oscillations and both were abolished by inhibitors of phospholipase C (U73122, 10 microM), phospholipase A(2) (AACOCF(3), 30 microM) and protein kinase C (chelerythrine chloride, 5 microM, and myristoylated protein kinase C peptide, 10 microM). Inhibition of vasomotion by the dual lipoxygenase and cyclo-oxygenase inhibitor, NDGA (10 microM), the lipoxygenase inhibitor, ETI (1 microM) but not by the cyclo-oxygenase inhibitors, aspirin (10 microM) and indomethacin (10 microM), or the cytochrome P450 inhibitor 17-ODYA (10 microM), suggested an involvement of the lipoxygenase pathway. The observations suggest that vasomotion of iris arterioles is voltage independent and results from the cyclical release of calcium from IP(3)-sensitive stores which are activated by cross talk between the phospholipase C and phospholipase A(2) pathways in vascular smooth muscle.