Regulatory Peptides, 42 (1992) 1-13
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© 1992 Elsevier Science Publishers B.V. All fights reserved 0167-0115/92/$05.00
R E G P E P 01232
Review
Modulation of electrical activity in gastrointestinal smooth muscle by peptides Allen W. Mangel and Ian L. Taylor Division of Gastroenterology, Duke University Medical Center and The Durham VA Medical Center, Durham, NC (USA) (Received 1 July 1992; revised version received 28 July 1992; accepted 30 July 1992)
Key words." Gastrointestinal smooth muscle; Electrical activity; Peptide; Review Contents I. ln~oduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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II. Excitation contraction coupling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Slow waves and spikes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Activity in calcium free solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Electrical activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Depolarization activated contractions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Peptide-induced contractile activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2 2 3 3 3 4
III. Modulation of electrical activity by peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . A. General comments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Esophagus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Stomach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Small intestine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Colon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5 5 5 6 7 7
IV. Regulation of ion channels by intracellulax pathways . . . . . . . . . . . . . . . . . . . . . . A. Stomach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Small intestine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Colon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8 8 9 9
V. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Correspondence to: A. W. Mangel, Division of Gastroenterology, Duke University Medical Center, P. O. Box 3913, Durham, NC 27710, USA.
I. Introduction This review discusses modulation of gastrointestinal smooth muscle electrical activity by peptides. A rise in intracellular calcium is the signal that triggers contraction in gastrointestinal smooth muscle, and activation of plasma membrane electrical events are often responsible for the increase in calcium. Changes in electrical activity frequently are correlated with peptide-induced modulation of mechanical activity.
II. Excitation contraction coupling 11-A. Slow waves and spikes The plasma membrane of gastric, small intestinal and colonic smooth muscle cells shows slow rhythmic changes in membrane potential. These changes have been denoted as slow waves, basic electrical rhythms, action potentials or electrical control activity (Fig. 1). In this review, we use the term slow wave. The waveform, amplitude, and/or duration o f slow waves are dependent upon the region of the gastrointestinal tract studied and no one specific hypothesis can explain the ionic basis o f slow wave generation in all regions of the gut [ 1 ]. It is likely that different control mechanisms exist in the different gastrointestinal regions and in different species. Regardless o f the mechanism responsible for slow wave generation, membrane depolarization associated with the upstroke o f the slow wave brings the membrane potential to threshold for activation of calcium conductance changes, which leads to an increase in intracellular calcium [ 1-3]. Most studies in gastrointestinal smooth muscle indicate an important role for L-type calcium channels [ 4 - 6 ] which are activated at a threshold between - 4 0 and - 5 0 mV.
5 sec
0
"'"
-40
---
10
sec
Fig. I. Voltage profile of electrical activity in small intestinal smooth muscle. Shown in the upper panel are spontaneous slow waves and spikes recorded with an intraceUularmicroelectrodefrom a segment of cat small intestinal smooth muscle. Three slow waves are shown with spikes triggered by the depolarization associated with the upstroke of the slow wave. In cat small intestinal muscle, slow waves average 27 mV from a resting potential of -67 mV [ 7]. Spikes approach but rarely overshoot 0 mV. Followingincubation of smooth muscle segments in calcium free solution an alternative form of electrical activity developes [ 9-13 ]. Shown in the lower panel are three spontaneously appearing electrical waves in calcium free solution. Characteristically, in calcium free solution, the resting membrane potential depolarizes to approx. -40 mV [9,11 ] and waves of membrane depolarization approach, but do not overshoot 0 mV.
In some tissue types, activation of voltage-dependent calcium channels results in the generation of a second electrical event known as the spike (Fig. 1). Alternatively, activation of calcium channels may be associated with enhancement of the plateau phase of the slow wave without the development of spiking. Due to the small size of gastrointestinal smooth muscle cells, the rise in intracellular calcium associated with activation of voltage-gated calcium channels is believed to be adequate by itself to activate the contractile machinery. Furthermore, measurements of changes in calcium levels during the course of slow waves show an oscillation in intracellular calcium [7], that is sufficient to activate contractions in the absence of spiking [ 8 ]. I1-B. Activity in calcium free solution II-B.I. Electrical activity Although the increase in intracellular calcium associated with membrane potential changes may be adequate to activate contractions, release of intracellular calcium also participates in activation of the contractile machinery in some muscle type. Incubation of gastrointestinal smooth muscle in solutions with no added calcium containing 2-5 mM EGTA (calcium free solution, hereafter), leads to the reversible elimination of normal slow waves and spikes with replacement by an alternate form of electrical activity [9]. These latter events are characterized by slow rhythmic membrane depolarizations lasting appreciably longer than normal slow waves or spikes (see Fig. 1). The resting membrane potential depolarizes from approx. -70 mV to approx. -40 mV in calcium-free solution [9,11,13], with peak amplitude of these spontaneous electrical events approaching 0 mV. II-B.2. Depolarization activated contractions In the absence of extracellular calcium, depolarization of the plasma membrane results in activation of contractions in a variety of gastrointestinal smooth muscle preparations [10-14]. In muscle preparations bathed in calcium free solutions, contractile activity could be observed after depolarization of the plasma membrane with high potassium solutions or in one-to-one correspondence with the rhythmic membrane potential changes described above. This contractile activity is observed in intact muscle segments containing both longitudinal and circular muscle layers, as well as in thin sheets of longitudinal muscle [11]. One would not expect a 'protected' extracellular calcium source to be present in thin sheets of longitudinal muscle indicating a role for mobilization of intracellular calcium. In contrast to these studies, others [ 15-20] have reported a failure of depolarization to induce contractile activity in gastrointestinal smooth muscle during incubation in calcium free solution. Explanations that could account for the differences between studies include: (1) species differences or different tissue types may explain the lack of activation by depolarization in some studies. (2) Several studies which used either small muscle bundles or isolated smooth muscle cells may not have had the critical mass of tissue necessary for the development of electrical and mechanical activity in calcium free solution [9,12]. (3)Finally, in some studies, inadequate concentrations of potas-
sium were used to cause 'depolarization'. To illustrate this latter point, in calcium containing solutions addition o f 10-20 m M K o causes depolarization of the membrane potential from approx. - 7 0 mV (see Table I). However, in calcium free solution, the resting membrane potential of intestinal muscle reversibly depolarizes from approx. - 7 0 mV to - 4 0 mV [9,11,13 ]. Thus, the potassium equilibrium potential (EK) is still negative to the resting potential (Fro) when only modest changes ( 1 0 - 3 0 raM) in Ko are made. As illustrated in Table I, at least 40 m M potassium needs to be added to the extraceUular solution to cause a 10 mV depolarization in cells with a resting potential positive to - 4 0 mV. Thus, when the resting potential is partially depolarized (as occurs in calcium free solution) a greater increase in extracellular potassium concentration is required to further depolarize the plasma membrane.
11-B.3. Peptide-induced contractile activity The effects of peptides on gastrointestinal smooth muscle contractile activity have been examined during incubation in calcium free solution. As expected, results differ depending on species and tissue type. Cholecystokinin ( C C K ) has been shown to induce contraction o f guinea pig and human, but not cat, gastric smooth muscle in calcium free solution [14,15,17]. In calcium free solution, substance-P, but not neurotensin, stimulates contraction of cat gastric smooth muscle [14]. Pentagastrininduced contractile activity o f canine gastric smooth muscle is markedly reduced by removal of extraceUular calcium [18].
TABLE I Effects of changes in Ko on EK Ki (mM)
Ko (raM)
130 (Ref. 65)
5 10 13 20 30 40 80 5
162 (Ref. 66) 10
13 20 30 40 80
EK (mV) - 82 65 - 58 - 47 - 37 - 30 - 12
-
- 88 70 - 64 - 53 - 42 - 35 - 18
-
Effects of changes in extraceUular potassium (Ko) on the potassium equilibrium potential (EK). Data are shown for two values ofintraceUularpotassium (K[). In cells with a resting membrane potential of ~ -70 mV, modest changes (~ 20 mM) in Ko depolarizes the resting potential by ~ 20 inV. In calcium free solution where significantdepolarization of the resting potential to ~ -40 mV has occurred, a greater increase in Ko is needed to further depolarize the membrane.
In small intestinal smooth muscle, CCK initiates contractile activity in guinea pig and human circular muscle in calcium free solution, but has no effect on the longitudinal muscle layer [16]. In contrast, substance-P, neurotensin, and CCK do not induce contraction of cat small intestinal smooth muscle during incubation in calcium free solution [ 14]. In the longitudinal muscle layer of guinea pigs, substance-P stimulates contractile activity which is reduced compared to that seen in calcium containing solutions [21]. Motilin has been shown to induce only small amplitude contractions in rabbit small intestinal muscle segments during incubation in calcium-deficient solutions [22,23]. In the distal, but not proximal colon of cats, substance-P stimulates contractile activity while CCK and neurotensin have no effect in either region [14]. In rabbit colonic smooth muscle, neurotensin also does not activate contractions during exposure to calcium free solution [20]. Although peptide-induced release of intracellular calcium may play a role in the excitation-contraction process in some gastrointestinal smooth muscles, our overall impression is that the influx of calcium through membrane associated L-type channels represents the predominant source of activator calcium that causes smooth muscle contraction. Therefore, this review will focus on the modulation of gastrointestinal smooth muscle electrical activity by peptides. This review will not address modulation of neurogenically-activated events by peptides.
IlL Modulation of electrical activity by peptides IliA.
General commen~
A detailed analysis of the ionic currents responsible for generation of electrical activity in gastrointestinal smooth muscle is beyond the scope of this review. In general, the stimulated inward current associated with the spike is primarily calcium-mediated, and the outward current is produced by an increase in calcium-dependent and voltageactivated potassium channels. The resting membrane potential primarily reflects contributions by potassium conductance and an electrogenic sodium pump. The ionic basis of slow wave activity is much more varied than that of spikes. However, during slow wave depolarization a stimulation of voltage-dependent calcium channels occurs. Electrical events serve to activate contractile activity. With depolarization, voltagegated calcium channels are activated, calcium enters the cell and contraction is induced. We will, therefore, focus on changes in gastrointestinal electrical activity induced by peptides as a tool to understanding changes in contractile activity. III-B. Esophagus
Classically, the esophagus has been considered a neurally-activated tissue. Spontaneous, atropine-sensitive slow waves and spikes have been recorded from segments of esophageal smooth muscle in organ baths [24,25]. In quiescent preparations, electrical activity can be induced by addition of cholinergic agonists. These findings suggest
that release of acetylcholine from intrinsic neurons may be responsible for initiation of esophageal slow waves. To date, we have not identified studies addressing the effects of peptides on spontaneous esophageal slow wave or spike activity.
III-C. Stomach Szurszewski and colleagues [26,28-31] have examined the effects of CCK, pentagastrin and vasoactive intestinal polypeptide (VIP) on the electrical activity in the canine antrum. Three forms of CCK (CCK-8, CCK-33 and CCK-39) were tested and all increased slow wave frequency, and the amplitude and duration of the plateau phase of the antral circular muscle slow wave. No significant change in resting potential or action potential upstroke occurs [26]. The atropine insensitivity of this response demonstrates that CCK does not exert its action via release of acetylcholine from intrinsic nerves but, rather, is a result of a direct action of CCK on smooth muscle cells [26]. In strips of cat antral muscle, bombesin depolarizes the resting membrane potential, shortens the slow wave plateau duration, and increases slow wave frequency [27]. The amplitude and frequency of spikes also increases. The effects of bombesin on slow waves are independent of extracellular calcium. Recordings from antral longitudinal muscle show pentagastrin to increase the frequency of slow waves and decrease the amplitude and duration of the plateau potential [28]. In most preparations, prepotential activity is induced and a decrease in the amplitude of the spike potential is observed. Overall, no significant change in resting membrane potential occurs. In other studies, pentagastrin, little gastrin and big gastrin were examined on intact antral muscle segments [29] or strips of antral circular muscle [30]. All three agents increase the amplitude and duration of the slow wave plateau phase and the frequency of spontaneous slow waves. Pentagastrin also induces prepotential activity in the antral circular muscle layer [28]. No significant change in slow wave upstroke or in resting potential is observed. In the corpus, pentagastrin increases the amplitude and duration of the plateau potential and depolarizes the resting potential [31]. In single gastric smooth muscle cells isolated from Bufo marinus, substance-P produces a depolarization of the resting potential which is associated with a decrease in membrane conductance [32]. Voltage-clamp experiments confirm that this effect resuits primarily from supression of an outward potassium current, similar to the Mcurrent [33]. This leads to the development of a net inward current and the production of spikes and contractions. Whole-cell patch clamp recordings reveal that substance-P enhances a slowly-inactivating, high threshold current [33]. A second calcium current which is characterized by low threshold and fast inactivation kinetics is not increased by substance-P. In intact gastric muscle segments from Bufo marinus substance-P increases the frequency of slow waves with a decrease in slow wave upstroke dV/dt and amplitude and only a small depolarization of the plasma membrane [34]. Although VIP decreases the force of contraction of canine antral muscle segments, no change in spontaneous slow wave activity has been reported [ 35 ]. Furthermore, VIP does not alter acetylcholine-mediated increases in the slow wave plateau amplitude or
duration. However, VIP does inhibit pentagastrin-mediated increases in the plateau potential amplitude without altering the resting potential. Different effects of VIP have been noted in the rat stomach [36]. Dose-dependent hyperpolarization of up to 10 mV is seen in the fundus. In the rat antrum VIP inhibits spikes and, at high doses, decreases slow wave amplitude. A small hyperpolarization of the antral resting potential occurs. III-D. Small intestine
A limited number of studies have addressed the effects of peptides on the electrical activity of small intestinal smooth muscle. In rabbit duodenal muscle segments, motilin depolarizes the resting potential by approximately 11 mV, decreases both membrane resistance and slow wave amplitude, and induces minute rhythm type activity with spiking [37]. In whole-cell patch clamp studies with isolated guinea pig ileal smooth muscle cells, Nakazawa et al. [38] found that substance-P induces inward currents at negative holding potentials. This current, unlike the activity induced by acetylcholine, is increased following hyperpolarization of the membrane to values negative to -50 inV. Ion substitution studies indicate sodium to be the principal ion mediating the inward current. III-E. Colon
Because of the proposed relationship between cholecystokinin and irritable bowel syndrome [39,40], the effects of cholecystokinin on colonic electrical activity have been extensively investigated. Recordings of electrical activity from irritable bowel syndrome patients and normal controls show a 3 and 6 cycle per minute slow wave rhythm [40,41]. Compared to control subjects, patients with irritable bowel syndrome have increased 3 cycle per minute rhythms in the basal state [40]. Intravenous administration of CCK or pentagastrin increases the relative frequency of 3 cycle per minute slow wave activity [40]. In normal controls, the infusion of either CCK or gastrin I, cause an increase in electrical spiking [42]. Parallel in vitro studies with segments of cat colonic smooth muscle show an increase in colonic spike activity with addition of either the octapeptide of CCK (CCK-8) or gastrin I [43-45]. However, higher doses of both peptides caused partial interruption in slow wave activity. The effects of CCK were unaffected by muscarinic or adrenergic blockade. In longitudinal smooth muscle cells isolated from the rabbit colon, substance-P causes calcium-dependent activation of a 200-210 pS potassium channel [46,47]. Substance-P mediated activation is inhibited by the L-type calcium channel blocker nifedipine. Higher concentrations of the peptide results in transient channel activation followed by sustained inhibition. Potassium channel activation by the higher concentration of substance-P is nifedipine-insensitive, but sensitive to removal of bath calcium. It was concluded that substance-P activates two distinct calcium influx pathways in rabbit colonic smooth muscle. Substance-P depolarizes the plasma membrane of canine colonic circular muscle, intermittantly prolongs the duration of slow waves, and induces prepotential-like activity and spiking [48]. In another study with canine colonic circular muscle,
substance-P was found to initially increase slow wave duration, followed by short duration slow waves with occasional long duration events [45]. After a long duration slow wave the upstroke velocity of the next slow wave is decreased. By analogy with the effects of acetylcholine on canine colonic slow wave activity, long duration slow waves may result from prolonged activation of L-type calcium channels during the plateau phase of the slow wave [49]. Substance-P did not induce spiking in this latter study. In the distal colon of rabbits, neurotensin produces a reversible, dose-dependent depolarization of the resting potential without noticeable change in slow wave activity [50]. An increase in membrane conductance, mediated via calcium influx, is associated with depolarization of the resting potential. VIP hyperpolarizes the plasma membrane of guinea pig taenia coli [51 ]. An inhibition of spike discharge is noted in some preparations without change in the resting potential. With high concentrations of VIP, hyperpolarization and inhibition of spike discharge are routinely seen.
IV. Regulation of ion channels by intracellular pathways Receptor-mediated changes in ion channel activity are coupled through intracellular second messenger system in a variety of cell types. In gastrointestinal smooth muscle, similar pathways appear to modulate electrical activity.
IV-A. Stomach In isolated gastric smooth muscle cells from Bufo marinus, two distinct calcium currents have been identified [52]; a low threshold, rapidly inactivating current and a high threshold, slowly inactivating current. Addition of sn-l,2-dioctanoyl-glycerol, a synthetic diacylglycerol compound which activates protein kinase C (PKC), increases the high threshold current. In contrast, a different structural congener, 1,2-dioctanoyl3-thioglycerol does not activate PKC nor calcium conductance. These studies indicate that PKC is involved in the activation of the high threshold current. As acetylcholine activates the same current, it was postulated that PKC may also mediate the effects of acetylcholine on this class of calcium channels. While the activity of an outwardly rectifying potassium channel is independent of ATP, GTP, NADPH or calcium in Bufo marinus it is increased by several fatty acids including oleic and myristic acid [53,54]. This activation occurs in excised patches suggesting direct activation of channel activity independent of cytosolic mediators. In intact cells this current is also activated by an indirect pathway following the conversion of arachodonic acid to its oxygenated metabolites [53,54]. The phosphatase inhibitors, calyculin A and okadalc acid, decrease the amplitude and duration of canine gastric slow waves primarily via effects on the plateau phase of the slow wave [55]. These changes are associated with a decrease in L-type calcium channel activity and phosphorylation of calcium channels may, therefore, inhibit calcium currents. An additional control mechanism for calcium channels is linked to
increases in intracellular calcium levels which produces calcium-dependent inactivation of channel activity [56]. IV-B. Small intestine
In isolated rabbit small intestinal cells a transient inward current is activated by carbachol. This current is insensitive to addition of non-hydrolyzable analogues of GTP or GDP [57,58]. In these cells, spontaneous transient outward currents are calcium-dependent and are blocked by GTP analogues in intact cells, but not in excised patches [59,60]. The effects of G-proteins on potassium conductance are thought to be linked to changes in accessibility of intracellular calcium stores, and are not the result of a direct coupling of G-proteins to the potassium channel. IV-C. Colon
Phosphatase inhibitors also decrease slow wave amplitude and duration in the canine colon by changing the slow wave plateau [55]. Phosphorylation of calcium channels in colonic smooth muscle is presumed to lead to inhibition of calcium channel activity. In isolated rabbit colonic smooth muscle cells, a large conductance chloride channel is present [61 ]. Channel activity is voltage dependent but insensitive to changes in extracellular calcium. Channel activity is increased by non-hydrolyzable analogues of GTP and reversible inhibited by non-hydrolyzable analogues of GDP. As these effects occur in inside-out patches, they are independent of changes in any cytosolic mediator and can be attributed to a direct G-protein effect on the channel.
V. Summary A rise in intracellular calcium is the predominant signal that leads to the activation of the contractile machinery in gastrointestinal smooth muscle. The primary sources of activating calcium are illustrated in Fig. 2. Voltage- and peptide-mediated release of intracellular calcium contribute to activation of some gastrointestinal smooth muscles. However, the primary source of activating calcium appears to be an influx of calcium across the plasma membrane. The degree of modulation of electrical activity by peptides varies depending upon the region of the gastrointestinal tract studied. Second messenger systems are undoubtly involved in the transduction pathway for receptor-mediated changes in ion channel activity in gastrointestinal smooth muscle. However, in comparison to other excitable cell types, little is known about the coupling mechanisms whereby peptide-receptor binding alters ion channel activity in gastrointestinal smooth muscle. This represents one of the challenging areas to be studied in the field of gastrointestinal smooth muscle. One disease in which a better appreciation of the regulation of ion channel activity could lead to therapeutic benefit is irritable bowel syndrome. A coupling of smooth muscle electrical activity to hypermotility in irritable bowel syndrome has been re-
t Ca;2 Fig. 2. Sources
of calcium
for activation
cium (Ca” + ) in gastrointestinal stored potential
calcium.
Arrow
thickness
or voltage operated
has been adapted vating calcium
of gastrointestinal
indicates
channels
relative smooth
to be influx of calcium
muscle through
tissues differences
muscle.
influx pathways
importance
while ROC indicates
from studies with vascular
is presumed
smooth
smooth muscle occurs through
Increases
of the respective
receptor
operated
[67]. As shown, voltage-gated
in intracellular
cal-
and release of intracellularily pathway.
channels.
the primary
channels,
POC denotes
This terminology source of acti-
although
species and
may exist.
ported. CCK increases the level of spike activity which triggers hyperrnotility [40]. It would follow that inhibition of calcium influx should reduce spiking and, therefore, hypermotility. In fact, the calcium channel blockers nifedipine and nicardipine have been shown to decrease colonic motility in irritable bowel syndrome patients [ 62-641. As our understanding of gastrointestinal smooth muscle ion channels expands, development of a gastrointestinal selective calcium channel blocker may be possible. This class of agents would be effective in the treatment of irritable bowel syndrome and potentially other peptide-related spastic smooth muscle disorders.
Acknowledgements
Support from NIH DK08452, DK38216; Glaxo Institute for Digestive Health Basic Research Award; a Glaxo Cardiovascular Discovery Grant; and VA funds are acknowledged.
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of the
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ac-
11 5 Vogalis, F., Publicover, N.G., Hume, J.R. and Sanders, K.M., Relationship between calcium current and cytosolic calcium in canine gastric smooth muscle cells, Am. J. Physiol., 260 (1991) C1012-1018. 6 Vivaudou, M. B., Singer, J.J. and Walsh, J. V., Multiple types of Ca 2 ÷ channels in visceral smooth muscle cells, Pflugers Arch., 418 (1991) 144-152. 7 Mangel, A.W., Connor, J.A. and Prosser, C.L., Effects of alterations in calcium levels on cat small intestinal slow waves, Am. J . Physiol., 243 (1982) C7-13. 8 Sanders, K.M., Excitation-contraction coupling without Ca 2 ÷ action potentials in small intestine, Am. J. Physiol., 244 (1983) C356-361. 9 Prosser, C.L., Kreulen, D.L., Weigel, R.J. and Yau, W., Prolonged potentials in gastrointestinal muscle induced by calcium chelation, Am. J. Physiol., 233 (1977) C19-24. 10 Mangel, A.W., Nelson, D.O., Connor, J.A. and Prosser, C.L., Contractions of cat small intestinal smooth muscle in calcium-free solution, Nature, 281 (1979) 582-583. 11 Mangel, A. W., Nelson, D. O., Rabovsky, J. L., Prosser, C. L. and Conner, J. A., Depolarization-induced contractile activity of smooth muscle in calcium-free solution, Am. J. Physiol., 242 (1982) C36-40. 12 Mangel, A.W., Voltage and receptor mediated contractile activity of colonic smooth muscle in calciumfree solution, Eur. J. Pharm., 102 (1984) 165-168. 13 Mangel, A.W., Mangel, C.P. and Sanders, K.M., Modulation of intestinal electrical and mechanical activity by calcium. In Calcium regulations in smooth muscle, INSERM, 124 (1984) 111-118. 14 Mangel, A.W., Fitz, J.G. and Taylor, I.L., Peptides do not induce contractions in gastrointestinal smooth muscle in calcium-free solution, Gen. Pharm., 22 (1991) 1135-1137. 15 Bitar, K. N., Bradford, P., Putney Jr., J.W. and Makhlouf, G.M., Cytosolic calcium during contraction of isolated mammalian gastric muscle cells, Science, 232 (1986) 1143-1145. 16 Bitar, K.N., Burgess, G.M., Putney Jr., J.W. and Makhlouf, G.M., Source of activator calcium in isolated guinea pig and human gastric muscle cells, Am. J. Physiol., 250 (1986) G280-286. 17 Collins, S.M., Calcium utilization by dispersed canine gastric smooth muscle cells, Am. J. Physiol., 251 (1986) G181-188. 18 Gilder, J.R. and Makhlouf, G.M., Contraction mediated by Ca 2÷ release in circular and Ca 2 ÷ influx in longitudinal intestinal muscle cells, J. Pharm. Exp. Ther., 244 (1988) 432-437. 19 Lee, K.Y., Biancani, P. and Behar, J., Calcium sources utilized by cholecystokinin and acetylcholine in the cat gallbladder muscle, Am. J. Physiol., 256 (1989) G785-788. 20 Shape Jr., W.J., Hyman, P.E., Mayer, E.A., Sevy, N., Kao, H.W. and Root, D., Calcium dependence of neurotensin stimulation of circular colonic muscle of the rabbit, Gastroenterology, 93 (1987) 823-828. 21 Holzer, P. and Lippe, I. Th., Substance P can contract the longitudinal muscle of the guinea-pig small intestine by releasing intracellular calcium, Br. J. Pharm., 82 (1984) 259-267. 22 Matthijs, G., Peeters, T.L. and Vantrappen, G., The role of intraceUular calcium stores in motilin induced contractions of the longitudinal muscle of the rabbit duodenum, Naunyn-Schmiedeberg's Arch. Pharm., 339 (1989) 332-339. 23 Adachi, H., Toda, N., Hatashi, S., Noguchi, M., Suzuki, T., Torizuka, K., Yajima, H. and Koyama, K., Mechanism of the excitatory action of motilin on isolated rabbit intestine, Gastroenterology, 80 (1981) 783-787. 24 Christensen, J. and Daniel, E.E., Electric and motor effects of autonomic drugs on longitudinal esophageal smooth muscle, Am. J. Physiol., 211 (1966) 387-394. 25 Nelson, D.O. and Mangel, A.W., Acetylcholine induced slow waves in cat esophageal smooth muscle, Gen. Pharm., 10 (1979) 19-20. 26 Morgan, K.G., Schmalz, P.F., Go, V. L.W. and Szurszewski, J.H., Electrical and mechanical effects of molecular variants of CCK on antral smooth muscle, Am. J. Physiol., 235 (1978) E324-329. 27 Milusheva, A., Bonev, A., Velkova, V., Boev, K. and Papasova, M., Bombesin-induced changes in membrane potential-dependent phasic contractions of cat gastric muscle, Gen. Physiol. Biophys., 7 (1988) 253-262. 28 Szurszewski, J.H., Mechanism of action of pentagastrin and acetylcholine on the longitudinal muscle of the canine antrum, J. Physiol., 252 (1975) 335-361. 29 Morgan, K.G., Schmalz, P.F., Go, V.L.W. and Szurszewski, J.H., Effects of pentagastrin, GI7 , and G 34 on the electrical and mechanical activities of canine smooth muscle, Gastroenterology 75, (1978) 405-412.
12 30 E1-Sharkawy, T.Y. and Szurszewski, J.H. Modulation of canine antral circular smooth muscle by acetylcholine, noradrenaline and pentagastrin, J. Physiol., 279 (1978) 309-320. 31 Morgan, K.G. and Szurszewski, J.H., Mechanisms of phasic and tonic actions of pentagastrin on canine gastric smooth muscle, J. Physiol., 301 (1980) 229-242. 32 Sims, S.M., Walsh Jr., J. V. and Singer, J.J., Substance P and acetylcholine both surpress the same K + current in dissociated smooth muscle cells, Am. J. Physiol., 251 (1986) C580-587. 33 Clapp, L. H., Vivaudou, M. B., Singer, J. J. and Walsh Jr., J. V., Substance P, like acetylcholine, augments one type of Ca 2+ current in isolated smooth muscle cells, Pflugers Arch., 413 (1989) 565-567. 34 Shonnard, P.Y. and Sanders, K.M., Effect of acetylcholine and substance P on electrical activity of intact toad gastric muscles, Am. J. Physiol., 258 (1990) G12-15. 35 Morgan, K.G., Schmalz, P.F. and Szurszewski, J.H., The inhibitor effects of vasoactive intestinal polypeptide on the mechanical and electrical activity of canine antra/ smooth muscle, J. Physiol., 282 (1978) 437-450. 36 lto, S., Kurokawa, A., Ogha, A., Ohga, T. and Sawabe, K., Mechanical, electrical and cyclic nucleotide responses to peptide VIP and inhibitory nerve stimulation in rat stomach, J. Physiol., 430 (1990) 337353. 37 Riemer, J., Kolling, K. and Mayer, C.J., The effect of motilin on the electrical activity of rabbit circular duodenal muscle, Pflugers Arch., 372 (1977) 243-250. 38 Nakazawa, K., Inoue, K., Fujimori, K. and Takanaka, A., Difference between substance P- and acetylcholine-induced currents in mammalian smooth muscle cells, Eur. J. Pharm., 179 (1990) 453-456. 39 Harvey, R.F. and Read, A. E., Effect of cholecystokinin on colonic motility and symptoms in patients with the irritable-bowel syndrome, Lancet, 2 (1973) 1-3. 40 Shape Jr., W.J., Carlson, G.M., Matarazzo, S.A. and Cohen, S., Evidence that abnormal myoelectrical activity produces colonic motor dysfunction in the irritable bowel syndrome, Gastroenterology, 72 (1977) 383-387. 41 Taylor, I. and Duthie, H.L. and Smallwood, R., The effect of stimulation on the myoelectrical activity on the rectosigmoid in man, Gut, 15 (1974) 599-607. 42 Snape Jr., W.J., Matarazzo, S.A. and Cohen, S., Effect of eating and gastrointestinal hormones on human colonic myoelectrical and motor activity, Gastroenterology, 75 (1978) 373-378. 43 Shape Jr., W.J., Interaction of the octapeptide of cholecystokinin and gastrin I with bethanechol in the ecology, stimulation of feline colonic smooth muscle, Gastroenterology, 84 (1983) 58-62. 44 Shape Jr., W.J., Effect of calcium on neurohumoral stimulation of feline colonic smooth muscle, Am., J. Physiol., 243 (1982) G134-140. 45 Snape Jr., W.J. and Cohen, S., Effect of bethanechol, gastrin I, or cholecystokinin on myoelectrical activity, Am. J. Physiol., 236 (1979) E458-463. 46 Mayer, E.A., Loo, D. D.F., Kodner, A. and Reddy, S.N., Differential modulation of Ca 2 + -activated K + channels by substance P, Am. J. Physiol., 257 (1989) G887-897. 47 Mayer, E.A., Loo, D.D.F., Snape Jr., W.J. and Sachs, G., The activation of calcium and calciumactivated potassium channels in mammalian colonic smooth muscle by substance P, J. Physiol., 420 (1990) 47-71. 48 Huizinga, J.D., Chang, G., Diamant, N.E. and EI-Sharkaway, T.Y., Electrophysiological basis of excitation of canine colonic circular muscle by cholinergic agents and substance P, J. Pharm. Exp. Ther., 231 (1984) 692-699. 49 Keel, K.D., Ward, S.M., Stevens, R.J., Frey, B.W. and Sanders, K.M. Electrical and mechanical effects of acetylcholine and substance P in subregions of canine colon, Am. J. Physiol., 262 (1992) G298307. 50 Snape Jr., W.J., Tan, S.T., Kao, H.W. and Hyman, P.E., Mechanism of neurotensin depolarization of rabbit colonic smooth muscle,, Regul. Pept., 18 (1987) 287-297. 51 Hills, J.M., Collins, C.S. and Burnstock, G., The effects of vasoactive intestinal polypeptide on the electrical activity of guinea-pig intestinal smooth muscle, Eur. J. Pharm., 88 (1983) 371-376. 52 Vivaudou, M.B., Clapp, L. H., Walsh Jr., J.V. and Singer, J.J., Regulation of one type of Ca 2 + current in smooth muscle cells by diacylglycerol and acetylcholine, FASEB J., 2 (1988) 2497-2504. 53 Ordway, R.W., Walsh Jr., J.V. and Singer, J.J., Arachidonic acid and other fatty acids directly activate potassium channels in smooth muscle cells, Science, 244 (1989) 1176-1179.
13 54 Ordway, R.W., Singer, J.J. and Walsh Hr., J.V., Direct regulation of ion channels by fatty acids, Trends Neurosci., 14 (1991) 96-100. 55 Ward, S.M., Vogalis, F., Blondfield, D.P., Ozaki, H., Fusetani, N , Uemura, D., Publicover, N.G. and Sanders, K.M., Inhibition of electrical slow waves and Ca 2 + currents of gastric and colonic smooth muscle by phosphatase inhibitors, Am. J. Physiol., 261 (1991) C64-70. 56 Vogalis, F., Publicover, N.G. and Sanders, K.M., Regulation of calcium currents by voltage and cytoplasmic calcium in canine gastric smooth muscle, Am. J. Physiol., 262 (1992) C691-700. 57 Lim, S.P. and Bolton, T.., A calcium-dependent rather than a G-protein mechanism is involved in the inward current evoked by muscarinic receptor stimulation in dialysed single smooth muscle cells of small intestine, Br. J. Pharrn., 95 (1988) 325-327. 58 Komori, S. and Bolton, T.B., Inositol triphosphate releases stored calcium to block voltage-dependent calcium channels in single smooth muscle cells, Pflugers. Arch., 418 (1991) 437-441. 59 Bolton, T.B. and Limn, S.P., Properties of calcium stores and transient outward currents in single smooth muscle cells of rabbit intestine, J. Physiol., 409 (1989) 385-401. 60 Komori, S. and Bolton, T.B., Role of G-proteins in muscarinic receptor inward and outward currents in rabbit jejunal smooth muscle, J. Physiol., 427 (1990) 395-419. 61 Sun, X.P., Supplisson, S., Torres, R., Sachs, G. and Mayer, E., Characterization of large-conductance chloride channels in rabbit colonic smooth muscle, J. Physiol., 448 (1992) 355-382. 62 Narducci, F., Bassotti, G., Gaburri, M. Farroni, F. and Morelli, A., Nifedipine reduces the colonic motor response to eating in patients with the irritable colon syndrome, Am. J. Gastroenterol., 80 (1985) 317-319. 63 Prior, A., Hams, S.R. and Whorwell, P.J., Reduction of colonic motility by intravenous nicardipine in irritable bowel syndrome, Gut, 28 (1987) 1609-1612. 64 Sun, W.M., Edwards, C.A., Prior, A., Rao, S.S.C. and Read, N.W., Effect of oral nicardipine on anorectal function in normal human volunteers and patients with irritable bowel syndrome, Dig. Dis. Sci., 35 (1990) 885-890. 65 Scheid, C.R. and Fay, F. S., Control of ion distribution in isolated smooth muscle cells. I. Potassium, J. Gen. Physiol., 75 (1980) 163-182. 66 Makhlouf, G. M., Smooth muscle of the gut. In T. Yamada (Ed.), Textbook of Gastroenterology, Vol. 1, J.B. Lippincott Co., Philadelphia, 1991, pp. 61-84. 67 Van Breemen, C., Mangel, A., Fahim, M. and Meisheri, K. Selectivity of calcium antagonistic action in vascular smooth muscle, Am. J. Cardiol., 49 (1982) 507-510.
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© 1992 Elsevier Science Publishers B.V. All fights reserved 0167-0115/92/$05.00
R E G P E P 01232
Review
Modulation of electrical activity in gastrointestinal smooth muscle by peptides Allen W. Mangel and Ian L. Taylor Division of Gastroenterology, Duke University Medical Center and The Durham VA Medical Center, Durham, NC (USA) (Received 1 July 1992; revised version received 28 July 1992; accepted 30 July 1992)
Key words." Gastrointestinal smooth muscle; Electrical activity; Peptide; Review Contents I. ln~oduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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II. Excitation contraction coupling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Slow waves and spikes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Activity in calcium free solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Electrical activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Depolarization activated contractions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Peptide-induced contractile activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2 2 3 3 3 4
III. Modulation of electrical activity by peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . A. General comments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Esophagus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Stomach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Small intestine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Colon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5 5 5 6 7 7
IV. Regulation of ion channels by intracellulax pathways . . . . . . . . . . . . . . . . . . . . . . A. Stomach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Small intestine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Colon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8 8 9 9
V. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Correspondence to: A. W. Mangel, Division of Gastroenterology, Duke University Medical Center, P. O. Box 3913, Durham, NC 27710, USA.
I. Introduction This review discusses modulation of gastrointestinal smooth muscle electrical activity by peptides. A rise in intracellular calcium is the signal that triggers contraction in gastrointestinal smooth muscle, and activation of plasma membrane electrical events are often responsible for the increase in calcium. Changes in electrical activity frequently are correlated with peptide-induced modulation of mechanical activity.
II. Excitation contraction coupling 11-A. Slow waves and spikes The plasma membrane of gastric, small intestinal and colonic smooth muscle cells shows slow rhythmic changes in membrane potential. These changes have been denoted as slow waves, basic electrical rhythms, action potentials or electrical control activity (Fig. 1). In this review, we use the term slow wave. The waveform, amplitude, and/or duration o f slow waves are dependent upon the region of the gastrointestinal tract studied and no one specific hypothesis can explain the ionic basis o f slow wave generation in all regions of the gut [ 1 ]. It is likely that different control mechanisms exist in the different gastrointestinal regions and in different species. Regardless o f the mechanism responsible for slow wave generation, membrane depolarization associated with the upstroke o f the slow wave brings the membrane potential to threshold for activation of calcium conductance changes, which leads to an increase in intracellular calcium [ 1-3]. Most studies in gastrointestinal smooth muscle indicate an important role for L-type calcium channels [ 4 - 6 ] which are activated at a threshold between - 4 0 and - 5 0 mV.
5 sec
0
"'"
-40
---
10
sec
Fig. I. Voltage profile of electrical activity in small intestinal smooth muscle. Shown in the upper panel are spontaneous slow waves and spikes recorded with an intraceUularmicroelectrodefrom a segment of cat small intestinal smooth muscle. Three slow waves are shown with spikes triggered by the depolarization associated with the upstroke of the slow wave. In cat small intestinal muscle, slow waves average 27 mV from a resting potential of -67 mV [ 7]. Spikes approach but rarely overshoot 0 mV. Followingincubation of smooth muscle segments in calcium free solution an alternative form of electrical activity developes [ 9-13 ]. Shown in the lower panel are three spontaneously appearing electrical waves in calcium free solution. Characteristically, in calcium free solution, the resting membrane potential depolarizes to approx. -40 mV [9,11 ] and waves of membrane depolarization approach, but do not overshoot 0 mV.
In some tissue types, activation of voltage-dependent calcium channels results in the generation of a second electrical event known as the spike (Fig. 1). Alternatively, activation of calcium channels may be associated with enhancement of the plateau phase of the slow wave without the development of spiking. Due to the small size of gastrointestinal smooth muscle cells, the rise in intracellular calcium associated with activation of voltage-gated calcium channels is believed to be adequate by itself to activate the contractile machinery. Furthermore, measurements of changes in calcium levels during the course of slow waves show an oscillation in intracellular calcium [7], that is sufficient to activate contractions in the absence of spiking [ 8 ]. I1-B. Activity in calcium free solution II-B.I. Electrical activity Although the increase in intracellular calcium associated with membrane potential changes may be adequate to activate contractions, release of intracellular calcium also participates in activation of the contractile machinery in some muscle type. Incubation of gastrointestinal smooth muscle in solutions with no added calcium containing 2-5 mM EGTA (calcium free solution, hereafter), leads to the reversible elimination of normal slow waves and spikes with replacement by an alternate form of electrical activity [9]. These latter events are characterized by slow rhythmic membrane depolarizations lasting appreciably longer than normal slow waves or spikes (see Fig. 1). The resting membrane potential depolarizes from approx. -70 mV to approx. -40 mV in calcium-free solution [9,11,13], with peak amplitude of these spontaneous electrical events approaching 0 mV. II-B.2. Depolarization activated contractions In the absence of extracellular calcium, depolarization of the plasma membrane results in activation of contractions in a variety of gastrointestinal smooth muscle preparations [10-14]. In muscle preparations bathed in calcium free solutions, contractile activity could be observed after depolarization of the plasma membrane with high potassium solutions or in one-to-one correspondence with the rhythmic membrane potential changes described above. This contractile activity is observed in intact muscle segments containing both longitudinal and circular muscle layers, as well as in thin sheets of longitudinal muscle [11]. One would not expect a 'protected' extracellular calcium source to be present in thin sheets of longitudinal muscle indicating a role for mobilization of intracellular calcium. In contrast to these studies, others [ 15-20] have reported a failure of depolarization to induce contractile activity in gastrointestinal smooth muscle during incubation in calcium free solution. Explanations that could account for the differences between studies include: (1) species differences or different tissue types may explain the lack of activation by depolarization in some studies. (2) Several studies which used either small muscle bundles or isolated smooth muscle cells may not have had the critical mass of tissue necessary for the development of electrical and mechanical activity in calcium free solution [9,12]. (3)Finally, in some studies, inadequate concentrations of potas-
sium were used to cause 'depolarization'. To illustrate this latter point, in calcium containing solutions addition o f 10-20 m M K o causes depolarization of the membrane potential from approx. - 7 0 mV (see Table I). However, in calcium free solution, the resting membrane potential of intestinal muscle reversibly depolarizes from approx. - 7 0 mV to - 4 0 mV [9,11,13 ]. Thus, the potassium equilibrium potential (EK) is still negative to the resting potential (Fro) when only modest changes ( 1 0 - 3 0 raM) in Ko are made. As illustrated in Table I, at least 40 m M potassium needs to be added to the extraceUular solution to cause a 10 mV depolarization in cells with a resting potential positive to - 4 0 mV. Thus, when the resting potential is partially depolarized (as occurs in calcium free solution) a greater increase in extracellular potassium concentration is required to further depolarize the plasma membrane.
11-B.3. Peptide-induced contractile activity The effects of peptides on gastrointestinal smooth muscle contractile activity have been examined during incubation in calcium free solution. As expected, results differ depending on species and tissue type. Cholecystokinin ( C C K ) has been shown to induce contraction o f guinea pig and human, but not cat, gastric smooth muscle in calcium free solution [14,15,17]. In calcium free solution, substance-P, but not neurotensin, stimulates contraction of cat gastric smooth muscle [14]. Pentagastrininduced contractile activity o f canine gastric smooth muscle is markedly reduced by removal of extraceUular calcium [18].
TABLE I Effects of changes in Ko on EK Ki (mM)
Ko (raM)
130 (Ref. 65)
5 10 13 20 30 40 80 5
162 (Ref. 66) 10
13 20 30 40 80
EK (mV) - 82 65 - 58 - 47 - 37 - 30 - 12
-
- 88 70 - 64 - 53 - 42 - 35 - 18
-
Effects of changes in extraceUular potassium (Ko) on the potassium equilibrium potential (EK). Data are shown for two values ofintraceUularpotassium (K[). In cells with a resting membrane potential of ~ -70 mV, modest changes (~ 20 mM) in Ko depolarizes the resting potential by ~ 20 inV. In calcium free solution where significantdepolarization of the resting potential to ~ -40 mV has occurred, a greater increase in Ko is needed to further depolarize the membrane.
In small intestinal smooth muscle, CCK initiates contractile activity in guinea pig and human circular muscle in calcium free solution, but has no effect on the longitudinal muscle layer [16]. In contrast, substance-P, neurotensin, and CCK do not induce contraction of cat small intestinal smooth muscle during incubation in calcium free solution [ 14]. In the longitudinal muscle layer of guinea pigs, substance-P stimulates contractile activity which is reduced compared to that seen in calcium containing solutions [21]. Motilin has been shown to induce only small amplitude contractions in rabbit small intestinal muscle segments during incubation in calcium-deficient solutions [22,23]. In the distal, but not proximal colon of cats, substance-P stimulates contractile activity while CCK and neurotensin have no effect in either region [14]. In rabbit colonic smooth muscle, neurotensin also does not activate contractions during exposure to calcium free solution [20]. Although peptide-induced release of intracellular calcium may play a role in the excitation-contraction process in some gastrointestinal smooth muscles, our overall impression is that the influx of calcium through membrane associated L-type channels represents the predominant source of activator calcium that causes smooth muscle contraction. Therefore, this review will focus on the modulation of gastrointestinal smooth muscle electrical activity by peptides. This review will not address modulation of neurogenically-activated events by peptides.
IlL Modulation of electrical activity by peptides IliA.
General commen~
A detailed analysis of the ionic currents responsible for generation of electrical activity in gastrointestinal smooth muscle is beyond the scope of this review. In general, the stimulated inward current associated with the spike is primarily calcium-mediated, and the outward current is produced by an increase in calcium-dependent and voltageactivated potassium channels. The resting membrane potential primarily reflects contributions by potassium conductance and an electrogenic sodium pump. The ionic basis of slow wave activity is much more varied than that of spikes. However, during slow wave depolarization a stimulation of voltage-dependent calcium channels occurs. Electrical events serve to activate contractile activity. With depolarization, voltagegated calcium channels are activated, calcium enters the cell and contraction is induced. We will, therefore, focus on changes in gastrointestinal electrical activity induced by peptides as a tool to understanding changes in contractile activity. III-B. Esophagus
Classically, the esophagus has been considered a neurally-activated tissue. Spontaneous, atropine-sensitive slow waves and spikes have been recorded from segments of esophageal smooth muscle in organ baths [24,25]. In quiescent preparations, electrical activity can be induced by addition of cholinergic agonists. These findings suggest
that release of acetylcholine from intrinsic neurons may be responsible for initiation of esophageal slow waves. To date, we have not identified studies addressing the effects of peptides on spontaneous esophageal slow wave or spike activity.
III-C. Stomach Szurszewski and colleagues [26,28-31] have examined the effects of CCK, pentagastrin and vasoactive intestinal polypeptide (VIP) on the electrical activity in the canine antrum. Three forms of CCK (CCK-8, CCK-33 and CCK-39) were tested and all increased slow wave frequency, and the amplitude and duration of the plateau phase of the antral circular muscle slow wave. No significant change in resting potential or action potential upstroke occurs [26]. The atropine insensitivity of this response demonstrates that CCK does not exert its action via release of acetylcholine from intrinsic nerves but, rather, is a result of a direct action of CCK on smooth muscle cells [26]. In strips of cat antral muscle, bombesin depolarizes the resting membrane potential, shortens the slow wave plateau duration, and increases slow wave frequency [27]. The amplitude and frequency of spikes also increases. The effects of bombesin on slow waves are independent of extracellular calcium. Recordings from antral longitudinal muscle show pentagastrin to increase the frequency of slow waves and decrease the amplitude and duration of the plateau potential [28]. In most preparations, prepotential activity is induced and a decrease in the amplitude of the spike potential is observed. Overall, no significant change in resting membrane potential occurs. In other studies, pentagastrin, little gastrin and big gastrin were examined on intact antral muscle segments [29] or strips of antral circular muscle [30]. All three agents increase the amplitude and duration of the slow wave plateau phase and the frequency of spontaneous slow waves. Pentagastrin also induces prepotential activity in the antral circular muscle layer [28]. No significant change in slow wave upstroke or in resting potential is observed. In the corpus, pentagastrin increases the amplitude and duration of the plateau potential and depolarizes the resting potential [31]. In single gastric smooth muscle cells isolated from Bufo marinus, substance-P produces a depolarization of the resting potential which is associated with a decrease in membrane conductance [32]. Voltage-clamp experiments confirm that this effect resuits primarily from supression of an outward potassium current, similar to the Mcurrent [33]. This leads to the development of a net inward current and the production of spikes and contractions. Whole-cell patch clamp recordings reveal that substance-P enhances a slowly-inactivating, high threshold current [33]. A second calcium current which is characterized by low threshold and fast inactivation kinetics is not increased by substance-P. In intact gastric muscle segments from Bufo marinus substance-P increases the frequency of slow waves with a decrease in slow wave upstroke dV/dt and amplitude and only a small depolarization of the plasma membrane [34]. Although VIP decreases the force of contraction of canine antral muscle segments, no change in spontaneous slow wave activity has been reported [ 35 ]. Furthermore, VIP does not alter acetylcholine-mediated increases in the slow wave plateau amplitude or
duration. However, VIP does inhibit pentagastrin-mediated increases in the plateau potential amplitude without altering the resting potential. Different effects of VIP have been noted in the rat stomach [36]. Dose-dependent hyperpolarization of up to 10 mV is seen in the fundus. In the rat antrum VIP inhibits spikes and, at high doses, decreases slow wave amplitude. A small hyperpolarization of the antral resting potential occurs. III-D. Small intestine
A limited number of studies have addressed the effects of peptides on the electrical activity of small intestinal smooth muscle. In rabbit duodenal muscle segments, motilin depolarizes the resting potential by approximately 11 mV, decreases both membrane resistance and slow wave amplitude, and induces minute rhythm type activity with spiking [37]. In whole-cell patch clamp studies with isolated guinea pig ileal smooth muscle cells, Nakazawa et al. [38] found that substance-P induces inward currents at negative holding potentials. This current, unlike the activity induced by acetylcholine, is increased following hyperpolarization of the membrane to values negative to -50 inV. Ion substitution studies indicate sodium to be the principal ion mediating the inward current. III-E. Colon
Because of the proposed relationship between cholecystokinin and irritable bowel syndrome [39,40], the effects of cholecystokinin on colonic electrical activity have been extensively investigated. Recordings of electrical activity from irritable bowel syndrome patients and normal controls show a 3 and 6 cycle per minute slow wave rhythm [40,41]. Compared to control subjects, patients with irritable bowel syndrome have increased 3 cycle per minute rhythms in the basal state [40]. Intravenous administration of CCK or pentagastrin increases the relative frequency of 3 cycle per minute slow wave activity [40]. In normal controls, the infusion of either CCK or gastrin I, cause an increase in electrical spiking [42]. Parallel in vitro studies with segments of cat colonic smooth muscle show an increase in colonic spike activity with addition of either the octapeptide of CCK (CCK-8) or gastrin I [43-45]. However, higher doses of both peptides caused partial interruption in slow wave activity. The effects of CCK were unaffected by muscarinic or adrenergic blockade. In longitudinal smooth muscle cells isolated from the rabbit colon, substance-P causes calcium-dependent activation of a 200-210 pS potassium channel [46,47]. Substance-P mediated activation is inhibited by the L-type calcium channel blocker nifedipine. Higher concentrations of the peptide results in transient channel activation followed by sustained inhibition. Potassium channel activation by the higher concentration of substance-P is nifedipine-insensitive, but sensitive to removal of bath calcium. It was concluded that substance-P activates two distinct calcium influx pathways in rabbit colonic smooth muscle. Substance-P depolarizes the plasma membrane of canine colonic circular muscle, intermittantly prolongs the duration of slow waves, and induces prepotential-like activity and spiking [48]. In another study with canine colonic circular muscle,
substance-P was found to initially increase slow wave duration, followed by short duration slow waves with occasional long duration events [45]. After a long duration slow wave the upstroke velocity of the next slow wave is decreased. By analogy with the effects of acetylcholine on canine colonic slow wave activity, long duration slow waves may result from prolonged activation of L-type calcium channels during the plateau phase of the slow wave [49]. Substance-P did not induce spiking in this latter study. In the distal colon of rabbits, neurotensin produces a reversible, dose-dependent depolarization of the resting potential without noticeable change in slow wave activity [50]. An increase in membrane conductance, mediated via calcium influx, is associated with depolarization of the resting potential. VIP hyperpolarizes the plasma membrane of guinea pig taenia coli [51 ]. An inhibition of spike discharge is noted in some preparations without change in the resting potential. With high concentrations of VIP, hyperpolarization and inhibition of spike discharge are routinely seen.
IV. Regulation of ion channels by intracellular pathways Receptor-mediated changes in ion channel activity are coupled through intracellular second messenger system in a variety of cell types. In gastrointestinal smooth muscle, similar pathways appear to modulate electrical activity.
IV-A. Stomach In isolated gastric smooth muscle cells from Bufo marinus, two distinct calcium currents have been identified [52]; a low threshold, rapidly inactivating current and a high threshold, slowly inactivating current. Addition of sn-l,2-dioctanoyl-glycerol, a synthetic diacylglycerol compound which activates protein kinase C (PKC), increases the high threshold current. In contrast, a different structural congener, 1,2-dioctanoyl3-thioglycerol does not activate PKC nor calcium conductance. These studies indicate that PKC is involved in the activation of the high threshold current. As acetylcholine activates the same current, it was postulated that PKC may also mediate the effects of acetylcholine on this class of calcium channels. While the activity of an outwardly rectifying potassium channel is independent of ATP, GTP, NADPH or calcium in Bufo marinus it is increased by several fatty acids including oleic and myristic acid [53,54]. This activation occurs in excised patches suggesting direct activation of channel activity independent of cytosolic mediators. In intact cells this current is also activated by an indirect pathway following the conversion of arachodonic acid to its oxygenated metabolites [53,54]. The phosphatase inhibitors, calyculin A and okadalc acid, decrease the amplitude and duration of canine gastric slow waves primarily via effects on the plateau phase of the slow wave [55]. These changes are associated with a decrease in L-type calcium channel activity and phosphorylation of calcium channels may, therefore, inhibit calcium currents. An additional control mechanism for calcium channels is linked to
increases in intracellular calcium levels which produces calcium-dependent inactivation of channel activity [56]. IV-B. Small intestine
In isolated rabbit small intestinal cells a transient inward current is activated by carbachol. This current is insensitive to addition of non-hydrolyzable analogues of GTP or GDP [57,58]. In these cells, spontaneous transient outward currents are calcium-dependent and are blocked by GTP analogues in intact cells, but not in excised patches [59,60]. The effects of G-proteins on potassium conductance are thought to be linked to changes in accessibility of intracellular calcium stores, and are not the result of a direct coupling of G-proteins to the potassium channel. IV-C. Colon
Phosphatase inhibitors also decrease slow wave amplitude and duration in the canine colon by changing the slow wave plateau [55]. Phosphorylation of calcium channels in colonic smooth muscle is presumed to lead to inhibition of calcium channel activity. In isolated rabbit colonic smooth muscle cells, a large conductance chloride channel is present [61 ]. Channel activity is voltage dependent but insensitive to changes in extracellular calcium. Channel activity is increased by non-hydrolyzable analogues of GTP and reversible inhibited by non-hydrolyzable analogues of GDP. As these effects occur in inside-out patches, they are independent of changes in any cytosolic mediator and can be attributed to a direct G-protein effect on the channel.
V. Summary A rise in intracellular calcium is the predominant signal that leads to the activation of the contractile machinery in gastrointestinal smooth muscle. The primary sources of activating calcium are illustrated in Fig. 2. Voltage- and peptide-mediated release of intracellular calcium contribute to activation of some gastrointestinal smooth muscles. However, the primary source of activating calcium appears to be an influx of calcium across the plasma membrane. The degree of modulation of electrical activity by peptides varies depending upon the region of the gastrointestinal tract studied. Second messenger systems are undoubtly involved in the transduction pathway for receptor-mediated changes in ion channel activity in gastrointestinal smooth muscle. However, in comparison to other excitable cell types, little is known about the coupling mechanisms whereby peptide-receptor binding alters ion channel activity in gastrointestinal smooth muscle. This represents one of the challenging areas to be studied in the field of gastrointestinal smooth muscle. One disease in which a better appreciation of the regulation of ion channel activity could lead to therapeutic benefit is irritable bowel syndrome. A coupling of smooth muscle electrical activity to hypermotility in irritable bowel syndrome has been re-
t Ca;2 Fig. 2. Sources
of calcium
for activation
cium (Ca” + ) in gastrointestinal stored potential
calcium.
Arrow
thickness
or voltage operated
has been adapted vating calcium
of gastrointestinal
indicates
channels
relative smooth
to be influx of calcium
muscle through
tissues differences
muscle.
influx pathways
importance
while ROC indicates
from studies with vascular
is presumed
smooth
smooth muscle occurs through
Increases
of the respective
receptor
operated
[67]. As shown, voltage-gated
in intracellular
cal-
and release of intracellularily pathway.
channels.
the primary
channels,
POC denotes
This terminology source of acti-
although
species and
may exist.
ported. CCK increases the level of spike activity which triggers hyperrnotility [40]. It would follow that inhibition of calcium influx should reduce spiking and, therefore, hypermotility. In fact, the calcium channel blockers nifedipine and nicardipine have been shown to decrease colonic motility in irritable bowel syndrome patients [ 62-641. As our understanding of gastrointestinal smooth muscle ion channels expands, development of a gastrointestinal selective calcium channel blocker may be possible. This class of agents would be effective in the treatment of irritable bowel syndrome and potentially other peptide-related spastic smooth muscle disorders.
Acknowledgements
Support from NIH DK08452, DK38216; Glaxo Institute for Digestive Health Basic Research Award; a Glaxo Cardiovascular Discovery Grant; and VA funds are acknowledged.
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J. H., Electrical
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(Ed.), Physiology in colonic
of the
electrical
ac-
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